CN110229818B - CpSNAC1 Gene Promoter of Prunus chinensis and its Application - Google Patents

Abstract

The invention relates to the field of genetic engineering, and in particular relates to a chimonanthus nitens CpSNAC1 gene promoter and application thereof. The invention aims to provide a new choice for improving the wintersweet resistance. The promoter of the chimonanthus nitens CpSNAC1 gene is obtained by amplification for the first time, 3 CpSNAC1 promoter deletion fragments are obtained by cloning, the expression characteristics of the CpSNAC1 gene are detected by a real-time fluorescent quantitative PCR technology, and the expression regulation and control characteristics of the CpSNAC1 gene promoter are preliminarily verified by methods such as a transgenic technology, GUS histochemical staining, GUS enzyme activity quantitative detection and the like. The result can provide reference for researching the related regulation and control of the wintersweet adversity stress.

Description

CpSNAC1 Gene Promoter of Prunus chinensis and its Application

technical field

The invention relates to the field of genetic engineering, in particular to a CpSNAC1 gene promoter of Prunus chinensis and its application.

Background technique

Chimonanthus praecox L., a deciduous bushy shrub, often semi-evergreen on warm ground, up to 3-4m in height, flowering from December to March of the second year, opening far before the leaves unfold, and the fruit matures around August . Wax plum is a highly ornamental tree species belonging to the genus Wax Prunus, known for its blooming in the cold winter and the twelfth lunar month. The flowers are elegant in color, fragrant and refreshing. In addition to the high ornamental value, there are also precious cultural value, medicinal value and economic value. With the development of modern molecular biotechnology, the research on Cannabis chinensis has shifted from traditional taxonomic research and some applied value research to molecular level research.

The NAC (NAM, ATAF1, ATAF2 and CUC2) transcription factor family is one of the largest transcription factor families in the plant genome and is also a plant-specific transcription factor. The names of the NAC family transcription factors are taken from the acronyms of the first three published genes with NAC domains, namely the NAM gene found in petunia and the ATAF1/2 and CUC2 genes found in Arabidopsis thaliana. Plant NAC transcription factors have multiple functions, and play an important role in the regulation of plant growth and development, the response of plants to stress, regulation of plant disease resistance, participation in plant secondary growth regulation, and hormone signal transduction.

At present, the research on NAC gene transcription factor has been extensive and thorough, but there are not many related researches on its promoter. Relevant studies have found that in addition to the basic components of promoters such as TATA-box and CAAT-box, the promoter sequence of NAC family genes also contains some unique cis-acting elements related to hormones, anti-stress, tissue and organ development, etc. , speculated to play a regulatory role in the activation and induction of NAC transcription factors.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a new choice for improving the resistance of the wax plum.

The present invention provides a CpSNAC1 gene promoter of Prunus chinensis, the nucleotide sequence of which is shown in positions 974-1437 of SEQ ID No.15.

Further, the nucleotide sequence of the CpSNAC1 gene promoter of the wax plum is shown in positions 727-1437 of SEQ ID No.15.

Further, the nucleotide sequence of the CpSNAC1 gene promoter of Prunus chinensis is shown in positions 215-1437 of SEQ ID No.15.

Further, the nucleotide sequence of the CpSNAC1 gene promoter of the wax plum is shown in SEQ ID No.15.

The present invention also provides the use of the CpSNAC1 gene promoter in the control of plant stress resistance.

Specifically, the plant is Arabidopsis thaliana or wax plum.

Specifically, the adversity is hormone, high salt, drought, high temperature or low temperature.

Specifically, the hormone treatment was abscisic acid treatment at a concentration of 50 μM·L ^-1 .

Specifically, the high salt is NaCl treatment with a concentration of 150 mM·L ^-1 .

Specifically, the drought is PEG treatment with a concentration of 10% by mass.

Specifically, the high temperature is 42°C.

Specifically, the low temperature is 4°C.

Beneficial effects of the present invention: the present invention clones the CpSNAC1 gene promoter of Prunus chinensis for the first time, and clones three CpSNAC1 promoter deletion fragments, and preliminarily verifies CpSNAC1 through transgenic technology, GUS histochemical staining, quantitative detection of GUS enzyme activity and other methods Expression regulation characteristics of gene promoters. Through research, it is found that the promoter in this application is closely related to the ability of regulating plants to cope with adversity (hormone, drought, high salt, high temperature, low temperature).

Description of drawings

Figure 1 CpSNAC1 gene promoter sequence.

Figure 2 is a sequence analysis of CpSNAC1pro. 5UTR Py-rich stretch: cis-acting element that increases transcription level; AAGAA-motif: cis-acting element involved in abscisic acid response; ABRE: cis-acting element involved in abscisic acid response; ACE: cis-acting element involved in light responsiveness ; ARE: anaerobic-induced essential cis-regulatory element; ATCT-motif: part of a conserved DNA module involved in light-responsiveness; AuxRR-core: cis-acting regulatory element involved in auxin response; Box 4: involved in light-responsiveness part of a sexually conserved DNA module; Box I: light-responsive element; Box-W1: fungal elicitor-responsive element; CAAT-box: common cis-acting element in promoter and enhancer regions; CAT-box: associated with cis-acting regulatory elements related to tissue expression; CATT-motif: partial light-responsive elements; CE3: cis-acting elements involved in ABA and VP1 responses; CGTCA-motif: cis-acting regulatory elements involved in MeJA responses; G-Box : cis-acting regulatory element involved in light-responsiveness; G-box: cis-acting regulatory element involved in light-responsiveness; GA-motif: partial light-responsive element; GARE-motif: gibberellin-responsive element; GCN4_motif: involved in endosperm Expressed cis-regulatory element; I-box: partial light-responsive element; MBS: MYB binding site involved in drought inducibility; O2-site: cis-acting regulatory element involved in the regulation of zein metabolism; P-box: erythroid Mycin-responsive element; Skn-1_motif: cis-acting regulatory element required for endosperm expression; Sp1: light-responsive element; TATA-box: transcription start point of core promoter element around -30; TC-rich repeats: involved in defense cis-acting element and stress response; TCA-element: cis-acting element involved in salicylic acid responsiveness; TGA-element: auxin-responsive element; TGACG-motif: cis-acting regulatory element involved in MeJA response; circadian : cis-acting regulatory elements involved in circadian control.

Figure 3 is a schematic diagram of the promoter structure of the CpSNAC1 gene, and the red arrow indicates the position of the deletion segment. TCA: cis-acting element involved in salicylic acid responsiveness; ABRE: cis-acting element involved in abscisic acid response; circadian: cis-acting regulatory element involved in circadian rhythm control; P-BOX: gibberellin response element; TC -rich: cis-acting elements involved in defense and stress responses; AuxRR-core: cis-acting regulatory elements involved in auxin responses; MBS: MYB binding site; CGTCA: cis-acting regulatory elements involved in MeJA responses; GARE : gibberellin response element; TGACG: cis-acting regulatory element involved in MeJA response; CAT-box: cis-acting regulatory element associated with meristem expression; CE3: cis-acting element involved in ABA and VP1 responses.

Figure 4 is a schematic diagram of each deletion segment of the CpSNAC1 gene promoter.

