CN1740190A - Gossypium barbadense lipoid transition protein and its coding gene and application - Google Patents

Abstract

The invention discloses a sea-island cotton lipid transfer protein, its coding gene and application. The purpose is to provide a sea-island cotton lipid transfer protein and its coding gene, and the application of the gene in breeding Verticillium wilt-resistant plants. The protein is a protein having one of the following amino acid residue sequences: 1) SEQ ID No.: 1 in the sequence listing; 2) the amino acid residue sequence of SEQ ID No.: 1 in the sequence listing through one to ten amino acid residues Substitution, deletion or addition of proteins with phospholipid transfer function. The prokaryotic expression product of the gene of the invention has strong antibacterial activity against Verticillium dahliae. Transgenic tobacco, Arabidopsis and cotton all showed strong tolerance to Verticillium dahliae toxin. The protein and its coding gene of the invention will play a huge role in the genetic breeding of Verticillium wilt resistant plants (especially cotton).

Description

A kind of sea island cotton lipid transfer protein and its coding gene and application

technical field

The invention belongs to the biological high-tech field of screening, isolation, gene transfer and functional research of plant disease-resistant genes, and in particular relates to a coding gene of a lipid transport protein of sea island cotton and its functional research, and the gene is used in cultivating anti-yellow Application in wilted plants.

Background technique

Verticillium wilt is one of the important diseases that threaten cotton production. It has become a major obstacle affecting cotton yield and quality because it occurs year after year and is difficult to control. According to statistics, the incidence area of cotton Verticillium wilt in my country has reached more than half of the entire cotton field at present. Breeding resistant varieties is the most economical means of controlling the disease. After research, sea-island cotton and other cotton species have high resistance to Verticillium wilt gene, but practice has proved that it is difficult to obtain stable high-disease-resistant and high-yield varieties by ordinary cross-breeding methods. The method of modern molecular genetics provides convenience for the isolation and transgenesis of disease resistance genes. At present, some disease resistance related genes have been isolated and obtained at home and abroad, and some antifungal genes have also been used to conduct transgenic research on cotton.

There have been reports at home and abroad on the transformation of antifungal genes into cotton. The transformed genes include: chitinase, β-1,3-glucanase, glucose oxidase (GO), gastrodia antifungal protein (gastrodia antifungalprotein , referred to as GAFP) and other genes. According to the analysis of the research results, after the transformation of the above-mentioned genes in upland cotton, the Verticillium wilt resistance of the transgenic plants can be improved to a certain extent, and even high-resistant plants or strains appear, but the promotion of finalized high-Verticillium wilt varieties has not yet been seen. . In addition, these genes belong to broad-spectrum antibacterial genes, not specific resistance genes against Verticillium dahliae, nor are they derived from disease-resistant cotton itself.

Liu Huijun et al. conducted an agronomic study on the glucose oxidase (GO) gene-transferred cotton lines B99261 (IV, VIII generations) and B99267 (VIII, IV generations) with the recipient Xinluzao 7 and Verticillium wilt-resistant control Zhongmian Institute 12. traits and disease resistance were compared. As a result, the plant type of the transgenic cotton became shorter, the fruit stalk became longer, and the number of bolls per plant and fiber quality were significantly improved. The resistance of B99261 to seedling anthracnose, blight and boll blight was significantly improved, but not to red rot; the resistance of B99267 to cotton boll blight was also significantly enhanced. The resistance of these two lines to Fusarium wilt and Verticillium wilt has been significantly improved, and the fourth generation B99261 has the best resistance to Verticillium wilt, reaching the level of disease resistance (Liu Huijun, Jian Guiliang, Zou Yafei. GO gene introduction has the greatest effect on cotton agronomic traits and disease resistance, Molecular Plant Breeding, 2003, Vol. 1, No. 5/6, pp. 669-672).

Liu Guizhen et al. introduced the bivalent plant expression vector pBLGC of β-1,3-glucanase and chitinase gene into cotton immature embryos by laser microbeam puncture method, transformed a generation of cotton seedlings, and used the root dipping method to resist Verticillium wilt. After screening, the surviving seedlings were transferred to the disease nursery for the determination of kanamycin (Kan 1%) resistance. Results Among the 29 transplanted seedlings, 9 plants showed obvious resistance to Kan, and 9 Kan-resistant plants were tested by PCR, and 7 of them were positive. After the T1 transgenic plants in the disease nursery experienced the peak of Verticillium wilt, 7 PCR-positive plants were obviously resistant to the disease, and had flowered and set bolls normally. The other T1 plants and the control plants all died due to the late disease. Preliminary proof that exogenous genes have been integrated into the cotton genome, and the transgenic plants show certain resistance to Verticillium wilt (Liu Guizhen, Lan Haiyan, Tian Yingchuan, Li Shucheng, Le Jinhua, Wang Lanlan, Chen Zhenghua, using laser micro-beam puncture method Research on obtaining Verticillium wilt-resistant transgenic cotton, China Laser, 27(3): 279-283). Le Jinhua et al. introduced the bivalent plant expression vector pBLGC of bean chitinase gene and tobacco β-1,3-glucanase gene into Xinjiang cotton main varieties (lines) 550, 822, and 1304 by pollen tube passage method. , Shi Yuan 321. Through kanamycin resistance detection, PCR, Southern hybridization verification and Verticillium wilt and fusarium wilt resistance screening, the transgenic cotton with good disease resistance and stable heredity was obtained. After 6 years and 10 generations of selective breeding, new genetically modified and disease-resistant lines resistant to Fusarium wilt and Verticillium wilt (Le Jinhua, Zhu Jianbo, Cui Baiming, Zhu Xinxia, Wang Aiying, Chen Zhenghua, Liu Gui, Li Guoying, Jian Guiliang, Wu Gang, Breeding New Cotton Disease-Resistant Varieties Using Target Gene Transformation Technology, Journal of Shihezi University (Natural Science Edition), Volume 6, Issue 3, September 2002).

