CN1225559C - Rice xanthomonad hrf3 gene, recombinant vector and method for plant transgenic breeding - Google Patents

Abstract

The invention discloses a rice xanthomonad hrf3 gene recombination carrier and a transgenic plant breeding method, which belong to the field of biotechnology. The present invention provides the hrf3 gene and uses the hrf3 gene as the target gene to construct the Ti-plasmid binary vector pBI121 expressed in plants to obtain the gene recombination vector, and transform the constructed recombinant vector pBI121::hrf3 into EHA105; Plants were screened for transformed regenerated plants (T ₀ generation) in the plant regeneration medium, and transformed lines were detected. The hrf3 gene transgenic plants enhanced the plant's broad-spectrum resistance to diseases and insect pests and improved other plant production traits. The picture shows the nucleotide sequence of the hrf3 gene.

Description

Rice xanthomonad hrf3 gene, recombinant vector and method for plant transgenic breeding

(1) Technical field

The invention "rice xanthomonad hrf3 gene, recombinant vector and method for plant transgenic breeding" belongs to the field of biotechnology, and is specially used to improve plant disease resistance and other beneficial production traits through plant genetic engineering technology.

(2) Technical background

The gene hrf exists in two pathogenic varieties (pv.oryzae and pv.oryzicola) of rice Xanthomonas oryzae and other Xanthomonas spp., and belongs to hypersensitive response and pathogenicity , hrp) gene cluster members. Most Gram-negative phytopathogenic bacteria have hrp gene clusters in which genes encoding proteins (Harpin) that induce allergic responses in plants vary. At present, the hrp gene clusters in four Gram-negative genera of plant pathogenic bacteria have been fully sequenced, and the four known hrp gene clusters have been classified according to the structure of the gene cluster, the composition of the operon, and the collinearity of the regulatory genes. The type hrp gene clusters were divided into two groups. Group I includes E. amylovora and Pseudomonas syringae pv. syringae; group II includes Ralstonia solanacearum and Xanthomonas solanacearum (Xanthomonas campestrispv. campestris). Regarding genes encoding harpins, hrpN in E. amylovora, hrpZ in Pseudomonas syringae, pop1 in Larsia solanacearum, X. oryzae pv. oryzae It is hrf3. Sequence comparison of genes encoding Harpins from different phytopathogenic bacteria found that the representative strain that obtained hrf3 was PXO86 of the pathogenic variety of Xanthomonas oryzae Bacteria blight, which is a known public strain (Chen Gongyou et al. Study on chemical mutagenesis of hrp gene mutants and related traits of pathogen (Xanthomonas oryzae pv. oryzae), Acta Phytopathology. 31(3): 199～207).

As a member of the hrp gene cluster, hrf1 has a regulatory effect on pathogenicity, but the protein product expressed in vitro has beneficial effects such as inducing disease resistance, insect resistance and promoting plant growth. The protein Harpin _Xo encoded by the Hrf1 gene has the characteristics of the Harpin protein family: the protein is hydrophilic, stable to heat, and the isoelectric point is about 4.3. The difference from the reported Harpins protein family members Harpin _Ea and Harpin _Pss is that Haprin _Xo contains a cysteine, and there are GGG-GG repeating units in the amino acid residues, and 3-5 μg/ml can induce plant programmed cell death (PCD ). The previous invention of our research group "a gene expression product encoding a plant growth regulator and its application" (patent application number 00135403.5; publication number CV 1300547A) has been used to treat the pathogenic variety of xanthomonad xanthomonas (X. oryzae pv.oryzae) and the direct application of the hrf1 gene and its expression product Harpin _Xoo in vitro, that is, soaking seeds, dressing seeds, dipping roots and spraying leaves in the form of preparations, and using the hrf1 gene to construct recombinant microorganisms have been patented.

Harpin _Xoo , the expression product of the hrf1 gene, can activate plant defense response-related enzymes, such as phenylalanine ammonia lyase (PAL), peroxidase (PO) and polyphenol oxidase ( PPO) activity, while inducing the expression of disease process-related protein genes such as PR-1b and PR-1a genes. Among them, PR-1b gene is regulated by jasmonic acid molecule, and PR-1a is regulated by salicylic acid molecule (Eyal et al., 1993, Ohshima et al., 1990). Among them, jasmonic acid and salicylic acid act as signal molecules in inducing plant resistance to insects and diseases. In addition, when used on tomato, it can also initiate pto-mediated signaling reactions, in which pti4/5/6 and effector genes PR-1a1, PR-1b1, NP24, and Pin2 have different degrees of enhanced expression at different times.

