CN109097370A - A method of regulation tea tree fine hair formation - Google Patents
A method of regulation tea tree fine hair formation Download PDFInfo
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Abstract
本发明提供一种调控茶树茸毛形成的方法，包括以下步骤：S1.对转录因子CsTTG1进行生物信息学分析，构建进化树;S2.将目标基因CsTTG1与载体质粒pBI121重组，构建了茶树CsTTG1基因的过表达载体PBI121‑CsTTG1;S3.将过表达载体PBI121‑CsTTG1并转导农杆菌GV1301中；S4.在农杆菌GV1301介导下，过表达载体pBI121‑CsTTG1转导到具有与转录因子CsTTG1同源性因子的茶树作物中。本发明通过CsTTG1基因在茶树种的特异性表达，从而获得对茶树茸毛形成的调控，为后期茶树分子育种、遗传改良及茶树转基因体系提供了理论基础和可行方法。The present invention provides a method for regulating the formation of tea tree hairs, comprising the following steps: S1. Carrying out bioinformatics analysis on the transcription factor CsTTG1 , and constructing an evolutionary tree; S2. Recombining the target gene CsTTG1 with the carrier plasmid pBI121, and constructing the CsTTG1 gene of the tea tree Overexpression vector PBI121‑ CsTTG1 ; S3. Overexpression vector PBI121‑ CsTTG1 is transduced into Agrobacterium GV1301; S4. Under the mediation of Agrobacterium GV1301, the overexpression vector pBI121‑ CsTTG1 is transduced into a gene with homology with the transcription factor CsTTG1 Sex factor in tea crops. The present invention regulates the formation of tea tree fuzz through the specific expression of CsTTG1 gene in tea tree species, and provides a theoretical basis and feasible method for later stage tea tree molecular breeding, genetic improvement and tea tree transgenic system.
Description
Technical field
The invention belongs to biological Cultivating techniques fields, and in particular to a method of regulation tea tree fine hair formation.
Background technique
Tea tree belong to Theaceae (Theaceae) Camellia (Camellia L.), it is perennial evergreen leaf plant, origin
In In Southwest China-Yunnan-Guizhou Plateau.Tea at least millennial history from eating to drinking, to a certain extent, tealeaves is
The label of China, it is various in style, cultivated area is big.The concentration of fine hair is the critically important economic characters of tea tree, is also simultaneously
Evaluate one of tealeaves shape and the great influence factor of taste quality, such as height famous with aobvious milli, good quality in many well-known teas in China
Grade keemun, Yunnan black tea are appeared with golden milli；Mount Huang Mao Feng, Xinyang Maojian Tea etc. are mainly appeared with pekoe；Junshan Silver Needle Tea covers and pushes up the masters such as yellow bud
It is significant with gold hair.However the growth and development process of fine hair is complex, at present both at home and abroad without being formed for tea tree fine hair
The research of mechanism and relevant molecule mechanism etc., the direction of research be concentrated mainly on the morphological feature of fine hair, Physiology and biochemistry and
Resistance etc..
The tender leaf of tea tree is the primary raw material of tea making, and the tea leaf quality of especially period in early spring picking is splendid, tea tree tender shoots
Contain higher amino acid and less polyphenols in fine hair, assigns the more delicate flavours of millet paste and less astringent taste.Tea tree fine hair
Quality and quantity be evaluate tea tree breed economy one of major traits, the fine hair of tea tree bud-leaf is more, shows tea tree germ
Ye Yue is tender, while its quality is also better.It can also be used as judging at the degree that shows there are quantity and in millet paste of tea milli in tea
The important indicator of tea leaf quality height.The chemical components such as tea polyphenols contained in fine hair, catechin and caffeine are all greatly
It is measured lower than contained in tealeaves；The crude fibre contained in fine hair is significantly higher than the content in tealeaves, in brewing process, only fine hair
Contained fiber can be dissolved into millet paste together with the fine hair to fall off, be absorbed by the body.
Tealeaves shape quality can be improved in fine hair in tealeaves.Many well-known teas include black tea, white tea etc. in the production process,
Require that tea milli can reveal, to improve dry tea shape quality and millet paste taste quality, such as advanced keemun, Yunnan black tea, Mount Huang
Mao Feng, silver tip pekoe, Xinyang Maojian Tea etc..The formation of tealeaves fine hair largely positive influence taste quality, in process
In, due to the external force for finishing and rubbing, the part fine hair in fresh leaf can be fallen off, and be attached on tealeaves surface.Brew it
Afterwards, be attached in tealeaves tea milli can be dissolved in millet paste completely, amino acid therein and it is other circulated in millet paste at branch, increase
The fragrance and flavour of millet paste, improve the taste quality of millet paste.Meanwhile fine hair is the biologic-organ with special heat insulation function,
The product of Evolution Development, be species to ecological environment natural selection as a result, can significantly increase to biology and abiotic stress
Resistance.Therefore, extremely important for the regulation of tea tree fine hair formation, theory can be provided for tea tree breed breeding and genetic improvement
Foundation, to improve the productivity effect of Tea Industry.
Existing research discovery finds homologous gene relevant with regulation arabidopsis epidermal hair growth and development in tea treeAtTTG1
Expression pattern is related, and is named asCsTTG1.Therefore, it is linearly closed by identification CsTTG1 gene with the growth and development of tealeaves fine hair
It is, isCsTTG1The relationship that the expression pattern of gene is developed with fine hair provides the foundation.
Summary of the invention
In view of this, the present invention provides a kind of method that regulation tea tree fine hair is formed, the present invention passes throughCsTTG1Gene exists
Tea tree kind it is specific expressed, to obtain the regulation that is formed to tea tree fine hair.
The technical solution of the present invention is as follows: a kind of method that regulation tea tree fine hair is formed, which is characterized in that including following step
It is rapid:
S1. to transcription factorCsTTG1Bioinformatic analysis is carried out, chadogram is constructed, other regulations is confirmed by chadogram
The homologous gene of fine hair confirms transcription factorCsTTG1 isTarget gene。
S2. by target geneCsTTG1It is recombinated with vector plasmid pBI121, constructs tea treeCsTTG1Gene crosses table
Up to carrier PBI121- CsTTG1 ;
S3. by over-express vector PBI121- CsTTG1And in the Agrobacterium GV1301 that transduces；
S4. under Agrobacterium GV1301 mediation, over-express vector pBI121- CsTTG1It transduces to having and transcription factorCsTTG1In the tea tree crop of homologous sex factor.
Further include step S5, specifically by after screening and culturing medium, take root and transplant lowerly, extract above-mentioned tea tree crop
DNA carries out PCR detection, while analyze using quantitative fluorescent PCR, verification result plasmid pBI121-CsTTG1Succeed
It is transferred to.
The chadogram is shown below:
。
In above formula, tea tree (Camellia sinensis (L.) O. Ktze):CsTTG1；Arabidopsis (Arabidopsis thaliana):AtTTG1(NM_180738.3)；Cucumber (Cucumis sativus):CsTTG1(EU215443.1)；Apple
(Malus domestica):MdTTG1(AF220203.1)；Rape (Brassica rapa):BrTTG1(XM_
009152730.2:154-1167)；Cabbage type rape (Brassica napus):BnTTG1(XM_013788228.2)；Wild cabbage
Type rape (Brassica napus):BnTTG1-2(NM_001316225.1) mulberries (Morus notabilis):MnTTG1
(XM_010101113.1)；Wild cabbage (Brassica oleracea):BoTTG1(XM_013743986.1)；Tobacco
(Nicotiana tabacum):NtCsTTG1(XM_019387918.1)；Upland cotton (Gossypium hirsutum):CsTTG1(NM_001327545.1)；Tree cotton: (Gossypium arboreum):GaTTG1(JQ005876.1).