Gastrodia antifungal protein (GAFP for short) is a protein with broad-spectrum antifungal activity isolated from the traditional Chinese medicine Gastrodia elata Bl. Pathogenic bacteria such as wilt have a strong in vitro inhibitory effect, so it has very important application value in the genetic engineering of plants against fungal diseases. Wang Yiqin and others transferred the gene gafp encoding GAFP into three Xinjiang colored cotton varieties by pollen tube passage method, and obtained transgenic plants with high resistance to Verticillium wilt through field disease resistance screening and molecular detection, and two Southern hybridization positive plants LB-5-8 and ZB-1-49 showed whole-plant immunity to Verticillium wilt. The results of RT-PCR showed that both LB-5-8 and ZB-1-49 had the correct transcription of gafp; the antibacterial experiments in vitro also showed that their crude protein extracts had significant effects on the pathogenic bacteria of Verticillium dahliae in cotton Inhibitory effect, indicating that gafp is correctly expressed in transgenic plants, and the translation product is active. After further selection and propagation, it was found that the offspring of transgenic colored cotton had stable and strong resistance to Verticillium wilt. This study provided a new way to control cotton Verticillium wilt through plant disease-resistant genetic engineering ( Wang Yiqin, Chen Dajun, Wei Xiaowei, Wu Minggang, Wang Dongmei, Yao Zhengpei, Huang Quansheng, Liu Fengjiang, Meili Guli, Li Renjing, Sun Yongru, research on the transformation of Gastrodia elata antifungal protein gene (gafp) into colored cotton, Botany Bulletin 2003, 20(6 ):703-712).

Wu Jiahe et al. used the rhizobacterium-mediated method to transfer the vascular bundle-specific promoter (from commelina yellow mottle virus, ComYMV) driven chitinase and CaMV 35S promoter driven β-1,3-glucose Glycanase chimeric bivalent gene was introduced into upland cotton varieties Jihe 321 and Zhongmiansuo 35. The kanamycin resistance of transgenic plants was preliminarily identified by kanamycin smear method, and then confirmed by PCR and Southern hybridization. The results showed that the bivalent resistance genes were integrated into the cotton genome in 1-2 copies. The seedlings of the transgenic line T3 were identified by the bottomless plastic pot fungus liquid watering method and the identification of the field disease nursery. The results showed that the 7 transformed lines showed different degrees of resistance or resistance to Verticillium wilt. The results of the seedling identification and the field disease nursery The survey results were basically consistent; the field disease index of the three transgenic homozygous lines, D9910, D9915 and D9919, were 6.5, 9.4 and 9.5, respectively, all reaching high resistance levels. The genetic analysis of the above three homozygous lines with high resistance to Verticillium wilt showed that the kanamycin resistance of the three resistant homozygous lines conformed to the segregation rule of a pair of dominant genes. Combined with the results of molecular hybridization verification, it can be considered that These three transgenic strains all contained one copy of the double resistance gene.

Higher plant lipid transfer proteins (lipid transfer proteins, LTP) is a kind of basic protein with small molecular weight (about 9kDa), which has been obtained from corn seedlings, spinach leaves, castor, barley, onion, cotton and Arabidopsis, etc. LTP has been purified from different plants, and the cDNA and gene encoding LTP have also been cloned from different plants. LTP is able to transport phospholipids between biomembranes, so it is believed that LTP is involved in the formation of intracellular biomembranes. After research, it was found that LTP has a signal peptide, which can be secreted from the inside of the cell to the outside of the cell, and is located on the cell wall, thus raising doubts about its ability to transport lipids in the cell. It has been reported that when plants are infected by pathogenic bacteria, LTP-like may promote long-distance transport through a lipid-dependent molecule (Ana M. Maldonado, Nature 419:26, September, 2002). There is also evidence that LTP is involved in the formation of cutin and wax, plant disease resistance and plant adaptation to environmental changes (temperature, salt, drought stress). In addition, the LTP gene was isolated from cotton, and the report of the research was only to link the function of the gene with the development of the fiber, while the LTP gene was isolated from the leaves of sea island cotton, and the gene was transformed into upland cotton, and obtained Plants with high resistance to Verticillium wilt have not been reported yet.

The assay method of LTP transport lipid activity is relatively complicated, it needs two kinds of biological membranes, namely donor membrane (donor) and acceptor membrane (acceptor). Donor membranes generally use liposomes formed by radiolabeled or fluorescently labeled phospholipids through ultrasonic treatment, while acceptor membranes mostly use mitochondria or other subcellular components that can be separated from liposomes by centrifugation. The ability of LTP to transfer lipids is expressed by co-incubating acceptor membranes with donor membranes before isolating them and measuring the amount of radiolabeled or fluorescently labeled phospholipids in the acceptor membrane. In the absence of LTP, the efficiency of phospholipid transfer between biological membranes is very low or nonexistent. In animal and plant LTP transport lipid tests, it was found that phospholipids exchanged bidirectionally between the donor membrane and the acceptor membrane, so LTP was called phospholipids exchange protein (PLEP), but it is now recognized that The naming is still LTP. Because LTP is a small molecular basic protein, the separation and purification of this protein is based on its biochemical characteristics, that is, it is separated by gel molecular sieve, ion exchange chromatography, reverse high pressure liquid chromatography and other techniques. During the monitoring of LTP activity during the purification process, it was found that there were multiple peaks of LTP translipid activity, which indicated the presence of isozyme components in plants.

