Rice blast resistance gene Pi-bd1 and application thereof

Abstract

The invention relates to a rice blast resistance gene Pi-bd1 and application of the rice blast resistance gene Pi-bd1 in gene engineering. The rice gene Pi-bd1 is composed of a Pi-bd1-1, a Pi-bd1-2 and a promoter, a cDNA (complementary deoxyribonucleic acid) sequence of the Pi-bd1-1 and a promoter sequence of the Pi-bd1-2 are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, and an amino acid sequence of the Pi-bd1-1 and an amino acid sequence of the Pi-bd1-2 are shown as SEQ ID NO.4 and SEQ ID NO.5. The rice gene Pi-bd1 and the rice gene Pi-bd1-2 have the advantages that the cDNA sequence of the Pi-bd1-1 and the promoter sequence of the Pi-bd1-2 are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3; the invention discloses a rice gene Pi-bd1 which is reported for the first time in rice, the gene is obtained through map-based cloning, and the resistance of a susceptible variety Suyunuo to seedling blast and panicle blast can be remarkably improved through co-expression of Pi-bd1-1 and Pi-bd1-2. Therefore, the gene can be used as a target gene to be introduced into a susceptible material to improve the plant disease resistance so as to improve the plant variety.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and relates to a rice blast resistance gene Pi-bd1 and its application in genetic engineering. Background Art

[0002] Rice (Oryza sativa L.) is one of the most important food crops in the world, providing staple food for more than half of the world's population. The global rice yield loss due to rice blast is about 10%-30% each year, posing a serious threat to rice yield and quality. Currently, using resistance genes to cultivate and plant disease-resistant varieties is the most economical, effective and environmentally friendly strategy to control rice blast.

[0003] At present, more than 35 R genes have been cloned. Except for pi21, Pi-d2, Ptr and Pi65, other R genes encode NBS-LRR (NLR) disease resistance proteins with nucleotide binding site (NBS) and leucine rich repeat (LRR) sequence domains. Some NLR disease resistance genes exist in pairs on chromosomes (PairNLR), and their resistance requires the joint participation of two genes, such as Pi5-1 / Pi5-2, Pia-1 / Pia-2, Pik1 / Pik2, Pikp1 / Pikp2, Pikm1 / Pikm2, Piks1 / Piks2, Pikh1 / Pikh2, and Pi1-5 / Pi1-6. Plant NLR genes can activate downstream immune responses and confer disease resistance to plants by recognizing non-toxic effector proteins secreted by pathogens. In nature, the Avr genes of pathogens mutate to escape the recognition of plant NLR genes. Similarly, plant NLR genes also have different allelic variations to cope with the mutations of Avr genes.

[0004] Due to the high variability of Avr genes in rice blast fungus, most R genes cannot provide durable resistance. Therefore, continuous identification of new R genes or alleles is of great significance for the prevention and control of rice blast. Summary of the invention

[0005] The purpose of the present invention is to disclose the rice gene Pi-bd1.

[0006] Another object of the present invention is to provide a genetic engineering application of the rice blast resistance gene Pi-bd1.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] The rice blast resistance gene Pi-bd1 is characterized in that the gene consists of two genes, Pi-bd1-1 and Pi-bd1-2, the cDNA sequence of the Pi-bd1-1 gene is shown in SEQ ID NO.1, and the cDNA sequence of the Pi-bd1-2 gene is shown in SEQ ID NO.2.

[0009] As a preferred embodiment of the present invention, the promoter sequence of the rice blast resistance gene Pi-bd1 is shown as SEQ ID NO.3.

[0010] The protein encoded by the rice blast resistance gene Pi-bd1 of the invention.

[0011] As a preferred embodiment of the present invention, the amino acid sequence of the protein encoded by the Pi-bd1-1 gene is shown as SEQ ID NO.4, and the amino acid sequence of the protein encoded by the Pi-bd1-2 gene is shown as SEQ ID NO.5.

[0012] The invention discloses an application of the rice blast resistance gene Pi-bd1 in improving the resistance of rice blast susceptible varieties to rice blast.

[0013] As a preferred embodiment of the present invention, the rice gene Pi-bd1 is introduced into susceptible rice varieties by engineering means to improve the resistance of susceptible rice varieties to rice blast.

[0014] As a preferred embodiment of the present invention, the rice gene Pi-bd1 is introduced into susceptible rice varieties by hybridization or backcrossing to improve the resistance of susceptible rice varieties to rice blast.