Figure 5 is a transient activity assay of CpSNAC1pro/CpSNAC1pro-D1/D2/D3. AgrobactertumGV3101 Blank: Agrobacterium GV3101 empty strain; 35S promoter control pcambial1305: pcambial vector containing CaMV35S promoter; CpSNAC1 promoter CpSNAC1pro: full-length CpSNAC1 promoter; CpSNAC1-D1promoter CpSNAC1pro-D1: CpSNAC1 promoter deletion fragment D1; -D2: CpSNAC1 promoter deletion fragment D2; CpSNAC1-D3 promoter CpSNAC1pro-D3: CpSNAC1 promoter deletion fragment D3.

Figure 6 is the chemical staining of CpSNAC1pro/CpSNAC1pro-D1/D2/D3 transgenic Arabidopsis 1-10d germination. WT: wild-type Arabidopsis thaliana; 1305: Arabidopsis thaliana transformed with pcambia empty vector.

Figure 7 is the histochemical staining of Arabidopsis thaliana after transgenic CpSNAC1 gene promoter and its deletion. Figures A to D are histochemical staining of CpSNAC1pro, CpSNAC1pro-D1, CpSNAC1pro-D2 and CpSNAC1pro-D3 transgenic Arabidopsis, respectively; A to L are flower clusters, flowers, petals, calyx, stamens, styles, stems, and pods, respectively , rosette leaves and stem leaves.

Figure 8 shows the expression analysis of GUS gene after CpSNAC1pro transgenic Arabidopsis was treated with abiotic stress.

Figure 9 is a BSA standard curve.

Figure 10 is a 4-MUG standard curve.

Figure 11 is the GUS enzyme activity assay of CpSNAC1pro, CpSNAC1pro-D1, CpSNAC1pro-D2 and CpSNAC1pro-D3 transgenic Arabidopsis thaliana after different induction stress treatments. A: 50 μM·L ^-1 ABA; B: 150 mM·L ^-1 Nac; C: 10% PEG; D: low temperature at 4°C; E: high temperature at 42°C.

Figure 12 is the GUS enzyme activity assay of CpSNAC1pro, CpSNAC1pro-D1, CpSNAC1pro-D2 and CpSNAC1pro-D3 transgenic Arabidopsis tissues.

Figure 13 shows the results of multiple sequence alignment of the protein encoded by CpSNAC1 and NAC proteins of other species. Sequences from NCBI (https://www.ncbi.nlm.nih.gov/): ZmNAC1 (GenBank ID: PWZ31933), GmNAC1 (GenBank ID: NP_001240958), ANAC072 (GenBank ID: OAO97067), ATAF1 (GenBank ID: CAA52771), ATAF2 (GenBank ID: AAM65967), GmNAC4 (GenBank ID: NP_001238424), NnNAC2 (GenBank ID: XP_010268987)

Figure 14 is a protein cluster analysis of proteins encoded by CpSNAC1.

Detailed ways

Example 1 Isolation of CpSNAC1 gene of Prunus chinensis

Total RNA was extracted from the leaves of Prunus chinensis by Trizol method, and cDNA was synthesized by reverse transcription. According to the known sequence fragments in the Prunus chinensis transcriptome database library, use the software Primer primer 5.0 to design specific primers for PCR amplification. The primer sequences are as follows:

NAC1-ORF-F: 5'-gttgtgaggcgcatttcttgcgt-3' (SEQ ID No. 3)

NAC1-ORF-R: 3'-gcttctgctctacatgctcttcctc-5' (SEQ ID No. 4)

Amplify the CpSNAC1 gene of Prunus chinensis using the cDNA of Prunus chinensis as the template. The PCR reaction system and reaction conditions are as follows: ddH ₂ O 17.8 μL, 10×HiFi Taq PCR Buffer II 2.5 μL, dNTP (10 mM) 1.5 μL, SNAC1-ORF-F ( 10 μM) 1 μL, SNAC1-ORF-R (10 μM) 1 μL, wax plum cDNA 1 μL, TaKaRa Ex Taq ^TM 0.2 μL. Pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 1 min, 28 cycles; extension at 72 °C for 10 min.

The PCR product was recovered and connected to the pMD19-T vector, transformed into E. coli competent cells Top10, and the recombinant was picked for sequencing.

RT-PCR Kit反转录试剂盒、ExTaq酶、HiFi Taq酶、pMD19-T载体均购自日本TaKaRa公司(大连)；胶回收试剂盒、质粒提取试剂盒、DNA分子量标准DL2000购自天根生化科技(北京)有限公司；大肠杆菌感受态Top10由西南大学园林花卉研究所自制。Note: Trizol reagent used in this step,

RT-PCR Kit reverse transcription kit, ExTaq enzyme, HiFi Taq enzyme, pMD19-T vector were purchased from Japan TaKaRa Company (Dalian); gel recovery kit, plasmid extraction kit, DNA molecular weight standard DL2000 were purchased from Tiangen Biochemical Science and Technology (Beijing) Co., Ltd;

The full-length cDNA of CpSNAC1 gene is 1008 bp, including the largest open reading frame of 891 bp, encoding 297 amino acids. Blastp comparison was performed in the NCBI database (Fig. 13), and the results showed that the protein encoded by the CpSNAC1 gene (SEQ ID No. 2) of Arabidopsis thaliana had high homology with the protein sequences of Arabidopsis ATAF1, ATAF2, soybean GmNAC2, and maize ZmNAC1. The phylogenetic tree analysis found (Fig. 14) that CpSNAC1 belongs to the SNAC subfamily. In terms of genetic distance, CpSNAC1 is closely related to soybean GmNAC2 and Arabidopsis ATAF1, and genes such as ATAF1, GmNAC2 and ZmNAC1 have been proved to have stress response. Function.

Example 2 Cloning of the CpSNAC1 promoter of Prunus chinensis

1 Extraction of the genomic DNA of Prunus chinensis

The total genomic DNA of Prunus chinensis was extracted by CTAB method.

(1) Take 0.1g of young leaves of Prunus chinensis, grind them into powder in liquid nitrogen, and transfer them into a 1.5mL centrifuge tube;

(2) Add 750 μL of CTAB extract solution preheated at 65°C, and vortex to mix quickly;

(3) 65°C water bath, lysing for 45min, shaking gently every 15min.

(4) Take out, add an equal volume of chloroform isoamyl alcohol (24:1) in a fume hood after cooling, gently invert and mix, and stand still for 10 min;

(5) Centrifuge at 12,000rpm and 4°C for 10min;

(6) Take the supernatant, add an equal volume of isopropanol pre-cooled at -20°C, mix well, and place it on ice for 30 minutes, and a white flocculent precipitate appears;

(7) Centrifuge at 12,000rpm and 4°C for 10min;

(8) Remove the supernatant, rinse with 75% ethanol for 2 to 3 times, and then rinse with 1 mL of absolute ethanol once;

(9) Dry the precipitation on an ultra-clean workbench or in an incubator below 64°C until it becomes translucent;

(10) Dissolve with 50 μL of TE or sterilized ultrapure water, take 1 μL of DNA to measure the concentration with a nucleic acid protein analyzer produced by a German company, and store at -20°C for later use.