The primary structure (amino acid sequence) of corn, wheat, castor and rice LTP has been determined, and the primary structure of LTP of some other plants can also be deduced from its cDNA sequence. LTP consists of 91-95 amino acid residues, has a signal peptide consisting of 21-27 amino acid residues, and also contains 8 highly conserved cysteine residues to form 4 disulfide bridges. The homology of its primary structure varies greatly among different species, and the homology of plant LTP amino acid residue sequence is between 30% and 70%. Despite this polymorphism, they all share the structural features and properties of LTP proteins. The three-dimensional structure of LTP was studied by X-ray crystallography and nuclear magnetic resonance (NMR), and the three-dimensional structure of LTP was drawn. Both methods are based on maize LTP, which contains 93 amino acid residues, constitutes 4 α-helixes (α-helix), forms 4 disulfide bonds near the C-terminus, and has an inset hydrophobic cavity ( Hydrophobic cavity), when LTP transports phospholipid molecules, the hydrophobic fatty chain of phospholipid molecules is embedded in the hydrophobic cavity of LTP. NMR results show that the hydrophobic cavity of LTP molecule is enough to accommodate the entire phospholipid molecule, but X-ray crystallography results show that only the acyl chain is inserted into the hydrophobic cavity of LTP molecule, and some specific LTPs, such as acyl-CoA binding protein (acyl The three-dimensional spatial structure of -CoA-binding protein (ACBP) is also similar to that of LTP. Regarding the mechanism of how LTP transports phospholipids between membranes, the accepted model is that LTP and phospholipids form LTP-phospholipid complexes. The two acyl chains of phospholipid molecules interact with LTP one strongly and the other weakly. When LTP carries phospholipid molecules to the membrane surface, this characteristic is conducive to the exchange of phospholipids. However, when dithiothreitol was used to reduce the alpha helix of LTP from 40% to 25%, the LTP translipid activity of maize was inhibited, indicating the importance of the disulfide bond of LTP to maintain its activity; adding NaCl and other ionic compounds It can also inhibit the activity of LTP, which indicates that the electrostatic balance between LTP and biomembrane is also very important to maintain the activity of LTP, and its activity is Ca ²⁺ dependent. The gene or cDNA encoding LTP has been cloned from spinach, tomato, barley, carrot, Arabidopsis and the like. About more than 100 expressed sequence tags (expressed sequence tag, EST) similar to LTP have been identified. These ESTs were all assigned to a class of tentative homologous sequences, the sequences of which are available on the Internet. But it remains to be confirmed whether they all have the function of the LTP gene. Southern blot analysis showed that there were multiple copies of the LTP gene in cotton, maize, and rice, and they were distributed on different chromosomes.

The expression of LTP gene has strong developmental and tissue specificity, and the expression characteristics of LTP gene in different species are not the same. However, studies on the expression of vegetative organs in most materials showed that there was often no expression of LTP genes in roots, and the expression of LTP genes in young tissues was stronger than that in aged tissues, and the expression in young inflorescences was stronger. The results of in situ hybridization experiments showed that the LTP genes of maize seedlings and carrot embryos were only expressed in epidermal cells, which had strong cell type specificity, while the LTP genes of rapeseed were expressed in all epicotyls, while the other The LTP gene is specifically expressed in the tapetum cells of rapeseed. In situ hybridization experiments also proved that the AtLTP1 gene of Arabidopsis thaliana was associated with the origin of the embryo, mainly expressed in epidermal cells, and it was believed that it might be related to the formation of the apical tissue of the embryo. And through the plant transgenic method, the promoter of LTP gene was combined with GUS gene to study the specificity of LTP gene expression.

The relationship between the exocrine function of LTP and somatic embryogenesis can be explained that it may be involved in the formation of the outer protective layer of young embryos. Recent studies also show that LTP is involved in the plant's disease resistance response and is a plant disease resistance protein. The purified plant LTP protein can inhibit the growth of bacteria and fungi in vitro. The LTP proteins isolated from corn, spinach, cauliflower, and Arabidopsis all showed different degrees of disease resistance function, and one LTP protein also showed induction expression by pathogenic fungi. However, the mechanism by which LTP participates in plant disease resistance is not clear, and one view is that it may affect the cell membrane of fungi as a polar protein. A disease-resistant protein of onion has homology to LTP, but it does not appear to have the function of transporting lipids. Other studies have shown that the expression of LTP is affected by environmental factors, such as temperature, salt and drought stress can induce the expression of LTP gene. NaCl, mannitol, and abscisic acid (ABA) can induce the expression of LTP gene in the stem of tomato, while the expression of LTP gene in barley and cotton can be induced by low temperature and ABA. However, whether their expression under adversity stress is related to the stress resistance of plants needs further study.

The expression regulation mechanism of LTP gene is still unclear. Specific LTP is a kind of protein that transports phospholipids with low specificity. It has the ability to transport different phospholipids, but there are also proteins that specifically bind to certain types of lipids in cells. For example, two types of proteins have been discovered recently. Proteins: one is acyl-CoA-binding protein (acyl-CoA-binding protein, ACBP), which has been isolated and purified from Arabidopsis thaliana. It is a kind of 92 amino acid residues, isoelectric point A basic protein with a value of 9, which can specifically bind to acyl-CoA, but is different from other lipids and acyl groups. Its basic characteristics are similar to LTP, but without signal peptide and 8 conserved cysteine residues of LTP, it is an intracellular protein. The ACBP cDNAs of cotton, rice and Arabidopsis have been cloned. Another type of specific lipid transfer protein (spLTP) is a type of soluble LTP, which has been found in animals and yeast, among them, the one that can specifically transport phosphatidylinositol (PI) is called PI- TP (phosphatidylinositol-transfer protein); LTP that can specifically transport phosphatidylcholine (PC) is called PC-TP (phosphatidylcholine-transfer protein).