As the same as hrpN, but its protein product is obviously different, the gene hrp of rice xanthomonad encoding Harpin _Xo may have application value as the target gene in plant genetic engineering. Plant genetic engineering uses exogenous genes with definite functions as the target genes. After transforming plants through a certain technical route, they are randomly integrated into the plant genome and expressed in plants to improve plant resistance to diseases, insects and other diseases. Beneficial traits. For example, Bacillus thuringiensis (Bacillus thuringiensis, Bt) insecticidal protein (ICP) can be used as a biological agent to spray plants for pest control. After genetically transformed cotton, transgenic cotton also has resistance to cotton bollworm (A 01N63/02CN 95113498.1A); antimicrobial peptide ( Cecropin) gene transformed tobacco, potato and rice, the transgenic plants showed resistance to bacterial diseases on these plants (Jia Shirong, 1993). The quality of crops such as storability, salt tolerance, amino acid quality and plant lipids can also be improved through transgenic technology. As a member of the Harpin gene family, the gene hrpN encoding Harpin _Ea from Erwinia amylovora has been successfully applied in the research and development of biopesticide Messenger in the United States. (Phytophthora infestans) and pear fire blight (Erwinia amylovora) were improved to varying degrees. But there is no report about the hrf3 gene and its application in plant transgenic breeding engineering technology. At present, there is no report about the hrf gene transgenic breeding method using the hrf gene as the target gene.

(3) Contents of the invention

Technical problem The purpose of the present invention is to provide rice xanthomonad hrf3 gene, recombinant vector and transgenic plant breeding method for the current situation that hrf3 gene and its application in plant transgenic breeding engineering technology have not been reported at present, and hrf3 gene is constructed in The transformation plasmid expressed by the plant is used to transform rice and tobacco to obtain transgenic plants. Its genetic recombination vectors are used as varieties and strains for commercial use or as genetic resources for direct use in production or for agricultural biological breeding and transgenic plants to improve crop disease resistance, insect resistance traits and other production traits of crops.

Technical solutions

The rice xanthomonad hrf3 gene sequence provided by the present invention is as follows:

As shown in the sequence table SEQ ID NO.1;

The above-mentioned rice xanthomonad hrf3 gene recombination vector is constructed by the following method:

Rice xanthomonad hrf3 gene sequence: as shown in the sequence table SEQ ID NO.1

The hrf3 gene was used as the target gene to construct the Ti-plasmid binary vector pBI121 for plant expression: the plasmid used to construct the binary transformation-expression vector was transformed from T-DNA, except for the T-DNA 25bp repeat sequence ( LB and RB), in addition to the promoter (P-nos) and terminator (T-nos) of the nopaline synthase gene, nptI used as a selection marker in prokaryotes (bacteria) and nptI used in eukaryotes (plants) ) used as a selectable marker, the antibiotics used for selection are Km and G-418; in addition, in pBI121, there are also the promoter 35S (p35S) and β-glucose of the cauliflower mosaic virus expressed in eukaryotes The aldolase (Gus) gene uidA; the hrf3 gene used as the target gene is inserted into the multi-enzyme cloning site between p35S and uid of the pBI121 plasmid, and the endonucleases used are XbaI and BamHI, which is the recombinant plasmid.

The transgenic plant breeding method of the rice xanthomonad hrf3 gene recombination carrier provided by the present invention is:

1) The constructed binary transformation recombinant vector plasmid pBI121::hrf3 can be directly transformed into the helper plasmid containing the vir region without Km

The resistant pTiBo542-derived plasmid was competent for Agrobacterium tumefaciens EHA105; E. coli DH5α containing pBI121::hrf3 could also be transferred to EHA105 by triparental mating in the presence of the helper plasmid pRK2013. EHA105 is a Km-sensitive derivative of EHA101 (C58/pTiBo542).

2) Agrobacterium tumefaciens EHA105 containing the helper plasmid pTiBo542 (Km ^s ) and transformation plasmid (pBI121::hrf3) at the same time is used for the transformation of plant cells, and the method of transforming plant cells with recombinant Agrobacterium tumefaciens uses the leaf disc method and embryo transformation method or pollen tube introduction method.