2. Amplification of upstream sequence of CpSNAC1 gene by chromosome walking method

(1) Primer design

The CpSNAC1 Unigene fragment was obtained by using the Prunus chinensis transcriptome database (SRA, http://www.ncbi.nlm.nih.gov/Traces/sra. Accession No. SRA106143) constructed by the laboratory, and obtained by RACE PCR according to the obtained Unigene sequence. 5'-UTR and 3'-UTR, after sequence splicing, obtain the complete cDNA sequence. The full-length cDNA of CpSNAC1 gene is 1008 bp, and the cDNA of this gene contains an open reading frame of 871 bp encoding 297 amino acids. Based on the 871bp open reading frame of the gene, using the principle of gene walking method to amplify unknown fragments, and using the software Primer Premier5.0, two downstream specific primers were designed for PCR amplification. The primer sequences are as follows:

NAC1-SP1: 5'-gctcttcctccatctcattctccctgc-3' (SEQ ID No. 5)

NAC1-SP2: 5'-gaacagcaatcggttgtgaggcg-3' (SEQ ID No. 6)

(2) Construction of genome walking enzyme digestion template

Genomic DNA walking of the library was performed according to the instructions in the Genome Walker ^™ Universal Kit. Take 1 μg of each quince genomic DNA, digest it with four blunt-end enzymes DraⅠ, EcoRⅤ, PvuⅡ and StuⅠ, and then purify the digested product, and then use T4-DNA ligase (TaKaRa, Dalian) to ligate the product with the kit's DNA. The upstream and downstream genome walking adapters were ligated overnight in a 16°C water bath to construct four genome walking libraries. The templates of DraⅠ, EcoRⅤ, PvuⅡ and StuⅠ were digested from the genomic DNA of Prunus chinensis.

(3) PCR reaction

Two rounds of nested Touch-Down PCR were used in a reaction system (25 μL).

The first round of PCR amplification was performed with NAC1-SP1 and primer AP1 (Universal Genome Walker ^™ 2.0 User Manual kit) for the adaptor sequence: 10×Buffer 2.5 μL, 2.5 mM dNTP 0.5 μL, primer AP1 (10 μmol/L) 0.5 μL, primer SP1 (10 μmol/L) 0.5 μL, Taq DNA polymerase 0.5 μL, template 1.0 μL, ddH ₂ O 19.5 μL.

The first-round product was diluted 50-fold and used as a template for the second-round reaction.

The second round of PCR amplification was performed with NAC1-SP2 and primer AP2 (Universal Genome Walker ^™ 2.0 User Manual kit) for the adapter sequence: 10×Buffer 2.5 μL, 2.5 mM dNTP 0.5 μL, primer AP2 (10 μmol/L) 0.5 μL, primer SP2 (10 μmol/L) 0.5 μL, Taq DNA polymerase 0.5 μL, template 1.0 μL, ddH ₂ O 19.5 μL.

PCR amplification conditions: 94°C for 25sec, 72°C for 3min, the first round of 7 cycles; 94°C for 25sec, 67°C for 3min, the first round of 32 cycles; 67°C for 10min. 94°C for 25sec, 72°C for 3min, the first round of 5 cycles; 94°C for 25sec, 67°C for 3min, the first round of 20 cycles; 67°C for 10min.

The PCR products were recovered and connected to T vector, transformed into E. coli competent cells, and the recombinants were picked for sequencing. The sequencing results were analyzed by DNAstar software.

The obtained upstream promoter sequence of the CpSNAC1 gene of Prunus chinensis was analyzed by the online promoter sequence analysis software PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html) and PLACE (http://www.dna.affrc .go.jp/PLACE) to search for potential transcriptional regulatory elements in the conserved regions of this promoter.

The results showed that the 1437 bp promoter sequence (SEQ ID No. 15) upstream of the CpSNAC1 gene was amplified from the Prunus chinensis genome by the chromosome walking method. The promoter sequence contains a large number of TATA-box, CAAT-box and other basic promoter elements and plant gene-related light-permissive elements, as well as 5'UTR Py-rich stretch that increases the transcription level, abscisic acid response elements ABRE, salicylic acid The response element TCA-element, the auxin response element AuxRR-core, the gibberellin response element GARE-motif, the MYB binding site MBS involved in drought, and the defense and stress response element TC-rich repeats, etc. (Fig. 2).

3 Cloning of the deletion fragment of the CpSNAC1 gene promoter

Deletion segmentation was performed based on cis-acting element analysis of the known promoter sequences of the CpSNAC1 gene (Figure 3). The forward primers were designed at about 1228bp, 742bp, and 471bp upstream of the start codon of the CpSNAC1 coding region. The reverse primers were the same as the reverse primers of the full-length sequence of the CpSNAC1 gene promoter in the previous section, named CpSNAC1-D1-F, CpSNAC1-D2 -F, CpSNAC1-D3-F, CpSNAC1-nco1-R.

The comparison between the obtained deletion sequence and the full-length sequence is shown in Figure 4, and the CpSNAC1 gene promoter-specific primers are designed as follows:

CpSNAC1-D1-F: aactgcagcaagtagggctaaataacccatttacc (Pst1 restriction site) (SEQ ID No. 7)

CpSNAC1-D2-F: aactgcagatgttgttgcacgagcgatcaat (Pst1 restriction site) (SEQ ID No. 8)

CpSNAC1-D3-F: aactgcaggttttccaacacgagccctctct (Pst1 restriction site) (SEQ ID No. 9)

CpSNAC1-nco1-R: catgccatgggaacagcaatcggttgtgaggc (Nco1 restriction site) (SEQ ID No. 10)

Using the pMD 19-T-CpSNAC1 plasmid as a template, PCR amplification was performed with Taq enzyme.

PCR reaction system The reaction system is as follows: ddH ₂ O 18.6 μL, 10×Toq Taq Buffer 2.5 μL, 2.5 mM dNTP 1.5 μL, CpNAC1-nco1-R (10 μmol/L) 1.0 μL, CpNAC1-D1/D2/D3-F (10 μmol /L) 1.0 μL, ToqTaq DNA polymerase 0.2 μL, plasmid 0.2 μL.

PCR amplification conditions: CpNAC1-D1: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30s, annealing at 58°C for 30s, extension at 72°C for 1min 20s, 28 cycles; extension at 72°C for 10 min. CpNAC1-D2: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, 28 cycles; extension at 72°C for 10 min. CpNAC1-D3: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 40 s, 28 cycles; extension at 72 °C for 10 min.

Note: The DNA extraction kit used in this step was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; the gene walking kit (Universal Genome Walker ^TM 2.0 User Manual), DraⅠ, EcoRⅤ, PvuⅡ and StuⅠ enzymes were purchased from TaKaRa, Japan Company (Dalian).

The PCR product was recovered and then connected to the T vector, transformed into E. coli competent cells, and the recombinants were picked for sequencing. After sequencing, the sub-sequences of CpSNAC1 gene promoter of 1223bp, 711bp and 464bp were finally obtained, which were named CpSNAC1pro-D1/D2/D3.