Contents of the invention

The purpose of the present invention is to provide a sea island cotton lipid transport protein and its coding gene.

The sea-island cotton lipid transport protein provided by the present invention, named AT7, is derived from Gossypium barbadense (Gossypium barbadense), and is a protein with one of the following amino acid residue sequences:

1) SEQ ID №: 1 in the sequence listing;

2) The amino acid residue sequence of SEQ ID №: 1 in the sequence listing has undergone one to ten amino acid residue substitutions, deletions or additions and has a phospholipid transfer function protein.

SEQ ID No. 1 in the sequence listing consists of 120 amino acid residues.

The gene (At7) encoding the lipid transport protein At7 of sea island cotton is one of the following nucleotide sequences:

1) The DNA sequence of SEQ ID №: 2 in the sequence listing;

2) The DNA sequence of SEQ ID №: 1 in the coding sequence list;

3) A nucleotide sequence that can hybridize to the DNA sequence defined by SEQ ID No. 2 in the sequence listing under high stringency conditions.

The highly stringent conditions are hybridization and membrane washing at 65°C in a solution of 0.1×SSPE (or 0.1×SSC), 0.1% SDS.

SEQ ID №: 2 in the sequence listing consists of 592 bases, and its coding sequence is the 80th-442th base from the 5' end, encoding a protein with the amino acid residue sequence of SEQ ID №: 1 in the sequence listing.

The expression vectors, transgenic cell lines and host bacteria containing the genes of the present invention all belong to the protection scope of the present invention.

Primer pairs for amplifying any fragment of At7 are also within the protection scope of the present invention.

Another object of the present invention is to provide a method for breeding plants resistant to Verticillium wilt.

The method for cultivating Verticillium wilt-resistant plants provided by the present invention is to introduce the sea island cotton lipid transporter gene At7 into plant tissues or cells to obtain Verticillium wilt-resistant plants.

The Gossypia sea-island lipid transporter gene At7 can be introduced into plant tissues or cells by a plant expression vector containing the Gossypium sea-island lipid-transporter gene At7; the starting vector for constructing the plant expression vector can be any double Agrobacterium vectors or vectors that can be used for plant microprojectile bombardment, such as pCAMBIA1301, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300 or pBI121, etc. When using At7 to construct a plant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription initiation nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, Ubiquitin (ubiquitin) gene promoter (pUbi), etc., they can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct a plant expression vector, enhancers, including translation enhancers, can also be used Or transcription enhancers, these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) Genes, etc.), antibiotic resistance markers (gentamycin markers, kanamycin markers, etc.) or chemical resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene.

Using pCAMBIA1301 as the starting vector, the constructed plant expression vector containing the G. gossypiae lipid transporter gene is pCAMBIA1301-At7.

The plant expression vector carrying At7 of the present invention can transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrical conduction, Agrobacterium-mediated, and the transformed Plant cells or tissues grown into plants. The transformed plant host can be various plants susceptible to Verticillium wilt (Verticillium wilt) such as cotton, tobacco, Arabidopsis, rice, wheat, alfalfa, soybean, tomato, eggplant and watermelon.

The invention provides a lipid transport protein At7 derived from sea-island cotton and its coding gene At7. Tests have proved that the prokaryotic expression product of At7 has strong antibacterial activity against Verticillium dahliae. Transgenic tobacco, Arabidopsis and cotton all showed strong tolerance to Verticillium dahliae toxin. The protein and its coding gene of the invention will play a huge role in the genetic breeding of Verticillium wilt resistant plants (especially cotton).

The present invention will be further described below in conjunction with specific embodiments.

Description of drawings

Fig. 1 is the detection result of the prokaryotic expression of IPTG-induced 1-4h At7 protein

Figure 2 is the inhibitory effect of At7 prokaryotic expression product At7 on Verticillium dahliae V ₉₉₁

Figure 3 is the inhibitory effect of At7 prokaryotic expression live bacteria (Escherichia coli BL21 transformed with pET-28a-At7) on Verticillium dahliae V ₉₉₁

Figure 3A is the physical map of the At7 overexpression vector pCAMBIA1301-At7

Figure 4 is the young shoots grown from the At7 transgenic tobacco callus

Figure 5 is At7 transgenic tobacco positive plants

Figure 6 is the result of RT-PCR detection of the transcription level of the target gene At7 in transgenic plants

Figure 7 is the result of acupuncture detection of Verticillium dahliae toxin on the wilting response of At7 transgenic tobacco positive strains

Figure 8 shows that the seeds of the T1 generation of At7 transgenic Arabidopsis thaliana were plated on the MS medium containing hygromycin for screening

Figure 9 is the plants after germination of the seeds of the T1 generation of At7 transgenic Arabidopsis positive and negative strains after antibiotic selection

Figure 10 is the PCR detection result of At7 transgenic Arabidopsis

Figure 11 is the detection result of At7 transgenic Arabidopsis resistance to Verticillium dahliae toxin

Figure 12 is the resistance ability of At7 transgenic positive plants to miscellaneous bacteria

Figure 13 is the cotton seedling just unearthed after transforming pCAMBIA1301-At7 into cotton

Fig. 14 is the situation that the seedling stage of At7 transgenic cotton planted in the diseased soil morbidity (the arrow points to the disease-resistant plant)

Figure 15 is the surviving plants of At7 transgenic cotton after selection of disease resistance

Figure 16 is the field performance of At7 transgenic positive plants obtained after disease resistance screening

Figure 17 is the result of PCR identification of At7 transgenic cotton plants

Detailed ways

The methods used in the following examples are conventional methods unless otherwise specified, and the primers used are all synthesized by Shanghai Sangong.

