Rice nb-arc protein osrpm-l1 and its coding gene in disease-resistant breeding

Abstract

The application discloses rice NB-ARC protein OsRPM-L1 and application thereof in disease-resistant breeding, and belongs to the technical field of genetic engineering. The application screens rice NB-ARC protein OsRPM-L1 and its coding gene OsRPM-L1, and the amino acid sequence of the rice NB-ARC protein OsRPM-L1 is shown in SEQ ID NO. 2. The application constructs a knockout plant and an overexpression plant of the OsRPM-L1 gene by taking Kittake rice as a background. The inoculation experiment proves that the disease susceptibility of the knockout plant of the OsRPM-L1 gene has no significant change, and the overexpression plant of the OsRPM-L1 gene is more resistant to diseases than the background plant; and it is indicated that the rice protein OsRPM-L1 positively regulates the disease resistance of plants, the coding gene OsRPM-L1 of the rice protein OsRPM-L1 can be used as a target gene for molecular breeding for improving the disease resistance of plants, has a wide application prospect, and has important significance for cultivating rice varieties with improved disease resistance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering, in particular to the application of rice NB-ARC protein OsRPM-L1 and its coding gene in disease resistance breeding. BACKGROUND

[0002] Fungal diseases such as rice blast cause large-scale yield reduction of crops, so cultivating disease-resistant crops is one of the main goals of planting industry. A number of genes related to plant disease resistance have been reported, including effector molecule genes and regulatory genes, which are derived from crops such as rice, wheat, corn, soybean and model plants such as Arabidopsis. Some of them have been used as target genes for crop disease resistance genetic engineering, and disease-resistant rice, wheat, corn, soybean, etc. have been successfully cultivated. However, due to the difference in genetic background, the application of some genes is hindered, so new related genes are still needed to meet the needs of breeding.

[0003] Rice is one of the most important food crops, and improving its disease resistance has important theoretical and practical significance. SUMMARY

[0004] The purpose of the present application is to provide the application of rice NB-ARC protein OsRPM-L1 and its coding gene in disease resistance breeding, in order to solve the problems existing in the prior art. The experimental results show that the rice blast disease characteristics of OsRPM-L1 gene knockout plants have no significant change, and the OsRPM-L1 gene overexpression plants are more resistant to rice blast, indicating that the OsRPM-L1 protein positively regulates the resistance of rice to rice blast, and can be used as a target gene for molecular breeding to improve plant disease resistance.

[0005] To achieve the above purpose, the present application provides the following scheme:

[0006] The present application provides an application of rice protein OsRPM-L1 in any of the following:

[0007] (1) in regulating the resistance of rice to rice blast;

[0008] (2) in cultivating transgenic rice with improved resistance to rice blast;

[0009] (3) in preparing a product for improving the resistance of rice to rice blast;

[0010] The amino acid sequence of the protein OsRPM-L1 is shown in SEQ ID NO. 2.

[0011] The present application also provides an application of the coding gene of the rice protein OsRPM-L1 in any of the following:

[0012] (1) in regulating the resistance of rice to rice blast;

[0013] (2) in the breeding of transgenic rice with improved resistance to rice blast;

[0014] (3) in the preparation of products for improving the resistance of rice to rice blast.

[0015] The nucleotide sequence of the coding gene is shown in SEQ ID NO. 1.

[0016] The application also provides an application of the recombinant vector comprising the coding gene in any of the following:

[0017] (1) in the regulation of the resistance of rice to rice blast;

[0018] (2) in the breeding of transgenic rice with improved resistance to rice blast;

[0019] (3) in the preparation of products for improving the resistance of rice to rice blast.

[0020] The application also provides an application of the engineered bacteria comprising the recombinant vector in any of the following:

[0021] (1) in the regulation of the resistance of rice to rice blast;

[0022] (2) in the breeding of transgenic rice with improved resistance to rice blast;

[0023] (3) in the preparation of products for improving the resistance of rice to rice blast.

[0024] Preferably, the expression amount of the rice protein OsRPM-L1 or the coding gene thereof is up-regulated in rice, and the resistance of the rice to rice blast is improved.