Example 3 Construction of the plant expression vector of the CpSNAC1 promoter of Prunus chinensis

1 Construction and verification of expression vector

According to the CpSNAC1 promoter sequence and the characteristics of the plant expression vector, the plant expression vector 1305 was selected, which is a binary vector containing the GUS gene system and the CaMV35S promoter. In order to study the function of the CpSNAC1 gene promoter, the 35S promoter in the plant expression vector 1305 was replaced with it, and it was fused with the GUS reporter gene to construct the plant expression vector. The CpSNAC1 gene promoter, each deletion fragment and the 1305 vector amplified by PCR were double digested with PstI and NcoI, respectively, and the digested products were recovered and ligated, and the ligated products were transformed into Escherichia coli TOP10. Cloned, sent for sequencing, and named pcambial1305-CpSNAC1, pcambial1305-CpSNAC1-D1/D2/D3. The positive plasmids were transferred into Agrobacterium GV3101 by electroporation, and the plasmids were extracted after preliminary identification of positive bacteria by PCR for restriction enzyme digestion identification.

Double digestion and ligation:

CpSNAC1, CpSNAC1-D1, CpSNAC1-D2, CpSNAC1-D3 intermediate vector and plant vector 1305 plasmids were double digested with PstI and NcoI restriction enzymes, respectively. Double enzyme digestion system: 2.5 μL of 10× Green Buffer, 0.5 μL of PstI, 0.5 μL of NcoI, 5 μL of CpSNAC1/1305, 11.5 μL of ddH ₂ O. After digestion at 37°C for 4 hours, inactivation at 80°C for 5 minutes. Agarose gel electrophoresis was performed to separate the digested fragments, and the target fragments were recovered with a gel recovery kit. Then, the recovered 1305 expression vector and CpSNAC1, CpSNAC1-D1, CpSNAC1-D2, and CpSNAC1-D3 fragments were ligated at a ratio of about 1:8 according to the instructions of T4 DNA ligase, and ligated overnight at 4°C. Ligation reaction system: 7.5 μL of 1305 expression vector fragment, 1.2 μL of CpSNAC1 promoter fragment, 1.0 μL of 10×T4DNA Ligase Buffer, and 0.3 μL of T4DNA Ligase.

The ligation product was transformed into E. coli TOP10 competent cells, spread on an LB plate containing 50 mg/L Kan, and incubated overnight at 37°C. Pick a single colony and shake it for culture, then carry out PCR identification, and activate the positive bacterial solution by electrophoresis. The plasmid was extracted and verified by double digestion. Finally, the positive plasmids with correct PCR identification and double-enzyme digestion verification were sent to sequencing, and after confirming no base mutation, they were named 1305-CpSNAC1, 1305-CpSNAC1-D1, 1305-CpSNAC1-D2, 1305-CpSNAC1-D3, namely Recombinant vector for plant expression of CpSNAC1 gene.

2. The expression vector transforms Agrobacterium

The expression vector was transformed into Agrobacterium GV3101 by electroporation.

(1) Soak the electric shock cup in 75% alcohol for 2 hours, then rinse with sterile water for 3 times, then soak and rinse with absolute ethanol for 3 times, and then place the electric shock cup on the ultra-clean workbench to dry it. Pre-chill on ice.

(2) Take out the Agrobacterium competent cell GV3101 from the -80°C refrigerator, and place it on ice to thaw.

(3) Add 1 μL of plasmid to 100 μL of thawed competent cells, mix it gently with a pipette tip, and let it stand on ice for 30 min.

(4) Assemble the electroconversion instrument, place it on the ultra-clean workbench, turn on the power switch of the instrument, and adjust the voltage to 2240V.

(5) Use a pipette to transfer all the mixture of competent cells and recombinant plasmids into the groove of the electric shock cup, and dry the outer wall.

(6) Put the electric shock cup into the electrotransformer, shock, and after hearing a beep, take out the electric shock cup, add 800 μL of pre-cooled anti-anti-YEB liquid medium, and gently stir and mix. Transfer it to a 1.5 mL sterile centrifuge tube with a pipette.

(7) Place in a shaker at 28°C, and incubate with shaking at 180 rpm in the shade for 3 to 4 hours.

(8) Draw 10 μL of bacterial liquid, spread it evenly on YEB (containing 50mg/L Kan and 50mg/L Gen) plate medium with a sterile coating stick, and invert at 28°C under constant temperature and dark conditions for 36-48h.

(9) Pick a single colony, place it on a shaker at 28°C for 24-36 hours in the dark, and carry out PCR identification. Positive clones can be used to transform tobacco or Arabidopsis, or add a final concentration of 20% glycerol and store in -80°C ultra-low temperature freezer.

Note: Agrobacterium-competent GV3101 and expression vector pcambia1305.1 used in this step were preserved by the Institute of Garden and Flowers, Southwest University; T4 DNA ligase, ExTaq enzyme, and Top polymerase were purchased from Japan TaKaRa Company (Dalian).

Example 4 Analysis of developmental spatiotemporal expression characteristics and inducible expression characteristics of CpSNAC1 promoter and its deletion sequence

1 Transformation of Arabidopsis thaliana by inflorescence infection

(1) Sowing and cultivation of Arabidopsis

Take an appropriate amount of Arabidopsis thaliana seeds into a 1.5mL centrifuge tube, add 16.7% sodium hypochlorite (NaClO) to the centrifuge tube for 8-10 minutes (full shaking during the period), and use a pipette tip to wash off the upper layer of NaClO solution, and then no Rinse the seeds with bacterial water for 4-6 times, and then use a pipette to sow the seeds of Arabidopsis thaliana on MS medium. After vernalization at 4 °C for 3 days, the plate is placed in 16h light, 8h dark, 2000Lux lighting conditions, and 22 hours. Cultivated in an environment of ℃ and 70% humidity, and transplanted Arabidopsis thaliana into a culture medium (earthworm soil: vermiculite: peat = 1:1:1) about 12 days, and waited for Arabidopsis to grow to a scape length of about 5cm Infection occurs when left and right.

(2) Transformation of Arabidopsis by inflorescence infection

①Activate the Agrobacterium containing the plant overexpression vector plasmid on the YEB+Kan50mg/L+Gen50mg/L medium plate, and cultivate it in the dark at 28°C for 36-48h.

②A single colony was picked and inoculated into 650 μL YEB (Kan: 50 mg/L, Gen: 50 mg/L) liquid medium, and cultured with shaking at 28° C. and 200 rpm for 24 h.

③ Carry out PCR identification of the cultured Agrobacterium liquid with specific primers. After confirming that it is correct, take 500 μL of the above bacterial liquid and inoculate it on 25 mL of YEB liquid medium without antibiotics, 28 ° C, 200 rpm shaking culture until the OD600 is between 0.8 and 1.0 for later use .

④Aspirate 10mL of the above bacterial solution into a sterile centrifuge tube, centrifuge at 28°C, 5000rpm for 15min, pour off the supernatant, collect the precipitate, and then reconstitute it with the currently prepared infection solution (sterile water + 5% sucrose + 0.5‰L-77). Suspended and diluted to an OD600 of about 0.8.