Embodiment 1: Cloning of sea island cotton lipid transporter gene At7

Plant material: Sea-island cotton AXMN with high resistance to Verticillium wilt.

(1) Cultivate the plant materials with sterile soil first. When the cotton seedlings grow 1-2 true leaves, take out the cotton seedlings and clean the root system with clean water, and then divide them into two groups, one of which is treated with strong pathogenicity Soak in 70-fold dilution of Verticillium dahliae V ₉₉₁ toxin, and take samples every 6 hours within 24 hours. cutting, divided into roots and aerial parts), weighed after sampling, and then immediately frozen with liquid nitrogen, and placed in a -80°C refrigerator for temporary storage; the other group was soaked in water and treated in the same way. (2) The samples treated with the toxin for 6-24 hours were mixed, and the total RNA of the aboveground leaves of cotton was extracted by the CTAB method, and the mRNA was purified by the mRNA purification kit of Promega Company; the control was also treated in the same way. (3) Obtain 10 toxin-induced specific expression fragments of Gossypium japonicus by SSH (Suppression Subtractive Hybridization, SSH) method, the specific method is as follows:

The PCR-SelectTM cDNA Subtraction Kit of Clontech Company was used and operated according to the instructions of the kit. Firstly, the mRNA of the aboveground leaves of cotton treated with the toxin for 6, 12, 18 and 24 hours was used as the template, and the one treated with water was used as the template. As a control, the first-strand cDNA was synthesized by reverse transcription; then the second-strand cDNA was synthesized using the first-strand cDNA as a template, and the synthesized cDNA was digested with the restriction endonuclease Rsal, and then the toxin-treated cDNA digestion fragments were respectively added Two kinds of adapters, use excess control cDNA fragments to anneal with two kinds of cDNA fragments with different adapters respectively (first hybridization), and then carry out the second hybridization of the two products after the first hybridization, and then through the second hybridization The product after the second hybridization is used as a template to carry out a common PCR and a nested PCR, and the second PCR product is directly separated by PAGE, and the obtained electrophoresis band is the gene fragment specifically expressed by Verticillium dahliae toxin-induced induction of sea island cotton. As a result, a total of 10 fragments were obtained, numbered At1-At10 respectively. Through sequence homology comparison, the seventh sequence was confirmed as lipid transport protein (LTP) gene, and the gene was named At7, specific primers were designed, and its full-length cDNA was amplified by PCR. The primer sequences were as follows:

Primer 1 (upstream primer): 5'-GATATTAATTTGTAGATAAAGTTAATATTACG-3';

Primer 2 (downstream primer): 5'-GACGACAATCAGCAATAGTAC-3'

Using the first-strand cDNA synthesized by reverse transcription of the above-mentioned mixed samples treated with Verticillium dahliae V ₉₉₁ toxin for 6, 12, 18 and 24 hours respectively as a template, under the guidance of primer 1 and primer 2, PCR amplified cotton resin For the full-length cDNA of the mass transporter gene At7, the PCR reaction conditions were as follows: first 94°C for 5 minutes; then 94°C for 30 s, 52°C for 30 s, and 72°C for 1 min, a total of 35 cycles; finally, 72°C for 10 min and stored at 4°C. After the reaction, the PCR product was detected by 1.0% agarose gel electrophoresis, and the target fragment of about 592bp was recovered and purified, cloned into the vector pGEM-T (Promega Company), screened for positive clones, and sequenced. The recombinant plasmid vector of the sequence is named At7-pGEM-T, and the sequencing results show that At7 has the nucleotide sequence of SEQ ID №: 2 in the sequence listing, and SEQ ID №: 2 in the sequence listing consists of 592 bases, and its encoding The sequence is bases 80-442 from the 5' end, encoding a protein with the amino acid residue sequence of SEQ ID No. 1 in the sequence listing.

Example 2: Detection of the antibacterial effect of At7 prokaryotic expression product on Verticillium dahliae

1. Prokaryotic expression of At7

The cDNA sequence of the sea island cotton lipotransporter gene At7 that embodiment 1 obtains is cloned in the vector pET-28a (Novagen Company), and the recombinant vector that contains the At7 cDNA sequence is named pET-28a-At7, then pET-28a- At7 was transformed into Escherichia coli BL21, and positive recombinants were obtained after screening. Pick a single colony containing pET-28a-At7, inoculate it in LB liquid medium, and induce expression at 37°C and 1 mmol/mL IPTG for 16 hours by conventional methods to transform Escherichia coli with pET-28a empty vector BL21 is a negative control, and samples are taken every 1 hour. The samples taken at 1 hour, 2 hours, 3 hours, and 4 hours are subjected to 6% SDS-PAGE electrophoresis detection. 1h, 2h, 3h, and 4h represent the prokaryotic expression of At7 protein induced by IPTG for _1h , _2h , _3h , and _4h , respectively. Negative control), the result was that the expression of At7 was the highest when induced by IPTG for 4h, and the expressed specific protein At7 had a molecular weight of 27.59kDa, was soluble in water when the pH was 9.8, and belonged to basic protein.