[0015] As a preferred embodiment of the present invention, a rice material containing the Pi-bd1 gene is used as the male parent, and a rice variety susceptible to rice blast is used as the female parent, and the F 1 Seeds, through systematic breeding to obtain rice materials containing the Pi-bd1 gene, or continue backcrossing and selfing, so as to obtain a stable pedigree. Each hybridization, backcrossing and selfing progeny uses molecular markers P7 and P8 or nucleic acid sequencing to detect the target gene, and then selects a single plant containing the target gene as the male parent for backcrossing or selfing.

[0016] A vector for co-expressing the rice genes Pi-bd1-1 and Pi-bd1-2.

[0017] As a preferred embodiment of the present invention, the vector is obtained by inserting the rice genes Pi-bd1-1 and Pi-bd1-2 into an expression vector.

[0018] The co-expression vector of the present invention is obtained by cloning the rice gene Pi-bd1 and inserting it into the pCAMBIA1300s vector linearized by restriction endonucleases HindIII and Kpn1 through homologous recombination.

[0019] As a further preferred embodiment of the present invention, the cDNA of the japonica rice variety Bodao is used as a template to amplify the cDNA sequences of rice genes Pi-bd1-1 and Pi-bd1-2 respectively, and the DNA of Bodao is used as a template to amplify the DNA sequence of the promoter, which is connected to the pCAMBIA1300s vector to construct a Pi-bd1 expression vector, and sequencing is performed to ensure that the cDNA sequences of Pi-bd1-1 and Pi-bd1-2 and the promoter sequence inserted into the vector are correct; the obtained Pi-bd1 expression vector verified to be correct by sequencing is transferred into Agrobacterium, and finally into the rice variety Su Yunuo susceptible to rice blast fungus; wherein the primer sequences for amplifying the Pi-bd1-1 sequence are P1 (SEQ ID NO.6) and P2 (SEQ ID NO.7), the primer sequences for amplifying the promoter sequence are P3 (SEQ ID NO.8) and P4 (SEQ ID NO.9), and the primer sequences for amplifying the Pi-bd1-2 sequence are P5 (SEQ ID NO.10) and P6 (SEQ ID NO.11).

[0020] The co-expression vector of the present invention is used for improving the resistance of rice blast susceptible varieties to rice blast.

[0021] Beneficial effects:

[0022] 1. The present invention discloses a rice gene Pi-bd1. The rice gene Pi-bd1 is reported for the first time in rice and is a rice blast resistance gene obtained through map-based cloning analysis. The gene can be introduced into susceptible varieties by hybridization, backcrossing and genetic engineering, thereby improving the disease resistance of rice.

[0023] 2. The present invention introduces Pi-bd1 into rice varieties susceptible to rice blast through hybridization or backcross breeding to cultivate rice blast-resistant varieties.

[0024] 3. The present invention constructs the Pi-bd1 expression vector, transforms it into rice, obtains transgenic plants, and cultivates varieties with rice blast resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1The figures are the ear blast and seedling blast phenotypes of Pi-bd1-1 and Pi-bd1-2 co-expressing complementary transgenic materials (HB-Pi-bd1). A: Photos of rice ears and statistical data of diseased grain rate (%) of Pi-bd1-1 and Pi-bd1-2 co-expressing complementary transgenic materials (HB-Pi-bd1) after inoculation with rice blast fungus Hoku1. The scale bar represents 2 cm, and the error bars represent mean ± standard error. **P<0.01. B: Leaves of Pi-bd1-1 and Pi-bd1-2 co-expressing complementary transgenic materials after inoculation with rice blast fungus Hoku17 days at the seedling stage. The scale bar represents 2 cm.

[0026] Figure 2 Figure 1 shows the ear blast and seedling blast phenotypes of the near-isogenic lines containing Pi-bd1. A, B: the resistant parent Bodao, the susceptible parent Suyunuo and the near-isogenic line of Pi-bd1 (Suyunuo Pi-bd1 ) Photos of rice ears after inoculation with Hoku1 (A) and statistical data on diseased grain rate (%) (B). The rice ears were photographed 15 days after inoculation. The middle line of each rectangular box represents the median, and the upper and lower edge lines of the rectangular box represent the upper and lower quartiles, respectively. Different letters represent significant differences (P<0.05). C: Leaves of the resistant parent Bodao, the susceptible parent Suyunuo, and the Pi-bd1 near-isogenic line after inoculation with Hoku1. The leaves were photographed 5 days after inoculation. DETAILED DESCRIPTION