⑤ For the first infection, cut off the flowers that have opened on the Arabidopsis plant, leaving only the white buds. In addition, you can water the day before treatment to maintain the humidity of the substrate. Before infection, you need to use a watering can to clean the four walls of the carton. Spray wet. Put the scape head about 0.5cm in length into the immersion dyeing solution for about 10 seconds, take it out and put it in a carton, protect it from light, incubate it at 22°C for 20h, take it out, and then transfer it to a normal growth environment.

⑥Secondary infection can be carried out after 1 week of infection to improve the transformation efficiency.

2 Screening and identification of hygromycin resistance in transgenic Arabidopsis

2.1 Transgenic Hygromycin Hyg Screening and PCR Detection

(1) One month after the transformation of Arabidopsis thaliana, the mature T0 generation seeds were collected, placed in a constant temperature oven at 37°C for about 15 days, and then stored in a 4°C refrigerator for subsequent experiments.

(2) After sterilizing the transgenic seeds of T0 generation, they were sown on MS plates containing 25 mg/L Hyg, sealed and vernalized in a refrigerator at 4°C for 3 days, and then transferred to a normal environment for cultivation. grows normally on the medium.

(3) Obtain several different lines, and continue to screen the T1 and T2 generation seeds of each line until the seeds harvested from all lines are green resistant seedlings after sowing, and then the follow-up treatment experiment can be carried out.

Using the DNA of the positive plant as a template, PCR was performed to confirm whether the target fragment was inserted in the transgenic plant.

2.2 GUS chemical staining detection of transgenic Arabidopsis

The transgenic Arabidopsis plants screened for hygromycin resistance were identified by GUS histochemical staining. The wild-type Arabidopsis plants were used as negative controls, and the transgenic Arabidopsis plants transformed with 1305 empty vector were used as positive controls.

(1) Staining: Take 12d seedlings of Arabidopsis thaliana in a 1.5mL centrifuge tube, add the prepared GUS staining solution to completely submerge the material, and place in a 37°C incubator for overnight staining in the dark.

(2) Decolorization: Take out the sample, suck off the dye solution, add 75% ethanol to decolorize, and decolorize 2 to 3 times until the negative control is white.

(3) Observation and identification: The GUS staining results of the transformed materials were observed with a solid microscope, and the results were recorded by photographing.

3 Transient expression of CpSNAC1 gene promoter and each deletion fragment in tobacco leaves

The GV3101 empty strain was used as the negative control, and the 1305 strain containing the CaMV35S promoter was used as the positive control.

(1) Preparation of Agrobacterium infection solution

① Take out the successfully transformed positive Agrobacterium strain, GV3101 empty strain and strain containing 1305 vector from the -80°C ultra-low temperature refrigerator. On the YEB solid medium plate containing 50mg/L Kan and 50mg/L Gen, placed at 28°C and cultured upside down in the dark for 36-48h.

②Pick a single colony of suitable size and inoculate it into 25 mL of YEB liquid medium containing 50 mg/L Kan and 50 mg/L Gen, and place it under constant temperature and dark conditions at 28°C for 24-36 h with shaking.

③ Take the above-mentioned cultured bacterial solution and inoculate it into YEB non-antibody liquid culture solution at 1:50, 28 ° C, 200 rpm dark-shaking culture, and perform secondary activation.

④When bacterial liquid OD600=0.6～0.8, stop shaking culture, take 8mL cultured bacterial liquid and 30mL MS1 liquid culture respectively based on aseptic culture bottle, mix evenly for later use, this mixed solution is used for infecting tobacco. infection fluid.

(2) Plant material preparation: Select the leaves of robust tobacco aseptic tissue culture seedlings, cut them into leaf pieces of about 1 cm with a sharp sterile surgical blade, and discard the midrib.

(3) Infection: The newly prepared leaf pieces were placed in the prepared three kinds of Agrobacterium infection solutions, respectively, and soaked in shading for 10 minutes. During this process, the tissue culture bottle was gently shaken to make the leaf pieces and the infection solution fully touch.

(4) Co-cultivation: Take out the leaf block and place it on sterile filter paper to absorb excess bacterial liquid. The infected leaf blocks were transferred to MS2 medium, the back of the leaf blocks was placed down, and incubated in the dark at 25°C for 2-3 days.

(5) Degermination: transfer the co-cultivated tobacco leaves to MS3 liquid degerming medium, and sterilize in the dark for 30 minutes. During this period, the culture bottle is gently shaken to make the leaf pieces fully contact with the sterilizing liquid, and then the leaves are sterilized. Place on sterile filter paper to remove excess fluid.

(6) Repeat step (5).

(7) Rinse the leaf block in sterile water for 3 times, 10 minutes each time, and then dry the excess liquid with sterile filter paper.

(8) GUS transient staining detection: randomly extract the treated leaf pieces for GUS staining analysis, place the plant tissue to be tested in a 1.5 mL centrifuge tube, add GUS staining solution, and place it in a 37°C oven overnight, then use 75% Decolorize with ethanol, 2-3 times for 10min each time, observe with a solid microscope when the negative control is white, and record the results by photographing.

4. GUS histochemical staining analysis of transgenic Arabidopsis

(1) The Arabidopsis thaliana germinated 1 to 10 days after sowing was placed in a 1.5 mL centrifuge tube, and the prepared histochemical GUS staining solution was added, and placed in a 37°C incubator for overnight staining. Destain with 75% ethanol for 2 to 3 times, then observe and take pictures under a stereomicroscope. At the same time, wild-type Arabidopsis is used as a negative control, and 1305 vector is used as a positive control.

(2) In order to study the expression specificity of CpSNAC1 promoter-driven GUS gene in each tissue of Arabidopsis plants, the roots, stems, leaves and flowers of adult transgenic Arabidopsis plants were collected for GUS histochemical staining analysis. (The method is the same as above). Fluorescence quantitative analysis of GUS gene in 5CpSNAC1pro transgenic Arabidopsis under abiotic stress

In order to study the response of the promoter to different inductions at the translation level, each line to be treated was sown in MS medium, and robust transgenic Arabidopsis thaliana with roughly the same growth was selected at 10 d, and then transferred into ABA containing 50 μM·L ^-1 respectively. , 150mM·L ^-1 NaCl, 10% PEG MS medium for 24h, select robust transgenic Arabidopsis with roughly the same growth and put them into 42℃ high temperature and 4℃ low temperature artificial climate box for 6h and 24h, each group of experimental settings 3 repetitions. After treatment, samples were taken to extract RNA, which was reversed to cDNA for fluorescence quantitative PCR detection to analyze whether the CpSNAC1 promoter was induced by hormones.

The Arabidopsis Actin gene was selected as the internal reference gene, and the internal reference gene and GUS gene were designed. The primer sequences are shown in Table 2.