2. Detection of the antibacterial effect of At7 prokaryotic expression product At7 on Verticillium dahliae

Take 1.5 mL of Escherichia coli BL21 transformed with pET-28a-At7 that has been induced by IPTG for 4 hours in step 1, collect the bacteria by centrifugation, add 100 μL of TE solution, and use the alternate freeze-thaw method (repeatedly at -70 ° C and room temperature for 5-6 Second) broken thalline, sucked supernatant and precipitated protein respectively and carried out the inhibition test of Verticillium dahliae V ₉₉₁ , with transforming the supernatant and precipitated protein of Escherichia coli BL21 induced by pET-28a empty vector as contrast, method is: The Verticillium dahliae was inoculated in the center of the PDA medium, and then 4 holes were drilled around the Verticillium dahliae, two of which were added with 100 μL of the supernatant that induced the expression of freeze-thawed products and some insoluble proteins. The other two wells were respectively added with the induced supernatant and precipitated protein of Escherichia coli BL21 transformed with pET-28a empty vector, placed at 28°C for 5 days to observe the antibacterial effect, the results are shown in Figure 2 (the treatment is the expression of At7 The inhibitory effect of protein on Verticillium dahliae; CK is the control, showing that the expression protein of Escherichia coli BL21 transformed with pET-28a empty vector has no inhibitory effect on Verticillium dahliae), indicating that At7 prokaryotic expression product At7 has inhibitory effect on Verticillium dahliae Bacterial activity, especially the antibacterial activity of precipitated protein is higher. In order to further identify the antibacterial activity of Escherichia coli BL2 transformed with pET-28a-At7, the antibacterial test was carried out with live bacteria, and the Escherichia coli BL21 transformed with pET-28a empty vector was used as a control, and the method was similar to that of the induced protein , but the supernatant and protein precipitation were replaced by Escherichia coli BL2 containing pET-28a-At7 and Escherichia coli BL21 with pET-28a empty vector, the results are shown in Figure 3 (At7-1 and At7-2 indicate transformation with pET- 28a-At7 Escherichia coli BL21, which were originally inoculated At7 prokaryotic expression protein (obtained by alternate freezing and thawing), and later reproduced and formed BL21 colonies due to the survival of some bacteria during freezing and thawing; CK1 and CK2 were used as controls), and transformed with pET- Compared with Escherichia coli BL21 with 28a empty vector, Escherichia coli BL2 transformed with pET-28a-At7 also had significant antibacterial activity against Verticillium dahliae.

Example 3: Detection of wilting response of Verticillium dahlia toxin to At7 transgenic tobacco positive strains

1. Obtaining At7 transgenic tobacco

1. Construction of At7 tobacco overexpression vector

After the cDNA sequence of the sea-island cotton lipotransporter gene At7 obtained in Example 1 was digested with restriction endonucleases BglII and BstE II, it was combined with the vector pCAMBIA1301 (CAMBIA, Australia) containing the CaMV 35S promoter through double digestion with the same enzyme. company) to obtain the overexpression vector of At7, named pCAMBIA1301-At7, and its physical map is shown in Figure 3A.

2. Obtaining and molecular detection of At7 transgenic tobacco

The At7 tobacco overexpression vector pCAMBIA1301-At7 constructed in step 1 was transformed into tobacco Shanxi Nicotiana using the Agrobacterium method, and the Agrobacterium used was GV3101. As a result, 75.6% of the transformed leaf segments grew calluses, and most of them differentiated There were green shoots (Fig. 4), and 30 seedlings finally emerged after transplanting, and the transgenic positive plants were as shown in Fig. 5 . Genomic DNA was extracted from the obtained transgenic plants, and PCR detection was carried out with specific primers Primer 1 and Primer 2. As a result, the target gene of about 600 bp was detected in 23 plants. In order to detect the transcription level of the target gene At7 in transgenic plants, the method for RT-PCR is used to detect, the results are shown in Figure 6 (swimming lanes 1-6 represent different transgenic plants), the target bands of swimming lanes 1-3 are brighter, It shows that At7 has a higher expression level in the 5th, 6th, and 7th plants tested, while the target bands in lanes 4-6 are darker, indicating that the 8th, 9th, and 13th plants have lower expression levels of At7.

2. Detection of wilting response of Verticillium dahlia toxin to At7 transgenic tobacco positive strains

For the At7 transgenic tobacco positive plants obtained in step 1, use leaf acupuncture to "inoculate" Verticillium dahliae V ₉₉₁ , select four different positions on each leaf, and inoculate the stock solution of V ₉₉₁ Verticillium dahliae filtrate (position 1), 30 times diluted Liquid (position 2), 100-fold dilution (position 3) and clear water (position 4), with wild-type tobacco leaves as a control, observed at 24, 48 and 72h after inoculation, the results are shown in Figure 7, At7 transformed tobacco Afterwards, the wilting effect of Verticillium dahliae V ₉₉₁ toxin on leaves was significantly reduced, while the control showed obvious scorching symptoms. Non-transgenic tobacco leaves were treated with the stock solution, 30-fold _dilution and 100-fold 24h after the treatment of the diluted solution showed obvious dry spot symptoms, and the whole leaf spot became more serious and the leaves turned yellow at 72h; while the control only showed a little dry spot at the acupuncture site 48h after inoculation, and showed obvious dry spot at the acupuncture site at 72h. A smaller dryness emerges.