[0025] Preferably, the method for up-regulating the expression amount of the rice protein OsRPM-L1 or the coding gene thereof in rice comprises the steps of constructing an overexpression vector of the coding gene of the rice protein OsRPM-L1, transforming the overexpression vector into Escherichia coli, and then infesting a rice plant.

[0026] The application also provides a method for improving the resistance of rice to rice blast, comprising the steps of up-regulating the expression level of the coding gene of the rice protein OsRPM-L1 in rice, and improving the resistance of the rice to rice blast.

[0027] The nucleotide sequence of the coding gene of the rice protein OsRPM-L1 is shown in SEQ ID NO. 1.

[0028] Preferably, the method for up-regulating the expression level of the coding gene of the rice protein OsRPM-L1 comprises over-expressing the coding gene of the rice protein OsRPM-L1 in rice.

[0029] Preferably, the method for over-expressing the coding gene of the rice protein OsRPM-L1 in rice comprises the following steps:

[0030] The over-expression vector of the coding gene of the rice protein OsRPM-L1 is constructed, the over-expression vector is transformed into E. coli, and then the rice plant is infected.

[0031] The application further provides a breeding method of rice with improved resistance to rice blast, comprising the following steps:

[0032] The coding gene of the rice protein OsRPM-L1 is over-expressed in rice cells, the rice cells are cultivated, and the rice cells are used to regenerate rice, so that the rice with improved resistance to rice blast is obtained.

[0033] The nucleotide sequence of the coding gene of the rice protein OsRPM-L1 is shown in SEQ ID NO. 1.

[0034] The application discloses the following technical effects:

[0035] The application takes Kittake rice as a background, and constructs a knock-out plant and an over-expression plant of the coding gene of the rice protein OsRPM-L1. The inoculation experiment proves that the influence of the knock-out of the OsRPM-L1 gene on the disease resistance of rice is not statistically significant, and the over-expression plant of the OsRPM-L1 gene is more resistant to diseases than the receptor plant; it is shown that the rice protein OsRPM-L1 positively regulates the disease resistance of plants, the coding gene OsRPM-L1 can be used as a target gene for molecular breeding of improving the disease resistance of plants, has a wide application prospect, and has important significance for cultivating rice varieties with improved disease resistance. BRIEF DESCRIPTION OF DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0037] Figure 1 The sequencing peak graph is detected for the knock-out plant;

[0038] Figure 2Figure 6 is a graph showing the phenotype of knockout plants and statistical results; wherein, A is the phenotype of wild type KT and knockout plant rpm1-l1 ko / KT after infection of Magnaporthe oryzae, the scale is 1 cm; B is the lesion length statistics on the leaves of wild type KT and knockout plant rpm1-l1 ko / KT after infection of Magnaporthe oryzae (n=11);

[0039] Figure 3 Figure 7 is a graph showing the expression level of RPM1-L1 in overexpression plant Ubi:RPM1-L1-Flag / KT;

[0040] Figure 4 Figure 8 is a graph showing the phenotype of overexpression plants and statistical results; wherein, A is the phenotype of wild type KT and overexpression plant Ubi:RPM1-L1-Flag / KT after infection of Magnaporthe oryzae, the scale is 1 cm; B is the lesion length statistics on the leaves of wild type KT and overexpression plant Ubi:RPM1-L1-Flag / KT after infection of Magnaporthe oryzae. DETAILED DESCRIPTION

[0041] The following detailed description of various example embodiments of the application will not be considered to limit the application to these specific embodiments but will be understood to describe certain aspects, features and embodiments of the application in more detail.

[0042] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. Each smaller range that falls within the broader ranges is also specifically included. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and each range is inclusive of its end points.

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference to the extent allowed by law. In the case of conflict between the description herein and the incorporated material, the description herein shall control.

[0044] In the description of the application specific embodiments, numerous specific details are set forth in order to provide a thorough understanding of the application. However, those of ordinary skill in the art will recognize that the application can be practiced without the specific details

[0045] As used herein, the terms “comprises”, “comprising”, “includes”, “including”, “has”, “having”, and the like are open-ended terms that are intended to permit but not limit possible elements or ingredients.