Table 2 Primer sequences

Supermix试剂盒(Bio-Rad)说明书进行，对转基因植株不同非生物胁迫下幼苗叶片中的表达情况进行实时荧光定量PCR分析。试验所得的数据用Bio-Rad Manager^TM Software(Versio n 1.1)分析，用

法计算CpSNAC1启动子启动下游GUS基因在不同材料中的相对表达量。反应体系如下：2×SsoFastEvaGreen Supermix 5μL，Primer-F(10μM)0.5μL，Primer-R(10μM)0.5μL，cDNA模板0.5μL，RNase Free dH₂O 3.5μL。Total RNA was extracted with Trizol reagent, and the first strand of cDNA was obtained by reverse transcription. Fluorescence quantitative PCR was performed using EvaGreen dye method with cDNA as template, and the technique was repeated three times. Experiments according to SsoFast ^TM

Supermix kit (Bio-Rad) was used to perform real-time quantitative PCR analysis on the expression of transgenic plants in seedling leaves under different abiotic stresses. The data obtained from the experiments were analyzed with Bio-Rad Manager ^™ Software (Version 1.1) using

The relative expression of the downstream GUS gene promoted by the CpSNAC1 promoter in different materials was calculated by the method. The reaction system is as follows: 5 μL of 2×SsoFastEvaGreen Supermix, 0.5 μL of Primer-F (10 μM), 0.5 μL of Primer-R (10 μM), 0.5 μL of cDNA template, and 3.5 μL of RNase Free dH ₂ O.

6 Quantitative detection of GUS protein in various tissues of transgenic Arabidopsis and under abiotic stress

The roots, stems, leaves, flowers and fruit pods of adult transgenic Arabidopsis thaliana plants were collected to detect GUS enzyme activity. At the same time, in order to study the response of the promoter to different induction, each line to be treated was sown in MS medium, and 10 days later Robust transgenic Arabidopsis thaliana with roughly the same growth vigor were selected and transferred into MS medium containing 50 μM·L ^-1 abscisic acid (ABA), 150 mM·L ^-1 NaCl, and 10% PEG (dry simulation) for 48 h. Robust transgenic Arabidopsis that were roughly the same were placed in an artificial climate chamber with a high temperature of 42 °C and a low temperature of 4 °C for 6 h and 24 h, and the quantitative detection of GUS protein was carried out respectively after treatment.

Using the Varioskan Flash full-wavelength scanning multi-function reader (multi-function microplate reader), the total protein content was determined according to the method of Bradford (1976), and the enzyme activity of GUS was determined by the method of Li et al. (2009).

(1) Preparation of protein standard curve

Use the modified Bradford method protein concentration determination kit to take seven 1.5mL centrifuge tubes, and prepare a BSA protein concentration gradient according to Table 3:

Table 3 Reaction system

Deionized water (μL) 1000 995 990 985 980 975 970 BSA protein standard solution (μL) 0 5 10 15 20 25 30 Final protein concentration (μg/mL) 0 5 10 15 20 25 30

After mixing, measure the light absorption value at 595nm on a multi-function microplate reader to make a standard curve.

(2) Preparation of 4-MU standard curve

Prepare 4-MU gradient concentration solution according to Table 4 with 4-MU and reaction stop solution:

Table 4 Reaction system

1μM 4-MU (μL) 0 62.5 125 250 500 1000 Reaction stop solution (μL) 1000 937.5 875 750 500 0 Final concentration (nM) 0 62.5 125 250 500 1000

After mixing, use a multifunctional microplate reader to measure the fluorescence value of each sample and draw a standard curve under the conditions of excitation light of 365 nm, emission light of 455 nm, and slit of 10 nm.

(3) GUS protein extraction

Take 100 mg of fresh plant samples, grind them into powder with liquid nitrogen, collect them immediately into a 1.5 mL centrifuge tube, add 600 μL of GUS extraction buffer, mix thoroughly and centrifuge at 12,000 rpm at 4 °C for 10 min, take the supernatant in Store in a 4°C refrigerator for later use.

(4) Determination of sample protein content

The specific operation steps refer to the instructions of the modified Bradford method protein concentration determination kit:

①Put 10 μL of protein supernatant from each sample into a 1.5 mL centrifuge tube, add water to 500 μL, and mix well.

② Take the 0-30 μg/mL protein solution prepared according to Table 3-3 and 150 μL of the sample diluent in ①, and add them to the wells of the ELISA plate in sequence.

③ Add 150 μL of Bradford reagent to each well, and immediately shake and mix.

④ After mixing, let it stand at room temperature for 10 minutes, during which time it is necessary to shake and mix 2 times.

⑤After 10min, measure the A595 value of each sample on the microplate reader.

⑥ Repeat steps ②～⑤ 2 times, and calculate the average value of A595 of each sample.

⑦ With the average value of A595 of 0-30 μg/mL protein solution as the ordinate, and the corresponding protein concentration as the abscissa, use Microsoft Excel software to draw the standard curve.

⑧According to the average value of A595 of the sample diluent, determine the protein concentration of each sample diluent on the standard curve.

⑨ Calculate the protein concentration of each sample according to the formula: sample protein concentration (μg/mL) = sample diluent protein concentration × 50 (sample dilution factor).

(5) Determination of GUS enzyme activity

Take 100 μL of GUS-containing supernatant, add it to 500 μL of MUG ( ₂ mmol/L) solution preheated at 37°C, and mix it quickly and thoroughly. ₃ ), as the 0 point of the enzymatic reaction. After that, 50 μL of the reaction solution was taken out at 15 min, 30 min, 45 min and 60 min, respectively, and added to 950 μL of stop solution to terminate the reaction. The fluorescence value of each sample at different time points was measured with a multifunctional microplate reader at the excitation wavelength at 365 nm and the emission wavelength at 455 nm. The wild-type Arabidopsis thaliana was used as a negative control, and the transgenic Arabidopsis thaliana transformed with the 1305 vector was used as a positive control. Three replicates were set for each sample, and the 4-MU content of each sample was detected on the 4-MU standard curve. The average value of the fluorescence value was taken.

(6) Calculation of enzyme activity

The GUS enzyme activity per unit time was calculated according to the light absorption value and corresponding fluorescence value of each sample, and the GUS enzyme activity was expressed as protease activity per milligram per minute (pmol·mg ^-1 ·min ^-1 ).

Note: The modified Bradford method protein concentration determination kit, X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid) used in the experiment were purchased from Shanghai Bioengineering Co., Ltd.; 4-MU, 4 -MUG is from Sigma Company; Varioskan Flash full-wavelength scanning multi-function reader (multi-function microplate reader) is from ThermoScientific Company. Tobacco (Nicotiana tabacum L) tissue culture seedlings used for transient expression transformation, and the genotype of Arabidopsis thaliana used for transformation were Columbia (Columbia, Co1) provided by the Institute of Garden and Flowers, Southwest University.

The results of transient expression in tobacco leaves showed that the tobacco leaves transfected with the CaMV35S promoter and transformed with pcambia1305-CpSNAC1, pcambia1305-CpSNAC1-D1, pcambia1305-CpSNAC1-D2, and pcambia1305-CpSNAC1-D3 had blue spots after GUS chemical histology staining. presented, while the negative control had no blue spots (Fig. 5), indicating that both the CpSNAC1 gene promoter and its deletion sequences CpSNAC1pro-D1, CpSNAC1pro-D2 and CpSNAC1pro-D3 can drive the expression of the reporter gene GUS.

GUS chemical staining analysis was performed on the CpSNAC1 gene promoter and its deletion sequence transgenic Arabidopsis seed germination stage and each tissue of mature plants. , stems, leaves and flowers can be dyed, but seeds cannot be dyed (Figure 7). The results show that both the CpSNAC1 promoter and its deletion sequence have the ability to drive GUS gene expression in the whole process from seed germination to growth of Arabidopsis thaliana, without tissue specificity.