[0027] Example 1 Localization of Pi-bd1

[0028] The resistance and genetic background of 124 RIL populations constructed by hybridization between Bodao and Suyunuo were identified, and Pi-bd1 was initially located at the end of chromosome 11. The RIL families containing Pi-bd1 were further backcrossed with Suyunuo and self-pollinated, resulting in 17,280 BC 5 F 6 Individuals were screened and the genotypes and resistance-susceptibility phenotypes of exchanged plants of different exchange types were identified. Finally, Pi-bd1 was located in the 184kb interval, which overlapped with the Pik locus. The five NLR genes in the candidate interval were selected as candidate genes and named P1-P5 in sequence. Protein sequence comparison revealed that P4 and P5 were new alleles of the Pik locus and were named Pi-bd1-1 and Pi-bd1-2 in sequence.

[0029] Example 2 Transgenic plant construction

[0030] 1) Cloning of the rice gene Pi-bd1

[0031] The rice blast-resistant japonica rice variety Bodao was selected, and samples were taken when the rice seedlings grew to the 3-4 leaf stage, and DNA was extracted using the CTAB method, and the quality of DNA was identified by electrophoresis. The japonica rice variety Bodao was selected, and when the rice grew to the 3-4 leaf stage, the leaves were immediately frozen with liquid nitrogen, ground with a mortar, and transferred into a 1.5 mL EP tube containing Trizol lysis solution (TRIzol Reagents, purchased from TIANGEN, CHINA), and after sufficient shaking, total RNA was extracted, and the quality of total RNA was identified by electrophoresis.

[0032] According to the genome sequence of rice, primers P1 and P2 with homology arms were designed. The first chain of cDNA synthesized by reverse transcription of the total RNA obtained in step 1) was used as a template, and PCR amplification was performed with a high-fidelity enzyme (purchased from Nanjing Novozymes Co., Ltd.) to obtain a cDNA sequence containing Pi-bd1-1.

[0033] P1:5'-ACGGCCAGTGCCAAGCTTCTAGCTAGTAGTTTCTGTTT-3'

[0034] P2:3'-ATGGAGGCGGCTGCCATGGCCG-5'

[0035] Design primers P3 and P4 with homology arms, use the DNA obtained in step 1) as a template, perform PCR amplification with a high-fidelity enzyme, and obtain a sequence containing the promoter.

[0036] P3:5'-ATGGCAGCCGCCTCCATCCCTACTCGCGCCTCGATCG-3'

[0037] P4:3'-CCTACCACCAACTCCATCTTGTTCTCTGTACTTCTCA-5'

[0038] Primers P5 and P6 with homology arms were designed, and the first strand of cDNA synthesized by reverse transcription of the total RNA obtained in step 1) was used as a template. PCR amplification was performed using a high-fidelity enzyme to obtain a cDNA sequence containing Pi-bd1-2.

[0039] P5:5'-ATGGAGTTGGTGGTAGGTGC-3'

[0040] P6:3'-TCTAGAGGATCCCCGGGTACCTCATGCAGTGACGAT-5'

[0041] The cDNA sequence of Pi-bd1-1, the cDNA sequence of Pi-bd1-2, the promoter sequence and the pCAMBIA1300S vector linearized by HindIII and Kpn1 obtained by the above amplification were homologously recombined using multi-fragment recombinase (purchased from Nanjing Novozymes Co., Ltd.), and sequencing was performed to ensure that the inserted sequence in the vector was correct. It was then transferred into Agrobacterium and further transferred into the rice variety Su Yunuo susceptible to rice blast fungus.

[0042] Example 3 Resistance identification of transgenic plants

[0043] When the transgenic plants constructed in Example 2 grow to the booting stage, the leaves of the ear covers to be inoculated are marked with a branch tying machine, and the spore solution of the rice blast pathogen is injected into the marked ear covers with a continuous syringe, so that the spore solution fills the entire ear covers. The diseased grain rate of the inoculated ears was counted 20 days after inoculation, and the results of the ear blast resistance and susceptible phenotype identification showed that the diseased grain rates of the four families co-expressing Pi-bd1-1 and Pi-bd1-2 transgenic materials were 12.88% ± 11.87% and 11.49 ± 5.63, respectively, which were significantly lower than those of the wild-type Su Yunuo (55.57% ± 19.15%), showing a rice blast resistance phenotype ( Figure 1 ).