[0046] The biological materials and reagents used in the present application are as follows:

[0047] Rice variety Kitaake (O. Sativa L. spp. japonica, var kitaake, AA genome, KT) belongs to the japonica subspecies, which is provided by Professor Chen Xuewei of Sichuan Agricultural University.

[0048] OsRPM-L1 knockout and overexpression transgenic rice are completed by Boyuan Biotechnology Co., Ltd.

[0049] Plant binary expression vector pVK005-01 is provided by Professor Wang Yonghong of Shandong Agricultural University.

[0050] Plant binary expression vector pTCK303 is provided by Professor Chen Xuewei of the State Key Laboratory of Sichuan Agricultural University.

[0051] Total RNA extraction kit: TRIzol, Invitrogen, USA, Catalog No. 15596026.

[0052] Reverse transcription kit: HiScript III RT SuperMix for qPCR (+gDNA wiper), Vanzyme, China, Catalog No. R323-01.

[0053] Homologous recombination kit: ClonExpress II One Step Cloning Kit, Vanzyme, China, Catalog No. C112-01.

[0054] Example 1 Rice NB-ARC gene OsRPM-L1 and cloning thereof

[0055] The present application screens a NB-ARC gene OsRPM-L1 in rice, and the primers are designed according to the rice KT genome reference gene sequence:

[0056] OsRPM-L1-F: ATGGCTGAGGCTGTGATTCT (SEQ ID NO. 3);

[0057] OsRPM-L1-R: TCAGAGAGCTCCATCTGTCT (SEQ ID NO. 4).

[0058] A DNA band of about 2.8 kb was obtained by amplification from the above primers with KT total cDNA as a template. After sequencing, it was confirmed to be OsRPM-L1 gene belonging to the NB-ARC protein family. After alignment of the sequence of LOC_Os10g21400 (OsRPM-L1) in the rice genome reference sequence and the cloned sequence obtained by amplification from KT total cDNA, it was shown that the cloned sequence obtained from KT was identical to the sequence of LOC_Os10g21400 (OsRPM-L1). The amino acid sequence of OsRPM-L1 protein is shown in SEQ ID NO. 2 in the sequence listing. The CDS sequence of the gene OsRPM-L1 encoding OsRPM-L1 protein (the amino acid sequence is shown as SEQ ID NO. 2) is shown as SEQ ID NO. 1.

[0059] SEQ ID NO. 1:

[0060]

[0061] SEQ ID NO. 2:

[0062] MAEAVILLAVKKIGVALGNEAINQATSYFKKFVTQLTELQGSMGRIKRELRLMHEFLSRMDVRN RNNQTYEIWVEEVRMLVHRIEDIVDDYLHLVGHKQDTGWGTYLKKGFKRPNVLFSLNRIAS SIKDAEANLVHLFQAKERWVWMAGGRATGSKSSSYIIETSRHLANISRSLDEDLVGVDENIR KLHEWLTSDELQREVIALHGMGGLGKTALAANVYRNEREKFECHAWVSISQTYSIKDVLKCL VTELDLKKKIQGNIGDMDTATLQNELKKFLMDQKYLIVLDDVWVPETVNDLFSIFVSNLKG SRVLVTTRIDGVAHLAFPDKRITLEPLSEKKSWELFCKTAFPRDKNHECPTKLTVLAQQIV SKCEGLPLAIVSVGRLLFVRDKTEEEFRRIQNQLDWELINNPSLEHVRNILYLSYIYLPTHL KSCFLYCSMFPEDYLITRKKLIRWWVAEGFIEERGGNTMEEVAEEYLKELVHRNMLQLIE MNGFGRIKSFRMHDIVRELAIDLCRKEHFGCSYNCENKHGKFLEGKDERRVVIHKLDKHIN QAILNECHSLRCLITLDEATPPSPCLLHLVADKCRYMSVLELTGLPIEKVPDAIGDLFNLRH LGLRGSKVKHLPNSIEKLSNLLTLDLNETEIQEVPNGIVKLKKLRHLFVEKMNELYGREFRP RTGVRIHKGLEKLNELQTLQGLEVQDEVSLRRLGELRQMRSIRIWGVKESYCESLCESLQQ MEFLSFLSVNASGKEEVLKLDGLNPLPPNLRKLNLRGILAEAGMLLGSPAAGDQNNHSLY SVHLSWSQLIEDPLPSLSRWSSLTDLMLTRAYVGEQFVFHQGWFPNLKELVLRDMPDLKR LEIHDGAMTSLQDLTLVNLSGLTEVPSGIELLSTLKNLGFWEITQDFLAALRQCHRIHHMQ WWYSVRGETDGAL.