3) The explants are selected in the plant regeneration medium containing Km for the transformed regeneration plant (T ₀ generation): when the T ₁ generation seeds are accelerated to germination, use

Soak in Km (150 μg/ml) for 12 hours to screen the transformed plants, and detect the transformation frequency and the expression of hrf3 gene in the T1 _generation plants according to the line.

4) Detection method of transformed strains: detection of hrf3 gene and P-35S promoter in leaves of transformed plants by PCR and Southern blot hybridization method, the expression of hrf3 genes was detected by quantitative RT-PCR method for the accumulation of RNA of hrf3 genes in leaves of plants .

Agrobacterium tumefaciens EHA105 is a known public strain, and the plant regeneration method of gene transformation and transformed cells is a conventional method for transgenic plants (Sambrook J, Russell DW, 2001, Molelcular cloning, A Laboratory Manual (3 ^rd ed), Spring Harbor Laboratory Press; Wen Weigang, Wang Jinsheng, 2001, Cloning and expression of the harpin gene of Xanthomonas oryzae, Acta Phytopathology, 31(4): 295-300).

Beneficial effects The present invention provides a rice xanthomonad hrf3 gene recombination vector and a transgenic plant breeding method, which breaks through the current situation that the hrf3 gene has not been applied in plant transgenic breeding engineering technology, and constructs the hrf gene in a plant-expressed plant for the first time Transform the plasmid, transform rice and tobacco with the method of binary transformation—expression vector pBI121::hrf3 to transform plants and express hrf3 gene in transgenic plants Agrobacterium tumefaciens-mediated method, and obtain transgenic plants. The disease resistance of the transgenic plants identified in the greenhouse and the field has significantly enhanced disease resistance to fungal diseases, bacterial diseases and viral diseases on the plants. The test showed that the T1 generation of hrf3 gene transgenic rice had 20% immunity to rice blast and 20% high resistance to rice blast, and about 15% of rice bacterial blight resistance. Compared with the control, the disease prevention effect 80-100% and 70-80% respectively. Rice transgenic plants with Harpin gene also exhibit excellent production traits such as low light tolerance, low temperature tolerance, dwarf plant type, and multiple tillers. More than 90% of the T1 generation plants of hrf3 gene transgenic tobacco showed resistance to tobacco mosaic virus (TMV), tobacco bacterial wilt bacteria (Ralstonia solanacearum) and tobacco red spot bacteria (Alternaria alternata), reaching high and medium resistance levels, The effects are 84%, 70% and 54%, respectively. The expression of defense-related genes such as GST1, PR-1a and PR-1b can be detected in the transgenic plant lines, but not expressed in the parental lines. hrf3 transgenic cotton, wheat, maize and tomato have been obtained, and the expression of hrf3 gene and related defense genes has been detected.

(4) Description of drawings

Figure 1 Nucleotide sequence of hrf3 gene

Figure 2 The amino acid sequence characteristics of the protein encoded by the hrf3 gene

Figure 3 Construction diagram of 35S::hrf3 gene recombination vector

H: Hind III; S: Sph I; P: Pst I; X: Xba I; B: BamH I; Sm: Sma I; Sa: Sac I; E: EcoR I

(5) Specific implementation methods

1. The binary transformation of expressing hrf3 gene in plants—the construction of expression vector pBIH (pBI121::hrf3)

The plasmid used to construct the binary transformation-expression vector is transformed from T-DNA, except for the T-DNA 25bp repeat sequence (LB and RB), the promoter (P-nos) and termination of the nopaline synthase gene In addition to T-nos, there are nptI used as a selection marker in prokaryotes (bacteria) and nptII used as a selection marker in eukaryotes (plants). The antibiotics used for selection are both Km and G- 418. Also in pBI121 are the promoter 35S (p35S) of the cauliflower mosaic virus expressed in eukaryotes and the uidA of the β-glucuronidase (Gus) gene. The hrf3 gene used as the target gene is inserted into the multi-enzyme cloning site between p35S and uid of the pBI121 plasmid ( FIG. 3 ), and the endonucleases used in the present invention are XbaI and BamHI. The binary vector pBI121 is a well-known public vector (Jefferson, R.A., Kavanash, T.A. & Bevan, 1987, M.W. EMBO J., 6, 3901-3907).