In the present invention, preferred means of the cDNA library as clone's correlated traits.Seminar is by tea where the present inventor
Construction cDNA library is set, and screens gene relevant to And Development of Tea Shoot fine hair growth and development, from the sequencing transcription of " purple Juan "
Group is found and arabidopsisAtTTG1Homologous WD repetitive proteins, therefore be named asCsTTG1.This chapter will be to the tea tree of acquisition
Young sprout fine hairCsTTG1The cDNA sequence of gene carries out bioinformatic analysis.Utilize ncbi database pairCsTTG1Sequence carries out
Search, analyzes the similitude of its nucleotide and amino acid；DNASTAR translation on line nucleotide sequence, Protparam meter
Calculate the relation analysis amount and theoretical isoelectric point of protein；And chadogram is constructed in MEGA 6.0, regulate and control for subsequent tea tree fine hair
It lays the foundation.Present inventor is by a large amount of creative realizations, eventually by transcription factorCsTTG1Carry out biological information
Credit analysis, constructs chadogram, and the homologous gene of other regulation fine hair is confirmed by chadogram, confirms transcription factorCsTTG1 isMesh
Mark gene。
Further, in step S2, over-express vector PBI121-The construction method of CsTTG1 includes:
A tail will be increased in the prior artCsTTG1Target fragment is connected with pBI121 plasmid vector using T4-DNA ligase
Connect, wherein linked system are as follows: according to target fragment: it is 15-30ul that overall reaction system, which is added, in the ratio that carrier segments are 5-2:1
In, it is specific as follows:CsTTG1Target fragment 250-60ng, pBI121 plasmid vector 50-30ng, T4-DNA ligase 0.2-
0.7ul, T4-DNA ligase Buffer 1-4ul mend sterile water ddH2O to 15-30ul, liquid-transfering gun, which is gently inhaled, plays mixing,
It is placed in PCR instrument, 16 DEG C of connections are overnight.
Further, in step S2, over-express vector PBI121-The construction method of CsTTG1 includes:
A tail will be increasedCsTTG1Target fragment is attached with pBI121 plasmid vector using T4-DNA ligase, wherein even
Junctor system are as follows: according to 3 target fragments: the ratios of 1 carrier segments is added overall reaction system and is in 20ul, specific as follows:CsTTG1
Target fragment 150ng, pBI121 plasmid vector 50ng, T4-DNA ligase0.5ul, T4-DNA ligase Buffer2ul,
Mend sterile water ddH2O to 20ul, liquid-transfering gun, which is gently inhaled, plays mixing, is placed in PCR instrument, and 16 DEG C of connections are overnight.
Further, in step S3 include in detail below operate: prepare connection product conversion bacillus coli DH 5 alpha competence,
Plasmid DNA extraction, the preparation of Agrobacterium GV3101 competence, recombinant plasmid transformed Agrobacterium competent cell.
Further, connection product conversion bacillus coli DH 5 alpha competence the following steps are included:
(1) by bacillus coli DH 5 alpha strain, gently cell is suspended after thawing completely on ice；
(2) after the mixing of 5ul connection product is added, it is placed in 30min on ice；
(3) 42 DEG C of water-baths are held successfully in advance, centrifuge tube is discharged water 90s in bath, is transferred quickly on ice, ice bath 2min；
(4) the fresh LB liquid medium of 800ul, 37 DEG C of shaking table culture 1h are added；
(5) 2min is centrifuged with 8000rmp, fresh 100ul LB culture solution suspension cell is added；
(6) appropriate bacterium solution is coated on the plate containing corresponding antibiotic, 37 DEG C of culture 12-16h；
(7) 12-16 h is cultivated in insulating box, is picked from the plate a single colonie with transfer needle, is inoculated into containing 10mL liquid
LB is cultivated in 50ml triangular flask, in 37 DEG C of shaking tables, 200 rpm shaken cultivation 10-12 h.
Further, the liquid LB includes 0.5-3% peptone, 0.2-0.7% yeast extract, 0.7-1.6% chlorination
Sodium, the pH of the liquid LB are 6.5-7.8.
Further, the Plasmid DNA extracting method is as follows: clone's bacterial plaque that picking has been identified is containing corresponding antibiosis
In the 5mL LB liquid medium of element, 37 DEG C, 200 rpm shaken cultivation 12-14 h, the bacterium solution shaken alkaline lysis method of extracting matter
Grain DNA, includes step in detail below:
(1) for the bacterium solution that absorption 2-4 m1 has shaken in 2mL centrifuge tube, 12000 rpm are centrifuged 1 min, reject supernatant；
(2) be added 200 μ L solution I, the solution I include 150 mmol/L glucose, 25 mmol/L Tris-HCl, pH8.0,
L0 mmol/L EDTA, pH8.0 acutely vibrate, and thallus is resuspended；
(3) be added the solution II that has just prepared of 200 μ L, the solution II includes 0.2 mol/L NaOH, 1% SDS, gently up and down
Centrifuge tube 3-5 times mixing of overturning, ice bath 5min；
(4) be added the solution III newly prepared of 200 μ L, the solution III include 5 M Kac 60mL, glacial acetic acid 11.5mL,
ddH228.5 mL of O gently overturns 3-5 mixing of centrifuge tube, 5 min of ice bath up and down；
(5) 12000 rpm are centrifuged 12 min；
(6) Aspirate supernatant is placed in new centrifuge tube, and isometric pre- cold isopropanol is added, mixes gently, is placed at room temperature for 2
Min, 12000 rpm are centrifuged 5 min, remove supernatant；
(7) the RNase-TE solution of 50 μ L, 10 μ g/mL, 37 DEG C of water-bath 30min are added；
(8) 50 μ L TE solution and isometric phenol/chloroform are added, 1min, 12000rpm are acutely vibrated
It is centrifuged 5 min；
(9) it takes and is suspended in the limpid liquid of top layer, isometric phenol/chloroform is added, 12000 rpm are centrifuged 2 min；
(10) supernatant is taken, the dehydrated alcohol of 1/10 volume, 3 M NaAC and the pre-cooling of 2 times of volumes is added, is stored at room temperature 2 min,
12000 rpm are centrifuged 2 min；
(11) supernatant is abandoned, then is precipitated 2-3 times with 70% ethanol washing, drying at room temperature 30min；
(12) 20-50 μ LTE or ddH is used2O dissolution precipitating, i.e. Plasmid samples, -20 DEG C of preservations.
Further, the Agrobacterium GV3101 competence preparation the following steps are included:
(1) the GV3101 bacterium solution of preservation is crossed on the YEP solid medium containing rifampin, training is inverted in 28 DEG C of insulating boxs
Support 24-36h；
(2) picking Agrobacterium GV3101 single colonie is inoculated into 2.0 mL YEP fluid nutrient mediums, 28 DEG C of shaking table 200rpm oscillations
Overnight incubation；
(3) bacterium solution for taking 500 μ L to shake is placed in 50 mL YEP culture solutions, 28 DEG C of shaken cultivation 5-6h
To OD600Value is 0.6；
(4) bacterium solution is poured into 50 mL centrifuge tubes of sterilization treatment, 5000 rpm are centrifuged 5 min and collect bacterium solution, abandon supernatant；
(5) thallus is resuspended in the NaCI of 10 mL, 0.15 mol/L, 5000 rpm are centrifuged 5 min；
(6) the 20 mM CaCl that thallus l ml is pre-chilled2Solution gently suspends, and 200 μ L packing, -80 DEG C save backup.
Recombinant plasmid transformed Agrobacterium competent cell the following steps are included:
(1) it takes the 100ul Agrobacterium competent cell of -80 DEG C of preservations to be placed on ice, cell gently suspends after being completely dissolved；
(2) 5 μ L recombinant plasmids are added, mixing, 30 min of ice bath are gently blown and beaten with pipette tips；
(3) centrifuge tube is put into quick-frozen 5 min in liquid nitrogen, heat shock 1min in 37 DEG C of water-baths goes to rapidly 2-3 min on ice；
(4) the YEP fluid nutrient medium of 1 mL antibiotic-free is added into centrifuge tube, mixes gently, is placed in 28 DEG C of insulating boxs and shakes
Swing 1 h；
(5) 4000 rpm are centrifuged 5 min, remove supernatant, and 100 μ L YEP liquid suspension cells are added；
(6) bacterium solution is coated in the YEP solid medium tablets containing corresponding antibiotic, 28 DEG C of culture 32-48 h；
(7) picking Agrobacterium single bacterium falls in YEP culture solution and sways 24 h for 28 DEG C, is identified by bacterium solution PCR, and result is the positive
Illustrate expression vector establishment success.