Example 4: Detection of resistance of At7 transgenic Arabidopsis to Verticillium dahliae toxin

1. Antibiotic screening and molecular detection of At7 transgenic Arabidopsis

The At7 overexpression vector pCAMBIA1301-At7 constructed in Example 3 was transformed into Arabidopsis thaliana by the Agrobacterium method, and the Agrobacterium used was GV3101, and the T1 generation seeds obtained after transformation were spread on MS medium containing 80 μg/mL hygromycin Vernalization was done for 3 days, and then placed in a light incubator to grow for 7 days. Most of the seeds could germinate (Fig. 8, the arrow points to the seeds of the transgenic positive plants), but the root growth of the transgenic negative plants was hindered, and the cotyledons could not be expanded. The positive transgenic plants have darker, flatter leaves, higher plant height, and thicker and longer roots (as shown in Figure 9, the arrow points to the transgenic positive plants). Transfer the positive plants to the MS medium without antibiotics, continue to grow to 4-5 true leaves, and then transplant them into the soil. Harvest the seeds of the T2 generation of the transgenic plants according to the line. As a result, a total of 41 T2 generation lines were obtained. . The Arabidopsis transgenic plants obtained through antibiotic screening were molecularly detected by PCR method, and the total genomic DNA of the positive plants was extracted and used as a template. Under the guidance of At7 cDNA specific primers primer 1 and primer 2, PCR amplification was carried out. Increase, after the reaction finishes, carry out 1.0% agarose gel electrophoresis detection to PCR product, detection result is as shown in Figure 10 (swimming lane M is molecular weight standard reference material, swimming lane 5,6 is positive transgenic strain, swimming lane 12,13 is For false positive transgenic lines, lane 35 is a positive control, and lane 14 is a negative control), those that can amplify a target fragment of about 600bp are positive transgenic plants, while false positive plants do not amplify fragments of corresponding size.

2. Detection of resistance of At7 transgenic Arabidopsis to Verticillium dahliae toxin

Treat the T2 seedlings of the positive transgenic Arabidopsis 35th strain obtained in step 1 with Verticillium dahliae V ₉₉₁ bacterium solution with a concentration of 0.22 μg/mL, and observe the growth changes of the seedlings. The results are shown in Figure 11 (indicated by the long arrows) Refers to At7 transgenic positive plants, short arrows indicate non-transgenic plants), there were 30 plants in the treated culture dish, and 8 seedlings began to wilt within 10 minutes after treatment, and then the degree of wilting gradually increased to 2 hours reached the most serious level; the other 22 strains were not sensitive to the treatment of Verticillium dahliae toxin, and there was no wilting phenomenon from the beginning to 6 hours, that is, the phenotype had resistance to wilting. The ratio of wilting-resistant plants to wilting plants was counted, and the ratio of wilting-resistant plants to wilting plants was 22:8, which conformed to the rule of 3:1 dominant single gene inheritance, indicating that At7 may be a single-copy integration in transgenic positive plants . In addition, when transplanting Arabidopsis thaliana, a petri dish was accidentally contaminated by miscellaneous bacteria in the experiment. As a result, the At7 transgenic positive plants showed strong resistance to the miscellaneous bacteria, while the non-transgenic strains did not have resistance (Fig. 12 As shown, the arrow in the figure indicates that At7 is in the transgenic positive plant).

Embodiment 5: Detection of resistance of At7 transgenic cotton to Verticillium dahliae toxin

1. The acquisition and detection of At7 transgenic cotton adopts the pollen-tube pathway method (pollen-tube pathway) designed by Zhou Guangyu et al. based on DNA fragment hybridization theory (Zhou G.Y. et al., Introduction of exogenous DNA into cotton embryos, MethEnzymol, 1983, 101 : 433-481) the At7 overexpression carrier pCAMBIA1301-At7 that embodiment 3 constructs is transformed into cotton (cotton variety is 2003-006), and specific method is: 20-24h after cotton self-pollination, remove corolla, expose pistil, The top of the ovary was cut flat, and the ventricle was not exposed. The pCAMBIA1301-At7 plasmid was added dropwise on the cut surface of the ovary or superficially injected, and 5 μL was injected into each flower, and the imported flowers were marked. After cotton bolling, it is harvested in combination. The next generation is planted in combinations. The transgenic plants were then identified by the following methods:

1. Identification of disease resistance

The seeds of the transgenic plants harvested after the At7 overexpression vector pCAMBIA1301-At7 were transformed into cotton were planted in the diseased soil containing 3.0×10 ⁸ Verticillium dahliae spores, and the disease resistance was identified under the "high pressure" of the pathogen. Because the cotton variety used for At7 transgenic is a susceptible variety, after high-pressure selection, most cotton seedlings died of disease, and the remaining few plants had high disease resistance. As a result, when the cotton seedlings grew to 2 true leaves, only 15 cotton seedlings did not show symptoms of Verticillium wilt, accounting for about 0.4% of the total, and all the rest were diseased and withered (as shown in Figures 13-16).

2. Identification of anti-kanamycin

After the 15 At7 transgenic cotton plants that survived the identification of disease resistance in step 1 grew 2 true leaves, the kanamycin solution with a concentration of 1000ppm was dripped on the growth point of the seedlings, and the contact card of the new leaves of the seedlings was observed a few days later. For the response of the parts of the solution to kanamycin, the leaves without reaction are kanamycin-resistant positive plants, and the leaves whose leaves turn yellow are negative plants sensitive to kanamycin. For the initially screened kanamycin resistance-positive plants, the above-mentioned phenotype identification of the seedling growth point by dripping the kanamycin solution was repeated twice, and finally 11 At7 transgenic cotton plants with kanamycin resistance were obtained.