Binary transformation—expression vector pBI121::hrf3 transformed plants and expression of hrf3 gene in transgenic plants:

The constructed binary transformation vector plasmid pBI121::hrf3 can be directly transformed into the competent Agrobacterium tumefaciens EHA105 containing the pTiBo542 derived plasmid of the vir region helper plasmid without Km resistance. EHA105 is a Km-sensitive derivative of EHA101 (C58/pTiBo542). E. coli DH5α containing pBI121::hrf3 can also be transferred into EHA105 by triparental mating in the presence of helper plasmid pRK2013. Agrobacterium tumefaciens EHA105 containing both helper plasmid pTiBo542 (Km ^s ) and transformation plasmid (pBI121::hrf1) was used for transformation of plant cells. The method for transforming plant cells by recombining Agrobacterium tumefaciens uses leaf disk method, embryo transformation method or pollen tube introduction method. The explants were selected for transformed regenerated plants (T ₀ generation) in plant regeneration medium containing Km. The T ₁ generation seeds were soaked in Km (150 μg/ml) for 12 hours to select transformed plants when they germinated.

The transformation frequency and the expression of hrf3 gene in the _T1 generation plants were detected by line. The detection method of the transformed strain is to detect the hrf3 gene and the P-35S promoter in the leaf of the transformed plant by PCR and Southern blot hybridization method, and the RNA accumulation of the hrf3 gene in the leaf of the plant is detected by the quantitative RT-PCR method for the expression of the hrf3 gene.

Agrobacterium tumefaciens EHA105 is a known public bacterial strain, and the plant regeneration method of gene transformation and transformed cells is a conventional method for transgenic plants (Clark MS, 1997, Plant Molecular Biology, A Laboratory Manual, Springer, Berlin Germany).

2. Phenotypic characteristics of hrf3 gene transgenic rice

The T1 generation seeds of the hrf3 gene transgenic rice obtained in 1 were respectively sown in the greenhouse and the isolated field in the disease endemic area, and the resistance of the hrf3 gene transgenic rice lines to rice blast (Magnaporthegrisea) and rice white was determined by artificial inoculation and induced infection. A resistant form of leaf blight (Xanthomonas oryzae pv. oryzae). The artificial inoculation method uses conventional spraying (Pyricularia grisea) and leaf cutting inoculation (bacteria bacterial blight). The induced row infection method is to artificially spray and inoculate the susceptible variety Xianyou 63 with Magnaporthe grisea, and transplant it to the periphery and between the borders of the disease identification nursery after the onset of the disease. The width of the border is 1.8 meters, and the spacing between plants and rows is 15 centimeters. The spores on the diseased spots in the induced row rice can spread evenly and randomly with the wind and rain. 100% of the susceptible strains and starting varieties were infected. According to the identification results of about 3,000 individual plants of 34 transgenic rice lines, 20% of the individual plants are immune to rice blast and highly resistant to rice blast, and about 15% of the individual plants are moderately resistant to rice bacterial blight. In contrast, the disease prevention effects reached 80-100% and 70-80% respectively. The parental rice varieties used for transgenesis include high-quality japonica rice R109, indica rice 3103 and hybrid rice restorer line Minghui 63. In addition to the obvious improvement of the disease resistance phenotype, the transgenic lines also showed obvious characteristics such as resistance to low light, low temperature and dwarfing plant type. The transgenic rice line was named the transgenic Hrp gene-resistant rice (Harice).

The bacterial blight of rice and the rice blast fungus for disease resistance identification are known and public. The transgenic parent rice varieties R109, 3103 and Minghui 63 are varieties used in production and are known and used for public use.

3. Phenotypic characteristics of hrf3 gene transgenic tobacco

Sow the T1 generation seeds of the hrf3 gene transgenic tobacco obtained in 1 in a pot in the greenhouse, transplant a single plant at the 2-leaf stage into a pot with a diameter of 20 cm, and artificially inoculate tobacco mosaic virus when there are 4 to 5 true leaves (TMV), Ralstonia solanacearum and Alternaria alternata. TMV inoculation was inoculated by foliar rubbing, bacterial wilt was inoculated by stem injection of bacterial suspension, and Alternaria spp. was inoculated by spore suspension droplet method. More than 90% of the plants of T1 generation seeds of transgenic tobacco lines screened by Km (150 μg/ml) showed resistance to the above three diseases, reaching high and medium resistance levels, and the effects were 84%, 70% and 54% respectively. But other traits are the same as the parent tobacco variety Xanthi (nc). The tobacco line transgenic for Harpin was named Hartob transgenic Hrp gene.