Further, in step S4, over-express vector pBI121-CsTTG1'sTransduction comprises the steps of:
(1) pBI121- of the carrying target gene of -80 DEG C of preservations is takenCsTTG1Agrobacterium liquid thaws, and is dipped respectively with transfer needle
A small amount of bacterium solution is drawn on the solid LB plating medium that attached 30-70ug/ml rifampin and 80-115ug/ml kanamycins
Line is inverted dark culturing 36-48h in 28 DEG C of insulating boxs；
(2) picking positive monoclonal bacterium colony wherein contains 30-70ug/ml in LB culture solution into 0.5-1.2mL LB culture solution
Rifampin and 80-115ug/ml kanamycins, 28 DEG C of shaking table 130-220rpm oscillation activation 18-24h, until bacterium solution OD value reaches
0.4-0.8；
(3) it takes whole bacterium solutions in step (2) into 35-60ml fresh YEP culture solution, wherein contains in YEP culture solution
30-70ug/ml rifampin and 80-115ug/ml kanamycins, identical condition relays persistent oscillation 13-18h in shaking table, until bacterium
Liquid OD value reaches 0.4-0.8；
(4) 3500-6000rpm is centrifuged 10-20min, removes supernatant, collects bacterium solution；
(5) isometric infected liquid is added, bacterium solution is suspended and is mixed；
(6) SilwetL-77 to final concentration of 0.01-0.04% wt/vol is added before infecting, mixes well, transfer mixed liquor in
In 500ml beaker；
(7) handstand plant is completely immersed in inflorescence in Agrobacterium, shakes gently bacterium solution 30s, plant is removed from solution, with guarantor
Fresh film will infect portion envelops and live moisturizing, dry the liquid in base of the plant；
(8) plant is put on polybag, 22 DEG C of dark cultures are placed under normal lighting conditions for 24 hours and cultivate；
(9) it infects once, infects three times again after 7 days；
(10) about after two months, single plant collects mature seed for growth, is placed in 4 DEG C of refrigerators and saves.
Fine hair is the main feature of And Development of Tea Shoot, and the length of fine hair, density, rugosity are the main features of tea tree breed,
Quality components, the appearance and mouthfeel to tea such as tea polyphenols rich in, amino acid, caffeine form important shadow in tea milli
It rings, tea tree fine hair can improve the resistance to biology and abiotic stress, however the fine hair of tea tree is stopped in the area research at present
On biochemical characteristic, not to the research in terms of the related gene of tea tree fine hair formation, therefore inquires into regulation tea tree fine hair and formed
The molecule mechanism of development has important scientific meaning to the development of Tea Breeding.The present invention utilizes tea tree from genetic transformation level
The molecular mechanism that fine hair is formed, passes throughCsTTG1Gene is specific expressed tea tree kind, is formed to obtain to tea tree fine hair
Regulation, provide theoretical basis and feasible method for later period tea tree molecular breeding, genetic improvement and tea tree transformation system.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with embodiment, it is clear that described reality
Applying example is only a part of the embodiment of the present invention, instead of all the embodiments.
Embodiment 1
A method of regulation tea tree fine hair formation, which comprises the following steps:
S1. to transcription factorCsTTG1Bioinformatic analysis is carried out, chadogram is constructed;
S2. by target geneCsTTG1It is recombinated with vector plasmid pBI121, constructs tea treeCsTTG1The overexpression of gene carries
Body PBI121- CsTTG1 ;
S3. by over-express vector PBI121- CsTTG1And in the Agrobacterium GV1301 that transduces；
S4. under Agrobacterium GV1301 mediation, over-express vector pBI121- CsTTG1It transduces to having and transcription factorCsTTG1In the tea tree crop of homologous sex factor.
Further include step S5, specifically by after screening and culturing medium, take root and transplant lowerly, extract above-mentioned tea tree crop
DNA carries out PCR detection, while analyze using quantitative fluorescent PCR, verification result plasmid pBI121-CsTTG1Succeed
It is transferred to.
The chadogram is shown below:
。
In above formula, tea tree (Camellia sinensis (L.) O. Ktze):CsTTG1；Arabidopsis (Arabidopsis thaliana):AtTTG1(NM_180738.3)；Cucumber (Cucumis sativus):CsTTG1(EU215443.1)；Apple
(Malus domestica):MdTTG1(AF220203.1)；Rape (Brassica rapa):BrTTG1(XM_
009152730.2:154-1167)；Cabbage type rape (Brassica napus):BnTTG1(XM_013788228.2)；Wild cabbage
Type rape (Brassica napus):BnTTG1-2(NM_001316225.1) mulberries (Morus notabilis):MnTTG1
(XM_010101113.1)；Wild cabbage (Brassica oleracea):BoTTG1(XM_013743986.1)；Tobacco
(Nicotiana tabacum):NtCsTTG1(XM_019387918.1)；Upland cotton (Gossypium hirsutum):CsTTG1(NM_001327545.1)；Tree cotton: (Gossypium arboreum):GaTTG1(JQ005876.1).
Further, in step S2, over-express vector PBI121-The construction method of CsTTG1 includes:
By increase A tail in the prior artCsTTG1Target fragment and pBI121 plasmid vector are carried out using T4-DNA ligase
Connection, wherein linked system are as follows: according to target fragment: ratio that carrier segments are 3:1 is added overall reaction system and is in 20ul,
It is specific as follows:CsTTG1Target fragment 150ng, pBI121 plasmid vector 50ng, T4-DNA ligase0.5ul, T4-DNA
Ligase Buffer2ul mends sterile water ddH2O to 20ul, liquid-transfering gun, which is gently inhaled, plays mixing, is placed in PCR instrument, 16 DEG C of connections
Overnight.
Further, in step S3 include in detail below operate: prepare connection product conversion bacillus coli DH 5 alpha competence,
Plasmid DNA extraction, the preparation of Agrobacterium GV3101 competence, recombinant plasmid transformed Agrobacterium competent cell.
Further, connection product conversion bacillus coli DH 5 alpha competence the following steps are included:
(1) by bacillus coli DH 5 alpha strain, gently cell is suspended after thawing completely on ice；
(2) after the mixing of 5ul connection product is added, it is placed in 30min on ice；
(3) 42 DEG C of water-baths are held successfully in advance, centrifuge tube is discharged water 90s in bath, is transferred quickly on ice, ice bath 2min；
(4) the fresh LB liquid medium of 800ul, 37 DEG C of shaking table culture 1h are added；
(5) 2min is centrifuged with 8000rmp, fresh 100ul LB culture solution suspension cell is added；
(6) appropriate bacterium solution is coated on the plate containing corresponding antibiotic, 37 DEG C of culture 14h；
(7) 14 h are cultivated in insulating box.A single colonie is picked from the plate with transfer needle, is inoculated into and is trained containing 10mL liquid LB
It supports in 50ml triangular flask, in 37 DEG C of shaking tables, 200 rpm shaken cultivation, 11 h.
Further, the liquid LB includes 1% peptone, 0.4% yeast extract, 1.1% sodium chloride, the liquid LB
PH be 7.1.
Further, the Plasmid DNA extracting method is as follows: clone's bacterial plaque that picking has been identified is containing corresponding antibiosis
In the 5mL LB liquid medium of element, 37 DEG C, 200 rpm shaken cultivation 13h, the bacterium solution shaken alkaline lysis method of extracting plasmid
DNA includes step in detail below:
(1) for the bacterium solution that 3 m1 of absorption have shaken in 2mL centrifuge tube, 12000 rpm are centrifuged 1 min, reject supernatant；
(2) be added 200 μ L solution I, the solution I include 150 mmol/L glucose, 25 mmol/L Tris-HCl, pH8.0,
L0 mmol/L EDTA, pH8.0 acutely vibrate, and thallus is resuspended；
(3) be added the solution II that has just prepared of 200 μ L, the solution II includes 0.2 mol/L NaOH, 1% SDS, gently up and down
Centrifuge tube 4 times mixings of overturning, ice bath 5min；
(4) be added the solution III newly prepared of 200 μ L, the solution III include 5 M Kac 60mL, glacial acetic acid 11.5mL,
ddH228.5 mL of O gently overturns 3-5 mixing of centrifuge tube, 5 min of ice bath up and down；
(5) 12000 rpm are centrifuged 12 min；
(6) Aspirate supernatant is placed in new centrifuge tube, and isometric pre- cold isopropanol is added, mixes gently, is placed at room temperature for 2
Min, 12000 rpm are centrifuged 5 min, remove supernatant；
(7) the RNase-TE solution of 50 μ L, 10 μ g/mL, 37 DEG C of water-bath 30min are added；
(8) 50 μ L TE solution and isometric phenol/chloroform are added, 1min, 12000rpm are acutely vibrated
It is centrifuged 5 min；
(9) it takes and is suspended in the limpid liquid of top layer, isometric phenol/chloroform is added, 12000 rpm are centrifuged 2 min；
(10) supernatant is taken, the dehydrated alcohol of 1/10 volume, 3 M NaAC and the pre-cooling of 2 times of volumes is added, is stored at room temperature 2 min,
12000 rpm are centrifuged 2 min；
(11) supernatant is abandoned, then is precipitated 2-3 times with 70% ethanol washing, drying at room temperature 30min；
(12) it is dissolved and is precipitated with 35 μ LTE, is i.e. Plasmid samples, -20 DEG C of preservations.