Real-time fluorescence quantitative PCR was performed on the seedlings before and after each treatment with CpSNAC1pro sequence to detect the expression of sequence GUS gene. The results showed that GUS gene expression was up-regulated after ABA, NaCl and PEG treatments, and decreased after 4°C low temperature and 42°C high temperature treatments (Fig. 8).

According to the concentration gradient of the BSA protein standard solution prepared in Table 3 above, the light absorption value at 595 nm was measured on a multifunctional microplate reader by using the modified Bradford method protein concentration determination kit. With the concentration of BSA protein standard solution as the abscissa and the absorbance value of A595 as the ordinate, draw a protein concentration standard curve (Figure 9). The variance R ² of the protein concentration standard curve is 0.9859, which is close to 1, indicating that the protein concentration of the sample has a correlation with the light absorption value at 595 nm, and the protein concentration in the solution with a certain light absorption value can be calculated according to the formula on the standard curve. .

Fluorescence intensity at slit 10 nm according to Table 4. Taking the absorbance value as the ordinate and the 4-MU concentration (μM) as the abscissa, a standard curve was drawn (Fig. 10). R ² =0.9924 of the fluorescence standard curve, which is close to 1, indicating that the concentration of 4-MU corresponding to the relative fluorescence intensity can be calculated according to the formula of this standard curve.

The GUS protein extracted from seedlings treated with CpSNAC1 gene promoter and its deletion sequences ABA, NaCl, PEG, 4 ℃ low temperature and 42 ℃ high temperature was tested for enzymatic activity. The GUS enzyme activity increased after treatment, and the GUS enzyme activity decreased after high temperature and low temperature treatments; the GUS enzyme activity of the CpSNAC1pro-D2 fragment increased after five different stress treatments; the GUS enzyme activity of the CpSNAC1pro-D3 fragment increased after ABA treatment, The enzymatic activity was almost unchanged after NaCl treatment, and decreased after PEG, high temperature and low temperature treatments (Fig. 11).

The GUS enzyme activity was detected in the adult plants, roots, stems, leaves, flowers and fruits of the CpSNAC1 gene promoter and its various deletion sequences. enzymatic activity, and the activity was highest in roots. The CpSNAC1pro-D2 fragment had the highest GUS enzymatic activity among all fragments (Figure 12). The inhibition of CpSNAC1pro-D3 fragment under PEG stress is probably related to the fact that the fragment does not contain cis-acting elements related to the MYB drought-binding site, and the cis-acting elements in the CpSNAC1pro-D3 fragment are less stress-related elements. Only ABA-responsive elements do not contain the defense and stress-responsive elements TC-rich repeats, so GUS enzyme activity is elevated only under ABA treatment but not under NaCl treatment. Only the GUS enzyme activity of the CpSNAC1pro-D2 fragment increased at high and low temperature, and the GUS staining of this fragment was the darkest, and it was the fragment with the highest GUS enzyme activity. . Therefore, it can be speculated that the induction of CpSNAC1 gene promoter by stress can affect the expression level of downstream genes, and the CpSNAC1pro-D2 fragment has an important effect on the activity of CpSNAC1 gene promoter.