Tobacco mosaic virus, bacterial wilt bacteria and Alternaria rubella for identification of tobacco disease resistance often occur in the field and are known and used.

4. Characteristics of plant defense response initiated by transgenic plant lines expressing hrf3 gene

From about 70% of the single plants of the T1 plants of rice and 95% of the single plants of the T1 plants of tobacco, the full-length insertion sequence of the hrf3 gene and the exogenous p35S promoter can be detected by PCR and Southern blot hybridization methods. The T1 generation plants of transgenic rice and tobacco transgenic lines with hrf3 gene did not show the phenomenon of programmed cell death (PCD). Quantitative RT-PCR was used to measure the expression of hrf3 gene and genes related to disease resistance and defense. PCR, Southern blot hybridization and RT-PCR were routine research methods in molecular biology. The expression of defense-related genes such as GST1, PR-1a and PR-1b can be detected in the transgenic plant lines, but not expressed in the parental lines.

Using the same method, hrf3 transgenic cotton, wheat, corn and tomato have been obtained, and the expression of hrf3 gene and related defense genes has been detected.

Binary vector pBI121 and Agrobacterium tumefaciens EHA105, rice variety R109, Minghui 63, tobacco variety Xanthi (nc); public knowledge.

The source strain PXO86 of the hrf3 gene used as the target gene is known and public (Weiguang Zhu, et al. 2000, White identification of Two Novel hrp-Associated Genes in the hrp Gene Cluster of Xanthomonasoryzae pv.oryzae, Journal of Bacteriology, 182 (7) : 1844-1853).

Claims (3)

1, the yellow unit cell hrf3 of paddy rice gene: shown in sequence table SEQ ID NO.1.

2, the recombinant vectors of the yellow unit cell hrf3 of paddy rice according to claim 1 gene is to make up by the following method to form:

1) hrf3 gene order: shown in sequence table SEQ ID NO.1;

2) be structured on the Ti-plasmid binary vector pBI121 of expression of plants as goal gene with the hrf3 gene: be used to make up double base conversion-expression vector plasmid be to transform by T-DNA, wherein remove T-DNA 25bp tumor-necrosis factor glycoproteins LB and RB, outside the promotor P-non of rouge alkali synthetase gene and the terminator T-nos, also have nptI that uses as selective marker in prokaryotic organism, bacterium and the nptII that uses as selective marker in eukaryote, plant, the microbiotic that is used to select is Km and G-418; The promotor 35S and the β-polyacetals phytase gene uidA of Portugal that in pBI121, also have the cauliflower mosaic virus of expressing in addition eukaryote; The hrf3 gene that uses as goal gene is inserted in the p35S and the multienzyme between the uid of pBi121 plasmid and cuts on the cloning site, and used restriction endonuclease is XbaI and BamHI, is recombinant plasmid.

3, the transgenic plant breeding method of the yellow unit cell hrf3 of paddy rice according to claim 2 gene recombined vector is: the double base of structure transforms transfer vector plasmid pBI121 ∷ hrf3, can directly transform in containing vir district helper plasmid not derive with the pTiBo542 that blocks that resistance among the competence Agrobacterium tumefaciens EHA105 of plasmid; Also can helping plasmid pRK2013 to exist under the situation, transfer among the EHA105 with the bacillus coli DH 5 alpha that contains pBI121 ∷ hrf3 by triparental mating;

1) transformed plant cells: contain helper plasmid pTiBo 542 Km simultaneously ^sBe used for the conversion of vegetable cell with the Agrobacterium tumefaciens EHA105 that transforms plasmid pBI121 ∷ hrf3, the method for reorganization Agrobacterium tumefaciens transformed plant cells is with leaf dish method, embryo conversion method and pollen tube introduction method;

2) explant screens the regeneration plant T that is transformed in containing the plant regeneration substratum ₀Generation: T ₁Screened transformed plant in 12 hours with 150 μ g/ml Km immersion during for presprouting of seeds, to T ₁Detect hrf3 expression of gene in transformation frequency and the plant for plant by strain system;

3) detection of transformation plant: detect hrf3 gene and P-35S promotor in the transformed plant blade with PCR and Southern blot hybridization method, the hrf3 expression of gene detects the accumulation of the RNA of hrf3 gene in the plant leaf with quantitative RT-PCR method.