Further, the Agrobacterium GV3101 competence preparation the following steps are included:
(1) the GV3101 bacterium solution of preservation is crossed on the YEP solid medium containing rifampin, training is inverted in 28 DEG C of insulating boxs
Support 28h；
(2) picking Agrobacterium GV3101 single colonie is inoculated into 2.0 mL YEP fluid nutrient mediums, 28 DEG C of shaking table 200rpm oscillations
Overnight incubation；
(3) bacterium solution for taking 500 μ L to shake is placed in 50 mL YEP culture solutions, 28 DEG C of shaken cultivation 5.4h
To OD600Value is 0.6；
(4) bacterium solution is poured into 50 mL centrifuge tubes of sterilization treatment, 5000 rpm are centrifuged 5 min and collect bacterium solution, abandon supernatant；
(5) thallus is resuspended in the NaCI of 10 mL, 0.15 mol/L, 5000 rpm are centrifuged 5 min；
(6) the 20 mM CaCl that thallus l ml is pre-chilled2Solution gently suspends, and 200 μ L packing, -80 DEG C save backup.
Further, the recombinant plasmid transformed Agrobacterium competent cell the following steps are included:
(1) it takes the 100ul Agrobacterium competent cell of -80 DEG C of preservations to be placed on ice, cell gently suspends after being completely dissolved；
(2) 5 μ L recombinant plasmids are added, mixing, 30 min of ice bath are gently blown and beaten with pipette tips；
(3) centrifuge tube is put into quick-frozen 5 min in liquid nitrogen, heat shock 1min in 37 DEG C of water-baths goes to rapidly 2-3 min on ice；
(4) the YEP fluid nutrient medium of 1 mL antibiotic-free is added into centrifuge tube, mixes gently, is placed in 28 DEG C of insulating boxs and shakes
Swing 1 h；
(5) 4000 rpm are centrifuged 5 min, remove supernatant, and 100 μ L YEP liquid suspension cells are added；
(6) bacterium solution is coated in the YEP solid medium tablets containing corresponding antibiotic, 28 DEG C of culture 42h；
(7) picking Agrobacterium single bacterium falls in YEP culture solution and sways 24 h for 28 DEG C, is identified by bacterium solution PCR, and result is the positive
Illustrate expression vector establishment success.
Further, in step S4, over-express vector pBI121-CsTTG1'sTransduction comprises the steps of:
(1) pBI121- of the carrying target gene of -80 DEG C of preservations is takenCsTTG1Agrobacterium liquid thaws, and is dipped respectively with transfer needle
A small amount of bacterium solution is crossed on the solid LB plating medium that attached 50ug/ml rifampin and 100ug/ml kanamycins, in 28
Dark culturing 42h is inverted in DEG C insulating box；
(2) picking positive monoclonal bacterium colony is into 0.8mL LB culture solution, wherein in LB culture solution containing 50ug/ml rifampin and
100ug/ml kanamycins, 28 DEG C of shaking table 180rpm oscillation activation 20h, until bacterium solution OD value reaches 0.6；
(3) it takes whole bacterium solutions in step (2) into 35-60ml fresh YEP culture solution, wherein contains in YEP culture solution
50ug/ml rifampin and 100ug/ml kanamycins, identical condition relays persistent oscillation 13-18h in shaking table, until bacterium solution OD value
Reach 0.6；
(4) 4500rpm is centrifuged 13min, removes supernatant, collects bacterium solution；
(5) isometric infected liquid is added, bacterium solution is suspended and is mixed；
(6) SilwetL-77 to final concentration of 0.02% wt/vol is added before infecting, mixes well, shifts mixed liquor in 500ml
In beaker；
(7) handstand plant is completely immersed in inflorescence in Agrobacterium, shakes gently bacterium solution 30s, plant is removed from solution, with guarantor
Fresh film will infect portion envelops and live moisturizing, dry the liquid in base of the plant；
(8) plant is put on polybag, 22 DEG C of dark cultures are placed under normal lighting conditions for 24 hours and cultivate；
(9) it infects once, infects three times again after 7 days；
(10) about after two months, single plant collects mature seed for growth, is placed in 4 DEG C of refrigerators and saves.
Embodiment 2
A method of regulation tea tree fine hair formation, which comprises the following steps:
S1. to transcription factorCsTTG1Bioinformatic analysis is carried out, chadogram is constructed;
S2. by target geneCsTTG1It is recombinated with vector plasmid pBI121, constructs tea treeCsTTG1The overexpression of gene carries
Body PBI121- CsTTG1 ;
S3. by over-express vector PBI121- CsTTG1And in the Agrobacterium GV1301 that transduces；
S4. under Agrobacterium GV1301 mediation, over-express vector pBI121- CsTTG1It transduces to having and transcription factorCsTTG1In the tea tree crop of homologous sex factor.
Further include step S5, specifically by after screening and culturing medium, take root and transplant lowerly, extract above-mentioned tea tree crop
DNA carries out PCR detection, while analyze using quantitative fluorescent PCR, verification result plasmid pBI121-CsTTG1Succeed
It is transferred to.
The chadogram is shown below:
。
Further, in step S2, over-express vector PBI121-The construction method of CsTTG1 includes:
A tail will be increased in the prior artCsTTG1Target fragment is connected with pBI121 plasmid vector using T4-DNA ligase
Connect, wherein linked system are as follows: according to target fragment: it is tool in 30ul that overall reaction system, which is added, in the ratio that carrier segments are 5:1
Body is as follows:CsTTG1Target fragment 250ng, pBI121 plasmid vector 50ng, T4-DNA ligase 0.7ul, T4-DNA
Ligase Buffer 4ul mends sterile water ddH2O to 30ul, liquid-transfering gun, which is gently inhaled, plays mixing, is placed in PCR instrument, 16 DEG C of connections
Overnight.
Further, in step S3 include in detail below operate: prepare connection product conversion bacillus coli DH 5 alpha competence,
Plasmid DNA extraction, the preparation of Agrobacterium GV3101 competence, recombinant plasmid transformed Agrobacterium competent cell.
Further, connection product conversion bacillus coli DH 5 alpha competence the following steps are included:
(1) by bacillus coli DH 5 alpha strain, gently cell is suspended after thawing completely on ice；
(2) after the mixing of 5ul connection product is added, it is placed in 30min on ice；
(3) 42 DEG C of water-baths are held successfully in advance, centrifuge tube is discharged water 90s in bath, is transferred quickly on ice, ice bath 2min；
(4) the fresh LB liquid medium of 800ul, 37 DEG C of shaking table culture 1h are added；
(5) 2min is centrifuged with 8000rmp, fresh 100ul LB culture solution suspension cell is added；
(6) appropriate bacterium solution is coated on the plate containing corresponding antibiotic, 37 DEG C of culture 12-16h；
(7) 16 h are cultivated in insulating box.A single colonie is picked from the plate with transfer needle, is inoculated into and is trained containing 10mL liquid LB
It supports in 50ml triangular flask, in 37 DEG C of shaking tables, 200 rpm shaken cultivation, 12 h.
Further, the liquid LB includes 3% peptone, 0.7% yeast extract, 1.6% sodium chloride, the liquid LB
PH be 7.8.
Further, the Plasmid DNA extracting method is as follows: clone's bacterial plaque that picking has been identified is containing corresponding antibiosis
In the 5mL LB liquid medium of element, 37 DEG C, 200 rpm shaken cultivation, 14 h, the bacterium solution shaken alkaline lysis method of extracting plasmid
DNA includes step in detail below:
(1) for the bacterium solution that 4 m1 of absorption have shaken in 2mL centrifuge tube, 12000 rpm are centrifuged 1 min, reject supernatant；
(2) be added 200 μ L solution I, the solution I include 150 mmol/L glucose, 25 mmol/L Tris-HCl, pH8.0,
L0 mmol/L EDTA, pH8.0 acutely vibrate, and thallus is resuspended；
(3) be added the solution II that has just prepared of 200 μ L, the solution II includes 0.2 mol/L NaOH, 1% SDS, gently up and down
Centrifuge tube 3-5 times mixing of overturning, ice bath 5min；
(4) be added the solution III newly prepared of 200 μ L, the solution III include 5 M Kac 60mL, glacial acetic acid 11.5mL,
ddH228.5 mL of O gently overturns 3-5 mixing of centrifuge tube, 5 min of ice bath up and down；
(5) 12000 rpm are centrifuged 12 min；
(6) Aspirate supernatant is placed in new centrifuge tube, and isometric pre- cold isopropanol is added, mixes gently, is placed at room temperature for 2
Min, 12000 rpm are centrifuged 5 min, remove supernatant；
(7) the RNase-TE solution of 50 μ L, 10 μ g/mL, 37 DEG C of water-bath 30min are added；
(8) 50 μ L TE solution and isometric phenol/chloroform are added, 1min, 12000rpm are acutely vibrated
It is centrifuged 5 min；
(9) it takes and is suspended in the limpid liquid of top layer, isometric phenol/chloroform is added, 12000 rpm are centrifuged 2 min；
(10) supernatant is taken, the dehydrated alcohol of 1/10 volume, 3 M NaAC and the pre-cooling of 2 times of volumes is added, is stored at room temperature 2 min,
12000 rpm are centrifuged 2 min；
(11) supernatant is abandoned, then is precipitated 2-3 times with 70% ethanol washing, drying at room temperature 30min；
(12) 50L ddH is used2O dissolution precipitating, i.e. Plasmid samples, -20 DEG C of preservations.