sequence listing

<110> Southwest University

<120> CpSNAC1 gene promoter of Prunus chinensis and its application

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1007

<212> DNA

<213> Artificial Sequence

<400> 1

gcagtggcag actcacatag cagattatag aagaaagaag aagaagaaga agaagaagat 60

gagttgtaat ttgcagctgc ctcctggttt tagatttcac ccaacagatg aggaattggt 120

actgcactat ttatgcaaga aatgcgcctc acaaccgatt gctgttccta taattgcaga 180

gattgatctt tataaatatg atccttggca acttcctgga aaggctcttt atggagaaaa 240

ggaatggtac ttcttctctc cgagggaccg gaagtatccg aacgggtcaa ggcccaatcg 300

ggctgcagcg accggatatt ggaaggccac cggagccgat aagccaatta ggcccagtgg 360

aagtctaaag cctgttggaa tcaagaaggc cctggtcttt tatgctggaa aggccccaaa 420

gggtgaaaag tccaactgga tcatgcacga atacaggctt gcagatgttg atcgctctgc 480

taggaagaaa aatagtctaa ggctggacga ttgggtgttg tgtaggatct acaacaaaaa 540

atggggtttg gaaggaaagc aaccaaaatc cagcatcaaa tgcagggaga atgagatgga 600

ggaagagcat gtagagcaga agcctgaact tcttacaaat gcccatcaac ccatgactcc 660

cattctcaac gatttcacct acttcgactc ggccgattcc atacccaggc tacaactcac 720

cgattccagc tgctcggagc acgtggtttc ccccgagttc acctgcgaaa gggaggtcca 780

gagccagcca aagtggaaga gagactggaa caacaccctc cacatccctc ccattaacaa 840

catggatgcc accaatccct ttagtcagat ggacggtgtg gagctttctc cacttttccg 900

aattccgtca ttacaggaca tatccatgta cttgcagaag ccattctgat tgattctatg 960

ggccatccta tttctattct ttcatatgag ccaaccacac atatcac 1007

<210> 2

<211> 296

<212> PRT

<213> Artificial Sequence

<400> 2

Met Ser Cys Asn Leu Gln Leu Pro Pro Gly Phe Arg Phe His Pro Thr

1 5 10 15

Asp Glu Glu Leu Val Leu His Tyr Leu Cys Lys Lys Cys Ala Ser Gln

20 25 30

Pro Ile Ala Val Pro Ile Ile Ala Glu Ile Asp Leu Tyr Lys Tyr Asp

35 40 45

Pro Trp Gln Leu Pro Gly Lys Ala Leu Tyr Gly Glu Lys Glu Trp Tyr

50 55 60

Phe Phe Ser Pro Arg Asp Arg Lys Tyr Pro Asn Gly Ser Arg Pro Asn

65 70 75 80

Arg Ala Ala Ala Thr Gly Tyr Trp Lys Ala Thr Gly Ala Asp Lys Pro

85 90 95

Ile Arg Pro Ser Gly Ser Leu Lys Pro Val Gly Ile Lys Lys Ala Leu

100 105 110

Val Phe Tyr Ala Gly Lys Ala Pro Lys Gly Glu Lys Ser Asn Trp Ile

115 120 125

Met His Glu Tyr Arg Leu Ala Asp Val Asp Arg Ser Ala Arg Lys Lys

130 135 140

Asn Ser Leu Arg Leu Asp Asp Trp Val Leu Cys Arg Ile Tyr Asn Lys

145 150 155 160

Lys Trp Gly Leu Glu Gly Lys Gln Pro Lys Ser Ser Ile Lys Cys Arg

165 170 175

Glu Asn Glu Met Glu Glu Glu His Val Glu Gln Lys Pro Glu Leu Leu

180 185 190

Thr Asn Ala His Gln Pro Met Thr Pro Ile Leu Asn Asp Phe Thr Tyr

195 200 205

Phe Asp Ser Ala Asp Ser Ile Pro Arg Leu Gln Leu Thr Asp Ser Ser

210 215 220

Cys Ser Glu His Val Val Ser Pro Glu Phe Thr Cys Glu Arg Glu Val

225 230 235 240

Gln Ser Gln Pro Lys Trp Lys Arg Asp Trp Asn Asn Thr Leu His Ile

245 250 255

Pro Pro Ile Asn Asn Met Asp Ala Thr Asn Pro Phe Ser Gln Met Asp

260 265 270

Gly Val Glu Leu Ser Pro Leu Phe Arg Ile Pro Ser Leu Gln Asp Ile

275 280 285

Ser Met Tyr Leu Gln Lys Pro Phe

290 295

<210> 3

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 3

gttgtgaggc gcatttcttg cgt 23

<210> 4

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 4

gcttctgctc tacatgctct tcctc 25

<210> 5

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 5

gctcttcctc catctcattc tccctgc 27

<210> 6

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 6

gaacagcaat cggttgtgag gcg 23

<210> 7

<211> 35

<212> DNA

<213> Artificial Sequence

<400> 7

aactgcagca agtagggcta aataacccat ttacc 35

<210> 8

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 8

aactgcagat gttgttgcac gagcgatcaa t 31

<210> 9

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 9

aactgcaggt tttccaacac gagccctctc t 31

<210> 10

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 10

catgccatgg gaacagcaat cggttgtgag gc 32

<210> 11

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 11

catcctctgg gaaccactga ac 22

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 12

catcacattg ctcgcttcgt t 21

<210> 13

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 13

gttatggttg ggatgggaca gaaag 25

<210> 14

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 14

gggcttcagt aaggaaacag ga 22

<210> 15

<211> 1437

<212> DNA

<213> Artificial Sequence

<400> 15

gagagttgga tgatgagaag aagatatttc atctattggg gtccacattg gccattgtta 60

tttgtcgggc gttaatgtaa tctatgtggg cccacgtatg atgtcgttga attgaaagat 120

ctaaattcat gcatttgaaa ggagaaaaaa accaatgttg gccaaaagga taccttttct 180

tcaaagagta aaagctcttt cttcttttct ttgacaagta gggctaaata acccatttac 240

ccaattaaat aataacattc aaatccaacg atcaaatcat cctccacgta ggacatggcc 300

attaataaat tataattacc aattgatacg acctcagccc caattcggtc tcatgacaat 360

catattttac actttgcatt tcgtgattaa ctctttttta tctatattat ggatctaaaa 420

aaatacaaaa tgtcaaataa aataaagatt ttatcattta attcttttaa ttatatttcc 480

aaatggaccc atagtctaaa aaatatagaa ttaaaatgag attaattttt attttaataa 540

aataaaaact gatgttcaaa tcaatttctc caaatactca tgataagtca aattgaatag 600

ttagatagat ggatgtatag aaataacact aatcttaata tatgaataat aattaattta 660

ttaaagaata aaatttaaaa gaaactatct actataaaga ttagtgtgaa tatcatgaaa 720

tggcaaatgt tgttgcacga gcgatcaatg attatctaga ttaattatat aaaataaaaa 780

atataattat taaaataaat caaagaaata aaagtggagc ccaccacaac gtacagacgt 840

ccagttggta tgcaaggggc ggttctgagg atcaagtcac cgtcgggcgt caacagacta 900

tcctgacaca tacgagccta cgtggaaatc taatccgacg cccgcaattt cctagaaaaa 960

acaccggtcc aacgttttcc aacacgagcc ctctcttttt ccgccacgtg cgcctccatc 1020

caaaacttgt gacctaagca gtgacgaaaa ctgcccagcc aagtggcgca gcatcgacac 1080

gtgtacgaat gcggtcaact tgtcttcatg gatccaaagc cgattgacgg tccccacgcg 1140

tgtatttcta gagaaacctc gagccgactt agacacgtgt cgcgaatcca ccttggctcc 1200

cctctataaa ggccgaggtt tctctcgatt tccgagtttc aactttcaac cgccgaattc 1260

agttctccag atccgttttg cagtggcaga ctcacatagc agattataga agaaagaaga 1320

agaagaagat gagttgtaat ttgcagctgc ctcctggttt tagatttcac ccaacagatg 1380

aggaattggt actgcactat ttatgcaaga aatgcgcctc acaaccgatt gctgttc 1437

Claims (15)

1. The CpSNAC1 gene promoter of Prunus chinensis, characterized in that: its nucleotide sequence is shown in the 974th～1437th position of SEQ ID No.15. 2. The CpSNAC1 gene promoter of Prunus chinensis, characterized in that: its nucleotide sequence is shown in positions 727-1437 of SEQ ID No.15. 3. The CpSNAC1 gene promoter of the wax plum, characterized in that: its nucleotide sequence is shown in positions 215-1437 of SEQ ID No.15. 4. The CpSNAC1 gene promoter of Prunus chinensis, characterized in that: its nucleotide sequence is shown in positions 1 to 1437 of SEQ ID No.15. 5. The purposes of the Prunus chinensis CpSNAC1 gene promoter in regulating plant stress resistance according to claim 1, characterized in that: the stress is hormone, drought, high temperature or low temperature; the plant is Arabidopsis thaliana. 6. the purposes of the Prunus chinensis CpSNAC1 gene promoter described in any one of claim 2～4 in regulating and controlling plant adversity resistance, it is characterized in that: described adversity is hormone, high salt, drought, high temperature or low temperature; The plant described is Arabidopsis thaliana. 7 . The use of the CpSNAC1 gene promoter of Prunus chinensis as claimed in claim 5 in regulating plant stress resistance, wherein the hormone treatment is treatment with a concentration of 50 μM·L ⁻¹ abscisic acid. 8 . 8 . The use of the CpSNAC1 gene promoter of Prunus chinensis as claimed in claim 6 in regulating plant stress resistance, wherein the hormone treatment is treatment with a concentration of 50 μM·L ⁻¹ abscisic acid. 9 . 9. The purposes of the Prunus chinensis CpSNAC1 gene promoter in regulating plant stress resistance as claimed in claim 6, wherein the high salt is the NaCl treatment of the concentration of 150mM·L ^-1 . 10. The use of the CpSNAC1 gene promoter of Prunus chinensis as claimed in claim 5 in regulating plant stress resistance, wherein the drought is PEG treatment at a concentration of 10% by mass. 11. The use of the CpSNAC1 gene promoter of Prunus chinensis as claimed in claim 6 in regulating plant stress resistance, wherein the drought is PEG treatment with a concentration of 10% by mass fraction. 12 . The use of the CpSNAC1 gene promoter of Prunus chinensis as claimed in claim 5 in regulating plant stress resistance, wherein the high temperature is 42° C. 13 . 13 . The use of the CpSNAC1 gene promoter of Prunus chinensis as claimed in claim 6 in regulating plant stress resistance, wherein the high temperature is 42° C. 14 . 14. The use of the CpSNAC1 gene promoter of Prunus chinensis as claimed in claim 5 in regulating plant stress resistance, wherein the low temperature is 4°C. 15 . The use of the CpSNAC1 gene promoter of Prunus chinensis as claimed in claim 6 in regulating plant stress resistance, wherein the low temperature is 4° C. 16 .

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