3. PCR identification

Extract the total DNA of the leaves of 11 At7 transgenic cotton plants identified as positive through the above two steps and use this as a template to perform PCR amplification under the guidance of At7cDNA-specific primers Primer 1 and Primer 2. After the reaction, the PCR product Carry out 1.0% agarose gel electrophoresis detection, the detection result is as shown in Figure 17 (swimming lane M is molecular weight standard reference material, and swimming lane 1-4 is different transgenic plants), can amplify about 600bp target fragment is positive transgenic plant , while the false positive plants did not amplify fragments of corresponding size.

2. Detection of resistance of At7 transgenic cotton to Verticillium dahliae toxin

1. Identification of indoor diseased soil

Take soil from a diseased field where cotton has been planted for 8 consecutive years (the field is mixed with cotton blight and Verticillium wilt, and the seedling deficiency rate is as high as 67%), and then inoculated with artificially cultivated Verticillium wilt V ₉₉₁ at an inoculation concentration of 3.0×10 ⁸ For spores, the inoculation rate is 30 spores per gram of soil. Then the seeds of the transgenic plants harvested after transforming cotton with the At7 overexpression vector pCAMBIA1301-At7 obtained in step 1 were planted at a sowing rate of 1 m ² for 100 g of seeds. Because the amount of pathogenic bacteria is very large, a large number of cotton dies after emergence, and the diseases that occur include seedling disease, wilt and Verticillium wilt. The disease-resistant At7 transgenic plants were obtained through high-intensity screening.

2. Natural identification in the field

The At7 transgenic disease-resistant cotton seedlings that survived the indoor disease soil identification in step 1 were transplanted to the disease nursery, and the disease was investigated every 10 days after mid-July, and finally the disease-resistant At7 transgenic plants were obtained.

sequence listing

<160>2

<210>1

<211>120

<212>PRT

<213> Gossypium barbadense

<400>1

Met Ala Ser Ser Met Ser Leu Lys Leu Ala Cys Val Ala Val Leu Cys

1 5 10 15

Met Val Val Gly Ala Pro Leu Ala Gln Gly Ala Val Thr Cys Gly Gln

20 25 30

Val Thr Ser Ser Leu Ala Pro Cys Ile Gly Tyr Leu Thr Gly Asn Gly

35 40 45

Ala Gly Gly Val Pro Pro Gly Cys Cys Gly Gly Ile Lys Ser Leu Asn

50 55 60

Ser Ala Ala Gln Thr Thr Pro Asp Arg Gln Ala Ala Cys Lys Cys Ile

65 70 75 80

Lys Ser Ala Ala Ala Gly Ile Ser Gly Ile Asn Tyr Gly Ile Ala Ser

85 90 95

Gly Pro Pro Gly Lys Cys Gly Val Asn Ile Pro Tyr Lys Ile Ser Pro

100 105 110

Ser Thr Asp Cys Asn Ser Val Lys

115 120

<210>2

<211>592

<212>DNA

<213> Gossypium barbadense

<400>2

ttgggaagaa gcagcaatag tactactact ccaagcaggc attttcctta caagtttgtt 60

ctccttgtga ttaatcgata tggctagctc aatgtccctt aagcttgcat gtgtggcggt 120

gttgtgcatg gtggtgggtg cacccctggc tcaaggggcc gtaacctgtg gtcaagtcac 180

aagctccctc gcaccctgca ttggttactt gacagggaat ggtgctggtg gcgttccccc 240

aggttgctgc ggcggcataa aatctctcaa ctccgccgcc caaacaacac cagaccggca 300

agcagcttgc aaatgcatca aaagtgcggc cgccggcatt tctggcatca actatggtat 360

tgcaagcgga cccccaggca agtgcggtgt caacatccct tacaagatca gccctagcac 420

tgactgcaac agcgtcaagt gaagttttgg catggaaagt tcaccagcta gtggaagcca 480

aaataacgat agctacagaa taaatatgga tgttaaaatt ccagagttgt gggttgtgta 540

ctatgccgct ttatgcgact acgtaatatt aactttatct acaaattaat aa 592

Claims (10)

1, a kind of sea island cotton lipid transfer protein is the protein with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 1;

2) with SEQ ID № in the sequence table: 1 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein of phosphatide transhipment effect.

2, sea island cotton lipid transfer protein according to claim 1 is characterized in that: described albumen has SEQ ID № in the sequence table: 1 amino acid residue sequence.

3, the gene of the described sea island cotton lipid transfer protein of coding claim 1 is one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 2 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: 1 dna sequence dna;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.

4, the gene of sea island cotton lipid transfer protein according to claim 3 is characterized in that: described gene has SEQ ID № in the sequence table: 2 dna sequence dna.

5, contain the described expression carrier of claim 3, transgenic cell line and host bacterium.

6, a kind of method of cultivating the resisting verticillium plant is with the described sea island cotton lipid transfer protein of claim 3 gene transfered plant tissue or cell, obtains the plant of resisting verticillium.

7, method according to claim 6 is characterized in that: described sea island cotton lipid transfer protein gene imports plant tissue or cell by the plant expression vector that contains described sea island cotton lipid transfer protein gene.

8, method according to claim 7 is characterized in that: the carrier that sets out that is used to make up described plant expression vector is pCAMBIA1301, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300 or pBI121.

9, method according to claim 8 is characterized in that: described plant expression vector is pCAMBIA1301-At7.

10, method according to claim 6 is characterized in that: described plant host is cotton, tobacco, Arabidopis thaliana, paddy rice, wheat, clover, soybean, tomato, eggplant or watermelon.

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