Further, the Agrobacterium GV3101 competence preparation the following steps are included:
(1) the GV3101 bacterium solution of preservation is crossed on the YEP solid medium containing rifampin, training is inverted in 28 DEG C of insulating boxs
Support 36h；
(2) picking Agrobacterium GV3101 single colonie is inoculated into 2.0 mL YEP fluid nutrient mediums, 28 DEG C of shaking table 200rpm oscillations
Overnight incubation；
(3) bacterium solution for taking 500 μ L to shake is placed in 50 mL YEP culture solutions, 28 DEG C of shaken cultivation 6h
To OD600Value is 0.6；
(4) bacterium solution is poured into 50 mL centrifuge tubes of sterilization treatment, 5000 rpm are centrifuged 5 min and collect bacterium solution, abandon supernatant；
(5) thallus is resuspended in the NaCI of 10 mL, 0.15 mol/L, 5000 rpm are centrifuged 5 min；
(6) the 20 mM CaCl that thallus l ml is pre-chilled2Solution gently suspends, and 200 μ L packing, -80 DEG C save backup.
Recombinant plasmid transformed Agrobacterium competent cell the following steps are included:
(1) it takes the 100ul Agrobacterium competent cell of -80 DEG C of preservations to be placed on ice, cell gently suspends after being completely dissolved；
(2) 5 μ L recombinant plasmids are added, mixing, 30 min of ice bath are gently blown and beaten with pipette tips；
(3) centrifuge tube is put into quick-frozen 5 min in liquid nitrogen, heat shock 1min in 37 DEG C of water-baths goes to rapidly 3 min on ice；
(4) the YEP fluid nutrient medium of 1 mL antibiotic-free is added into centrifuge tube, mixes gently, is placed in 28 DEG C of insulating boxs and shakes
Swing 1 h；
(5) 4000 rpm are centrifuged 5 min, remove supernatant, and 100 μ L YEP liquid suspension cells are added；
(6) bacterium solution is coated in the YEP solid medium tablets containing corresponding antibiotic, 28 DEG C of 48 h of culture；
(7) picking Agrobacterium single bacterium falls in YEP culture solution and sways 24 h for 28 DEG C, is identified by bacterium solution PCR, and result is the positive
Illustrate expression vector establishment success.
Further, in step S4, over-express vector pBI121-CsTTG1'sTransduction comprises the steps of:
(1) pBI121- of the carrying target gene of -80 DEG C of preservations is takenCsTTG1Agrobacterium liquid thaws, and is dipped respectively with transfer needle
A small amount of bacterium solution is crossed on the solid LB plating medium that attached 70ug/ml rifampin and 115ug/ml kanamycins, in 28
Dark culturing 48h is inverted in DEG C insulating box；
(2) picking positive monoclonal bacterium colony is into 1.2mL LB culture solution, wherein in LB culture solution containing 70ug/ml rifampin and
115ug/ml kanamycins, 28 DEG C of shaking table 220rpm oscillations activate for 24 hours, until bacterium solution OD value reaches 0.8；
(3) it takes whole bacterium solutions in step (2) into 60ml fresh YEP culture solution, wherein contains in YEP culture solution
70ug/ml rifampin and 115ug/ml kanamycins, identical condition relays persistent oscillation 18h in shaking table, until bacterium solution OD value reaches
To 0.8；
(4) 6000rpm is centrifuged 10min, removes supernatant, collects bacterium solution；
(5) isometric infected liquid is added, bacterium solution is suspended and is mixed；
(6) SilwetL-77 to final concentration of 0.04% wt/vol is added before infecting, mixes well, shifts mixed liquor in 500ml
In beaker；
(7) handstand plant is completely immersed in inflorescence in Agrobacterium, shakes gently bacterium solution 30s, plant is removed from solution, with guarantor
Fresh film will infect portion envelops and live moisturizing, dry the liquid in base of the plant；
(8) plant is put on polybag, 22 DEG C of dark cultures are placed under normal lighting conditions for 24 hours and cultivate；
(9) it infects once, infects three times again after 7 days；
(10) about after two months, single plant collects mature seed for growth, is placed in 4 DEG C of refrigerators and saves.
Embodiment 3
A method of regulation tea tree fine hair formation, which comprises the following steps:
S1. to transcription factorCsTTG1Bioinformatic analysis is carried out, chadogram is constructed;
S2. by target geneCsTTG1It is recombinated with vector plasmid pBI121, constructs tea treeCsTTG1The overexpression of gene carries
Body PBI121- CsTTG1 ;
S3. by over-express vector PBI121- CsTTG1And in the Agrobacterium GV1301 that transduces；
S4. under Agrobacterium GV1301 mediation, over-express vector pBI121- CsTTG1It transduces to having and transcription factorCsTTG1In the tea tree crop of homologous sex factor.
Further include step S5, specifically by after screening and culturing medium, take root and transplant lowerly, extract above-mentioned tea tree crop
DNA carries out PCR detection, while analyze using quantitative fluorescent PCR, verification result plasmid pBI121-CsTTG1Succeed
It is transferred to.
The chadogram is shown below:
。
Further, in step S2, over-express vector PBI121-The construction method of CsTTG1 includes:
A tail will be increased in the prior artCsTTG1Target fragment is connected with pBI121 plasmid vector using T4-DNA ligase
Connect, wherein linked system are as follows: according to target fragment: it is tool in 15ul that overall reaction system, which is added, in the ratio that carrier segments are 2:1
Body is as follows:CsTTG1Target fragment 60ng, pBI121 plasmid vector 30ng, T4-DNA ligase 0.2ul, T4-DNA
Ligase Buffer 1ul mends sterile water ddH2O to 15ul, liquid-transfering gun, which is gently inhaled, plays mixing, is placed in PCR instrument, 16 DEG C of connections
Overnight.
Further, in step S3 include in detail below operate: prepare connection product conversion bacillus coli DH 5 alpha competence,
Plasmid DNA extraction, the preparation of Agrobacterium GV3101 competence, recombinant plasmid transformed Agrobacterium competent cell.
Further, connection product conversion bacillus coli DH 5 alpha competence the following steps are included:
(1) by bacillus coli DH 5 alpha strain, gently cell is suspended after thawing completely on ice；
(2) after the mixing of 5ul connection product is added, it is placed in 30min on ice；
(3) 42 DEG C of water-baths are held successfully in advance, centrifuge tube is discharged water 90s in bath, is transferred quickly on ice, ice bath 2min；
(4) the fresh LB liquid medium of 800ul, 37 DEG C of shaking table culture 1h are added；
(5) 2min is centrifuged with 8000rmp, fresh 100ul LB culture solution suspension cell is added；
(6) appropriate bacterium solution is coated on the plate containing corresponding antibiotic, 37 DEG C of culture 12h；
(7) 12 h are cultivated in insulating box, pick from the plate a single colonie with transfer needle, are inoculated into and are trained containing 10mL liquid LB
It supports in 50ml triangular flask, in 37 DEG C of shaking tables, 200 rpm shaken cultivation 10h.
Further, the liquid LB includes 0.5% peptone, 0.2% yeast extract, 0.7% sodium chloride, the liquid
The pH of LB is 6.5.