[0044] The seedling blast resistance identification was carried out in an artificial climate chamber or incubator, and spray inoculation was performed when the seedlings grew to two leaves and one heart. The spore suspension was evenly sprayed on the leaves of the seedlings with a spray bottle, and then carefully placed in the dark for 24 hours at 28°C and 99% relative humidity. The light cycle was then adjusted to 14 hours during the day / 10 hours in the dark. The phenotype was investigated after 7 days. The seedling blast resistance phenotype results showed that the transgenic material family co-expressing Pi-bd1-1 and Pi-bd1-2 did not develop rice blast lesions after inoculation with rice blast fungi, showing a rice blast resistance phenotype, while the wild-type Su Yunuo showed obvious rice blast lesions, showing a rice blast susceptible phenotype ( Figure 1 The above results indicate that the rice blast resistance gene Pi-bd1 can significantly increase the resistance of susceptible variety Su Yunuo to seedling blast and panicle blast.

[0045] Example 4: Introduction of Pi-bd1 by backcrossing

[0046] Example 4

[0047] The blast-resistant variety Bodao was used as the male parent and the blast-susceptible variety Suyunuo was used as the female parent. 1 Seeds. Backcross 3 generations to get BC 3 F 1 Seeds, BC 3The F1 seeds were self-pollinated 3-5 times continuously to obtain a stable family. The molecular markers P7 (3'-GCTGCCAAGATCTTCCGTGC-5') and P8 (3'-CATTCTTGTTTCTCTCCCAAC-5') were used to identify the target gene and rice blast resistance in each hybridization, backcrossing and selfing progeny, and then the single plant containing the target gene was selected as the male parent for backcrossing. The near-isogenic line containing Pi-bd1 (Suyunuo Pi-bd1 ) were used to identify seedling blast and ear blast resistance. The results showed that the near-isogenic lines containing Pi-bd1 significantly increased their resistance to seedling blast and ear blast ( Figure 2 ).

[0048] In summary, the Pi-bd1 gene provided by the inventors is a new allele isolated from rice, and its function is related to rice resistance to rice blast. The gene can be used to improve plant varieties, thereby improving the disease resistance of plants. Usually, the Pi-bd1 gene of the present invention is first used as a target gene, and the gene is introduced into a rice variety susceptible to rice blast by genetic engineering means or by hybridization or backcrossing, thereby cultivating a rice blast-resistant variety.

Claims

1. A rice blast resistance gene Pi-bd1, characterized in that: The gene consists of two genes, Pi-bd1-1 and Pi-bd1-2. The cDNA sequence of the Pi-bd1-1 gene is shown in SEQ ID NO.1, and the cDNA sequence of the Pi-bd1-2 gene is shown in SEQ ID NO.

2.

2. The rice blast resistance gene Pi-bd1 according to claim 1, characterized in that: The promoter sequence of the rice blast resistance gene Pi-bd1 is shown in SEQ ID NO.

3.

3. The protein encoded by the rice blast resistance gene Pi-bd1 according to claim 1.

4. The protein according to claim 3, characterized in that The amino acid sequence of the protein encoded by the Pi-bd1-1 gene is shown in SEQ ID NO.4, and the amino acid sequence of the protein encoded by the Pi-bd1-2 gene is shown in SEQ ID NO.

5.

5. Use of the rice blast resistance gene Pi-bd1 according to claim 1 or 2 in improving the resistance of rice blast susceptible varieties to rice blast.

6. The use according to claim 5, characterized in that: The rice gene Pi-bd1 is introduced into susceptible rice varieties by means of genetic engineering to improve the resistance of susceptible rice varieties to rice blast.

7. The use according to claim 5, characterized in that: The rice gene Pi-bd1 is introduced into susceptible rice varieties by backcrossing to improve the resistance of susceptible rice varieties to rice blast.

8. A vector for co-expressing the rice genes Pi-bd1-1 and Pi-bd1-2 as claimed in claim 1.

9. The co-expression vector according to claim 7, characterized in that: The vector is obtained by inserting the rice genes Pi-bd1-1 and Pi-bd1-2 into an expression vector.

10. Use of the co-expression vector according to claim 7 in improving the resistance of rice blast susceptible varieties to rice blast.

Images