Further, the Plasmid DNA extracting method is as follows: clone's bacterial plaque that picking has been identified is containing corresponding antibiosis
In the 5mL LB liquid medium of element, 37 DEG C, 200 rpm shaken cultivation 12h, the bacterium solution shaken alkaline lysis method of extracting plasmid
DNA includes step in detail below:
(1) for the bacterium solution that absorption 2m1 has shaken in 2mL centrifuge tube, 12000 rpm are centrifuged 1 min, reject supernatant；
(2) be added 200 μ L solution I, the solution I include 150 mmol/L glucose, 25 mmol/L Tris-HCl, pH8.0,
L0 mmol/L EDTA, pH8.0 acutely vibrate, and thallus is resuspended；
(3) be added the solution II that has just prepared of 200 μ L, the solution II includes 0.2 mol/L NaOH, 1% SDS, gently up and down
Centrifuge tube 3 times mixings of overturning, ice bath 5min；
(4) be added the solution III newly prepared of 200 μ L, the solution III include 5 M Kac 60mL, glacial acetic acid 11.5mL,
ddH228.5 mL of O gently overturns 3-5 mixing of centrifuge tube, 5 min of ice bath up and down；
(5) 12000 rpm are centrifuged 12 min；
(6) Aspirate supernatant is placed in new centrifuge tube, and isometric pre- cold isopropanol is added, mixes gently, is placed at room temperature for 2
Min, 12000 rpm are centrifuged 5 min, remove supernatant；
(7) the RNase-TE solution of 50 μ L, 10 μ g/mL, 37 DEG C of water-bath 30min are added；
(8) 50 μ L TE solution and isometric phenol/chloroform are added, 1min, 12000rpm are acutely vibrated
It is centrifuged 5 min；
(9) it takes and is suspended in the limpid liquid of top layer, isometric phenol/chloroform is added, 12000 rpm are centrifuged 2 min；
(10) supernatant is taken, the dehydrated alcohol of 1/10 volume, 3 M NaAC and the pre-cooling of 2 times of volumes is added, is stored at room temperature 2 min,
12000 rpm are centrifuged 2 min；
(11) supernatant is abandoned, then is precipitated 2 times with 70% ethanol washing, drying at room temperature 30min；
(12) with 20 μ LddH2O dissolution precipitating, i.e. Plasmid samples, -20 DEG C of preservations.
Further, the Agrobacterium GV3101 competence preparation the following steps are included:
(1) the GV3101 bacterium solution of preservation is crossed on the YEP solid medium containing rifampin, training is inverted in 28 DEG C of insulating boxs
It supports for 24 hours；
(2) picking Agrobacterium GV3101 single colonie is inoculated into 2.0 mL YEP fluid nutrient mediums, 28 DEG C of shaking table 200rpm oscillations
Overnight incubation；
(3) bacterium solution for taking 500 μ L to shake is placed in 50 mL YEP culture solutions, 28 DEG C of shaken cultivation 5h
To OD600Value is 0.6；
(4) bacterium solution is poured into 50 mL centrifuge tubes of sterilization treatment, 5000 rpm are centrifuged 5 min and collect bacterium solution, abandon supernatant；
(5) thallus is resuspended in the NaCI of 10 mL, 0.15 mol/L, 5000 rpm are centrifuged 5 min；
(6) the 20 mM CaCl that thallus l ml is pre-chilled2Solution gently suspends, and 200 μ L packing, -80 DEG C save backup.
Recombinant plasmid transformed Agrobacterium competent cell the following steps are included:
(1) it takes the 100ul Agrobacterium competent cell of -80 DEG C of preservations to be placed on ice, cell gently suspends after being completely dissolved；
(2) 5 μ L recombinant plasmids are added, mixing, 30 min of ice bath are gently blown and beaten with pipette tips；
(3) centrifuge tube is put into quick-frozen 5 min in liquid nitrogen, heat shock 1min in 37 DEG C of water-baths goes to rapidly 2 min on ice；
(4) the YEP fluid nutrient medium of 1 mL antibiotic-free is added into centrifuge tube, mixes gently, is placed in 28 DEG C of insulating boxs and shakes
Swing 1 h；
(5) 4000 rpm are centrifuged 5 min, remove supernatant, and 100 μ L YEP liquid suspension cells are added；
(6) bacterium solution is coated in the YEP solid medium tablets containing corresponding antibiotic, 28 DEG C of 32 h of culture；
(7) picking Agrobacterium single bacterium falls in YEP culture solution and sways 24 h for 28 DEG C, is identified by bacterium solution PCR, and result is the positive
Illustrate expression vector establishment success.
Further, in step S4, over-express vector pBI121-CsTTG1'sTransduction comprises the steps of:
(1) pBI121- of the carrying target gene of -80 DEG C of preservations is takenCsTTG1Agrobacterium liquid thaws, and is dipped respectively with transfer needle
A small amount of bacterium solution is crossed on the solid LB plating medium that attached 30ug/ml rifampin and 80ug/ml kanamycins, in 28
Dark culturing 36h is inverted in DEG C insulating box；
(2) picking positive monoclonal bacterium colony is into 0.5mL LB culture solution, wherein in LB culture solution containing 30ug/ml rifampin and
80ug/ml kanamycins, 28 DEG C of shaking table 130rpm oscillation activation 18h, until bacterium solution OD value reaches 0.4；
(3) it takes whole bacterium solutions in step (2) into 35ml fresh YEP culture solution, wherein contains in YEP culture solution
30ug/ml rifampin and 80ug/ml kanamycins, identical condition relays persistent oscillation 13h in shaking table, until bacterium solution OD value reaches
0.4；
(4) 3500rpm is centrifuged 20min, removes supernatant, collects bacterium solution；
(5) isometric infected liquid is added, bacterium solution is suspended and is mixed；
(6) SilwetL-77 to final concentration of 0.01% wt/vol is added before infecting, mixes well, shifts mixed liquor in 500ml
In beaker；
(7) handstand plant is completely immersed in inflorescence in Agrobacterium, shakes gently bacterium solution 30s, plant is removed from solution, with guarantor
Fresh film will infect portion envelops and live moisturizing, dry the liquid in base of the plant；
(8) plant is put on polybag, 22 DEG C of dark cultures are placed under normal lighting conditions for 24 hours and cultivate；
(9) it infects once, infects three times again after 7 days；
(10) about after two months, single plant collects mature seed for growth, is placed in 4 DEG C of refrigerators and saves.
Embodiment 4
A method of regulation tea tree fine hair formation, which comprises the following steps:
S1. to transcription factorCsTTG1Bioinformatic analysis is carried out, chadogram is constructed;
S2. by target geneCsTTG1It is recombinated with vector plasmid pBI121, constructs tea treeCsTTG1The overexpression of gene carries
Body PBI121- CsTTG1 ;
S3. by over-express vector PBI121- CsTTG1And in the Agrobacterium GV1301 that transduces；
S4. under Agrobacterium GV1301 mediation, over-express vector pBI121- CsTTG1It transduces to having and transcription factorCsTTG1In the tea tree crop of homologous sex factor.
Further include step S5, specifically by after screening and culturing medium, take root and transplant lowerly, extract above-mentioned tea tree crop
DNA carries out PCR detection, while analyze using quantitative fluorescent PCR, verification result plasmid pBI121-CsTTG1Succeed
It is transferred to.
The chadogram is shown below:
。
Further, in step S2, over-express vector PBI121-The construction method of CsTTG1 includes:
A tail will be increased in the prior artCsTTG1Target fragment is connected with pBI121 plasmid vector using T4-DNA ligase
Connect, wherein linked system are as follows: according to target fragment: it is tool in 20ul that overall reaction system, which is added, in the ratio that carrier segments are 4:1
Body is as follows:CsTTG1Target fragment 160ng, pBI121 plasmid vector 40ng, T4-DNA ligase 0.6ul, T4-DNA
Ligase Buffer 2ul mends sterile water ddH2O to 20ul, liquid-transfering gun, which is gently inhaled, plays mixing, is placed in PCR instrument, 16 DEG C of connections
Overnight.
Further, in step S3 include in detail below operate: prepare connection product conversion bacillus coli DH 5 alpha competence,
Plasmid DNA extraction, the preparation of Agrobacterium GV3101 competence, recombinant plasmid transformed Agrobacterium competent cell.
Further, connection product conversion bacillus coli DH 5 alpha competence the following steps are included:
(1) by bacillus coli DH 5 alpha strain, gently cell is suspended after thawing completely on ice；
(2) after the mixing of 5ul connection product is added, it is placed in 30min on ice；
(3) 42 DEG C of water-baths are held successfully in advance, centrifuge tube is discharged water 90s in bath, is transferred quickly on ice, ice bath 2min；
(4) the fresh LB liquid medium of 800ul, 37 DEG C of shaking table culture 1h are added；
(5) 2min is centrifuged with 8000rmp, fresh 100ul LB culture solution suspension cell is added；
(6) appropriate bacterium solution is coated on the plate containing corresponding antibiotic, 37 DEG C of culture 13h；
(7) 15 h are cultivated in insulating box, pick from the plate a single colonie with transfer needle, are inoculated into and are trained containing 10mL liquid LB
It supports in 50ml triangular flask, in 37 DEG C of shaking tables, 200 rpm shaken cultivation, 11.5 h.
Further, the liquid LB includes 1.1% peptone, 0.5% yeast extract, 0.9% sodium chloride, the liquid
The pH of LB is 6.9.
Further, the Plasmid DNA extracting method is as follows: clone's bacterial plaque that picking has been identified is containing corresponding antibiosis
In the 5mL LB liquid medium of element, 37 DEG C, 200 rpm shaken cultivation, 12.5 h, the bacterium solution shaken alkaline lysis method of extracting matter
Grain DNA, includes step in detail below:
(1) for the bacterium solution that absorption 2.5m1 has shaken in 2mL centrifuge tube, 12000 rpm are centrifuged 1 min, reject supernatant；
(2) be added 200 μ L solution I, the solution I include 150 mmol/L glucose, 25 mmol/L Tris-HCl, pH8.0,
L0 mmol/L EDTA, pH8.0 acutely vibrate, and thallus is resuspended；
(3) be added the solution II that has just prepared of 200 μ L, the solution II includes 0.2 mol/L NaOH, 1% SDS, gently up and down
Centrifuge tube 4 times mixings of overturning, ice bath 5min；
(4) be added the solution III newly prepared of 200 μ L, the solution III include 5 M Kac 60mL, glacial acetic acid 11.5mL,
ddH228.5 mL of O gently overturns 3-5 mixing of centrifuge tube, 5 min of ice bath up and down；
(5) 12000 rpm are centrifuged 12 min；
(6) Aspirate supernatant is placed in new centrifuge tube, and isometric pre- cold isopropanol is added, mixes gently, is placed at room temperature for 2
Min, 12000 rpm are centrifuged 5 min, remove supernatant；
(7) the RNase-TE solution of 50 μ L, 10 μ g/mL, 37 DEG C of water-bath 30min are added；
(8) 50 μ L TE solution and isometric phenol/chloroform are added, 1min, 12000rpm are acutely vibrated
It is centrifuged 5 min；
(9) it takes and is suspended in the limpid liquid of top layer, isometric phenol/chloroform is added, 12000 rpm are centrifuged 2 min；
(10) supernatant is taken, the dehydrated alcohol of 1/10 volume, 3 M NaAC and the pre-cooling of 2 times of volumes is added, is stored at room temperature 2 min,
12000 rpm are centrifuged 2 min；
(11) supernatant is abandoned, then is precipitated 2-3 times with 70% ethanol washing, drying at room temperature 30min；
(12) with 40 μ L ddH2O dissolution precipitating, i.e. Plasmid samples, -20 DEG C of preservations.
Further, the Agrobacterium GV3101 competence preparation the following steps are included:
(1) the GV3101 bacterium solution of preservation is crossed on the YEP solid medium containing rifampin, training is inverted in 28 DEG C of insulating boxs
Support 32h；
(2) picking Agrobacterium GV3101 single colonie is inoculated into 2.0 mL YEP fluid nutrient mediums, 28 DEG C of shaking table 200rpm oscillations
Overnight incubation；
(3) bacterium solution for taking 500 μ L to shake is placed in 50 mL YEP culture solutions, 28 DEG C of shaken cultivation 5.5h
To OD600Value is 0.6；
(4) bacterium solution is poured into 50 mL centrifuge tubes of sterilization treatment, 5000 rpm are centrifuged 5 min and collect bacterium solution, abandon supernatant；
(5) thallus is resuspended in the NaCI of 10 mL, 0.15 mol/L, 5000 rpm are centrifuged 5 min；
(6) the 20 mM CaCl that thallus l ml is pre-chilled2Solution gently suspends, and 200 μ L packing, -80 DEG C save backup.
Recombinant plasmid transformed Agrobacterium competent cell the following steps are included:
(1) it takes the 100ul Agrobacterium competent cell of -80 DEG C of preservations to be placed on ice, cell gently suspends after being completely dissolved；
(2) 5 μ L recombinant plasmids are added, mixing, 30 min of ice bath are gently blown and beaten with pipette tips；
(3) centrifuge tube is put into quick-frozen 5 min in liquid nitrogen, heat shock 1min in 37 DEG C of water-baths goes to rapidly 2-3 min on ice；
(4) the YEP fluid nutrient medium of 1 mL antibiotic-free is added into centrifuge tube, mixes gently, is placed in 28 DEG C of insulating boxs and shakes
Swing 1 h；
(5) 4000 rpm are centrifuged 5 min, remove supernatant, and 100 μ L YEP liquid suspension cells are added；
(6) bacterium solution is coated in the YEP solid medium tablets containing corresponding antibiotic, 28 DEG C of culture 39h；
(7) picking Agrobacterium single bacterium falls in YEP culture solution and sways 24 h for 28 DEG C, is identified by bacterium solution PCR, and result is the positive
Illustrate expression vector establishment success.
Further, in step S4, over-express vector pBI121-CsTTG1'sTransduction comprises the steps of:
(1) pBI121- of the carrying target gene of -80 DEG C of preservations is takenCsTTG1Agrobacterium liquid thaws, and is dipped respectively with transfer needle
A small amount of bacterium solution is crossed on the solid LB plating medium that attached 45ug/ml rifampin and 110ug/ml kanamycins, in 28
Dark culturing 40h is inverted in DEG C insulating box；
(2) picking positive monoclonal bacterium colony is into 0.9mL LB culture solution, wherein in LB culture solution containing 45ug/ml rifampin and
110ug/ml kanamycins, 28 DEG C of shaking table 145rpm oscillation activation 22h, until bacterium solution OD value reaches 0.7；
(3) it takes whole bacterium solutions in step (2) into 50ml fresh YEP culture solution, wherein contains in YEP culture solution
45ug/ml rifampin and 110ug/ml kanamycins, identical condition relays persistent oscillation 16h in shaking table, until bacterium solution OD value reaches
To 0.7；
(4) 5000rpm is centrifuged 15min, removes supernatant, collects bacterium solution；
(5) isometric infected liquid is added, bacterium solution is suspended and is mixed；
(6) SilwetL-77 to final concentration of 0.03% wt/vol is added before infecting, mixes well, shifts mixed liquor in 500ml
In beaker；
(7) handstand plant is completely immersed in inflorescence in Agrobacterium, shakes gently bacterium solution 30s, plant is removed from solution, with guarantor
Fresh film will infect portion envelops and live moisturizing, dry the liquid in base of the plant；
(8) plant is put on polybag, 22 DEG C of dark cultures are placed under normal lighting conditions for 24 hours and cultivate；
(9) it infects once, infects three times again after 7 days；
(10) about after two months, single plant collects mature seed for growth, is placed in 4 DEG C of refrigerators and saves.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.It is noted that the technical characteristic being not described in detail in the present invention, can pass through this
Field any prior art is realized.
Claims (10)
1. a kind of method that regulation tea tree fine hair is formed, which comprises the following steps:
S1. to transcription factorCsTTG1Bioinformatic analysis is carried out, chadogram is constructed;
S2. by target geneCsTTG1It is recombinated with vector plasmid pBI121, constructs tea treeCsTTG1The overexpression of gene carries
Body PBI121- CsTTG1 ;
S3. by over-express vector PBI121- CsTTG1And in the Agrobacterium GV1301 that transduces；
S4. under Agrobacterium GV1301 mediation, over-express vector pBI121- CsTTG1It transduces to having and transcription factorCsTTG1In the tea tree crop of homologous sex factor.
2. the method that regulation tea tree fine hair according to claim 1 is formed, which is characterized in that further include step S5, specifically
For by after screening and culturing medium, take root and transplant and extract the DNA of above-mentioned tea tree crop lowerly, carry out PCR detection, carry out simultaneously
It is analyzed using quantitative fluorescent PCR, verification result plasmid pBI121-CsTTG1It is successfully transferred to.
3. the method that regulation tea tree fine hair according to claim 1 is formed, which is characterized in that the chadogram such as following formula institute
Show:
。
4. the method that regulation tea tree fine hair according to claim 1 is formed, which is characterized in that in step S2, be overexpressed and carry
Body PBI121-The construction method of CsTTG1 includes:
A tail will be increasedCsTTG1Target fragment is attached with pBI121 plasmid vector using T4-DNA ligase, wherein even
Junctor system are as follows: according to target fragment: it is in 15-30ul, specifically such as that overall reaction system, which is added, in the ratio that carrier segments are 5-2:1
Under:CsTTG1Target fragment 250-60ng, pBI121 plasmid vector 50-30ng, T4-DNA ligase 0.2-0.7ul, T4-
DNA ligase Buffer 1-4ul mends sterile water ddH2O to 15-30ul, liquid-transfering gun, which is gently inhaled, plays mixing, is placed on PCR instrument
In, 16 DEG C of connections are overnight.
5. the method that regulation tea tree fine hair according to claim 1 is formed, which is characterized in that include following tool in step S3
Gymnastics is made: preparing connection product conversion bacillus coli DH 5 alpha competence, Plasmid DNA is extracted, Agrobacterium GV3101 competence
Preparation, recombinant plasmid transformed Agrobacterium competent cell.
6. the method that regulation tea tree fine hair according to claim 5 is formed, which is characterized in that the connection product conversion is big
Enterobacteria DH5 α competence the following steps are included:
(1) by bacillus coli DH 5 alpha strain, gently cell is suspended after thawing completely on ice；
(2) after the mixing of 5ul connection product is added, it is placed in 30min on ice；
(3) 42 DEG C of water-baths are held successfully in advance, centrifuge tube is discharged water 90s in bath, is transferred quickly on ice, ice bath 2min；
(4) the fresh LB liquid medium of 800ul, 37 DEG C of shaking table culture 1h are added；
(5) 2min is centrifuged with 8000rmp, fresh 100ul LB culture solution suspension cell is added；
(6) appropriate bacterium solution is coated on the plate containing corresponding antibiotic, 37 DEG C of culture 12-16h；
(7) 12-16 h is cultivated in insulating box, is picked from the plate a single colonie with transfer needle, is inoculated into containing 10mL liquid
LB is cultivated in 50ml triangular flask, in 37 DEG C of shaking tables, 200 rpm shaken cultivation 10-12 h；
The liquid LB include 0.5-3% peptone, 0.2-0.7% yeast extract, 0.7-1.6% sodium chloride, the liquid LB's
PH is 6.5-7.8.
7. the method that regulation tea tree fine hair according to claim 5 is formed, which is characterized in that the Plasmid DNA is extracted
Method is as follows: clone's bacterial plaque that picking has been identified is in the 5mL LB liquid medium containing corresponding antibiotic, and 37 DEG C, 200
Rpm shaken cultivation 12-14 h, the bacterium solution alkaline lysis method of extracting plasmid DNA shaken include step in detail below:
(1) for the bacterium solution that absorption 2-4 m1 has shaken in 2mL centrifuge tube, 12000 rpm are centrifuged 1 min, reject supernatant；
(2) be added 200 μ L solution I, the solution I include 150 mmol/L glucose, 25 mmol/L Tris-HCl, pH8.0,
L0 mmol/L EDTA, pH8.0 acutely vibrate, and thallus is resuspended；
(3) be added the solution II that has just prepared of 200 μ L, the solution II includes 0.2 mol/L NaOH, 1% SDS, gently up and down
Centrifuge tube 3-5 times mixing of overturning, ice bath 5min；
(4) be added the solution III newly prepared of 200 μ L, the solution III include 5 M Kac 60mL, glacial acetic acid 11.5mL,
ddH228.5 mL of O gently overturns 3-5 mixing of centrifuge tube, 5 min of ice bath up and down；
(5) 12000 rpm are centrifuged 12 min；
(6) Aspirate supernatant is placed in new centrifuge tube, and isometric pre- cold isopropanol is added, mixes gently, is placed at room temperature for 2
Min, 12000 rpm are centrifuged 5 min, remove supernatant；
(7) the RNase-TE solution of 50 μ L, 10 μ g/mL, 37 DEG C of water-bath 30min are added；
(8) 50 μ L TE solution and isometric phenol/chloroform are added, 1min, 12000rpm are acutely vibrated
It is centrifuged 5 min；
(9) it takes and is suspended in the limpid liquid of top layer, isometric phenol/chloroform is added, 12000 rpm are centrifuged 2 min；
(10) supernatant is taken, the dehydrated alcohol of 1/10 volume, 3 M NaAC and the pre-cooling of 2 times of volumes is added, is stored at room temperature 2 min,
12000 rpm are centrifuged 2 min；
(11) supernatant is abandoned, then is precipitated 2-3 times with 70% ethanol washing, drying at room temperature 30min；
(12) 20-50 μ LTE or ddH is used2O dissolution precipitating, i.e. Plasmid samples, -20 DEG C of preservations.
8. the method that regulation tea tree fine hair according to claim 5 is formed, which is characterized in that the Agrobacterium GV3101 sense
By state preparation the following steps are included:
(1) the GV3101 bacterium solution of preservation is crossed on the YEP solid medium containing rifampin, training is inverted in 28 DEG C of insulating boxs
Support 24-36h；
(2) picking Agrobacterium GV3101 single colonie is inoculated into 2.0 mL YEP fluid nutrient mediums, 28 DEG C of shaking table 200rpm oscillations
Overnight incubation；
(3) bacterium solution for taking 500 μ L to shake is placed in 50 mL YEP culture solutions, 28 DEG C of shaken cultivation 5-6h
To OD600Value is 0.6；
(4) bacterium solution is poured into 50 mL centrifuge tubes of sterilization treatment, 5000 rpm are centrifuged 5 min and collect bacterium solution, abandon supernatant；
(5) thallus is resuspended in the NaCI of 10 mL, 0.15 mol/L, 5000 rpm are centrifuged 5 min；
(6) the 20 mM CaCl that thallus l ml is pre-chilled2Solution gently suspends, and 200 μ L packing, -80 DEG C save backup.
9. the method that regulation tea tree fine hair according to claim 5 is formed, which is characterized in that recombinant plasmid transformed Agrobacterium
Competent cell the following steps are included:
(1) it takes the 100ul Agrobacterium competent cell of -80 DEG C of preservations to be placed on ice, cell gently suspends after being completely dissolved；
(2) 5 μ L recombinant plasmids are added, mixing, 30 min of ice bath are gently blown and beaten with pipette tips；
(3) centrifuge tube is put into quick-frozen 5 min in liquid nitrogen, heat shock 1min in 37 DEG C of water-baths goes to rapidly 2-3 min on ice；
(4) the YEP fluid nutrient medium of 1 mL antibiotic-free is added into centrifuge tube, mixes gently, is placed in 28 DEG C of insulating boxs and shakes
Swing 1 h；
(5) 4000 rpm are centrifuged 5 min, remove supernatant, and 100 μ L YEP liquid suspension cells are added；
(6) bacterium solution is coated in the YEP solid medium tablets containing corresponding antibiotic, 28 DEG C of culture 32-48 h；
(7) picking Agrobacterium single bacterium falls in YEP culture solution and sways 24 h for 28 DEG C, is identified by bacterium solution PCR, and result is the positive
Illustrate expression vector establishment success.
10. the method that regulation tea tree fine hair according to claim 1 is formed, which is characterized in that in step S4, be overexpressed and carry
Body pBI121-CsTTG1'sTransduction comprises the steps of:
(1) pBI121- of the carrying target gene of -80 DEG C of preservations is takenCsTTG1Agrobacterium liquid thaws, and is dipped respectively with transfer needle
A small amount of bacterium solution is drawn on the solid LB plating medium that attached 30-70ug/ml rifampin and 80-115ug/ml kanamycins
Line is inverted dark culturing 36-48h in 28 DEG C of insulating boxs；
(2) picking positive monoclonal bacterium colony wherein contains 30-70ug/ml in LB culture solution into 0.5-1.2mL LB culture solution
Rifampin and 80-115ug/ml kanamycins, 28 DEG C of shaking table 130-220rpm oscillation activation 18-24h, until bacterium solution OD value reaches
0.4-0.8；
(3) it takes whole bacterium solutions in step (2) into 35-60ml fresh YEP culture solution, wherein contains in YEP culture solution
30-70ug/ml rifampin and 80-115ug/ml kanamycins, identical condition relays persistent oscillation 13-18h in shaking table, until bacterium
Liquid OD value reaches 0.4-0.8；
(4) 3500-6000rpm is centrifuged 10-20min, removes supernatant, collects bacterium solution；
(5) isometric infected liquid is added, bacterium solution is suspended and is mixed；
(6) SilwetL-77 to final concentration of 0.01-0.04% wt/vol is added before infecting, mixes well, transfer mixed liquor in
In 500ml beaker；
(7) handstand plant is completely immersed in inflorescence in Agrobacterium, shakes gently bacterium solution 30s, plant is removed from solution, with guarantor
Fresh film will infect portion envelops and live moisturizing, dry the liquid in base of the plant；
(8) plant is put on polybag, 22 DEG C of dark cultures are placed under normal lighting conditions for 24 hours and cultivate；
(9) it infects once, infects three times again after 7 days；
(10) about after two months, single plant collects mature seed for growth, is placed in 4 DEG C of refrigerators and saves.
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