[0063] Construction of OsRPM-L1 plant knock-out vector (pVK005-sgRNA OsRPM-L1 ) of Example 2

[0064] The knock-out target sequence was designed as follows based on the genome of rice KT:

[0065] CGTGAGGTGATAGCGCTGCA (SEQ ID NO. 5);

[0066] The following primers were synthesized:

[0067] KO-F: TGTG CGTGAGGTGATAGCGCTGCA G (SEQ ID NO. 6);

[0068] KO-R: AAAAC TGCAGCGCTATCACCTCACG (SEQ ID NO. 7);

[0069] After denaturation at 95°C, the double-stranded DNA was formed. The pVK005-01 vector was digested by BsaI, and the linearized vector was recovered. The above DNA double-stranded and linearized vector were mixed and subjected to T4 DNA ligase ligation reaction at room temperature for 30 minutes. The system: DNA double-stranded fragment 0.5-4 μL, linearized vector 0.5-4 μL, T4 ligase 2 μL, and double distilled water was added to 10 μL.

[0070] Obtained pVK005-sgRNA OsRPM-L1 knock-out vector.

[0071] Transformed into DH5α competent cells, selected by kanamycin, and single colonies were picked for colony PCR identification. The positive colonies were extracted for plasmid and sequencing, and the correct pVK005-sgRNA OsRPM-L1 was confirmed, which could be genetically transformed.

[0072] The construction of OsRPM-L1 knock-out transgenic rice and the gene sequencing of OsRPM-L1 knock-out plants were completed by Boyuan Biotechnology Co., Ltd.

[0073] Example 3 Construction of OsRPM-L1 plant overexpression vector (pTCK303-35S:OsRPM-L1)

[0074] The cDNA obtained by reverse transcription of total RNA of rice KT was used as a template, and the cloning primer was:

[0075] OE-F: TATCCAGATCCAGTGGGATCC ATGGCTGAGGCTGTGATTCT (SEQ ID NO. 8);

[0076] OE-R:CGCACTAGTAAGCTTGGTACC TCAGAGAGCTCCATCTGTCT (SEQ ID NO. 9).

[0077] PCR amplification of OsRPM-L1 full-length cDNA, pTCK303-35S: eGFP-nosT vector was digested by BamHI and Kpn I, and linearized vector was recovered. The PCR recovery product OsRPM-L1 full-length cDNA and linearized vector were mixed and homologous recombination was performed according to the following system. System: DNA fragment 0.5-5 μL, linearized vector 0.5-5 μL, 5x CEII buffer 4 μL, Exnase II 2 μL, double distilled water to 20 μL.

[0078] Obtaining pTCK303-35S: OsRPM-L1 recombinant vector.

[0079] After transformation into DH5a competent cells and kanamycin screening, single colonies were picked for colony PCR identification. After positive colonies were extracted for plasmid and sequencing, it was confirmed that pTCK303-35S: OsRPM-L1 was correct and could be used for genetic transformation.

[0080] The construction of OsRPM-L1 overexpression transgenic rice and the determination of OsRPM-L1 expression in transgenic plants were completed by Boyuan Biotechnology Co., Ltd.

[0081] Example 4 OsRPM-L1 participates in regulating the disease resistance of plants

[0082] The inventors found that OsRPM-L1 plays an important role in regulating plant disease resistance, so they tested the effect of OsRPM-L1 on rice disease resistance.

[0083] Puncture inoculation treatment at seedling stage: the test materials were Kittake (KT), KT knockout lines OsRPM-L1-KO1 and OsRPM-L1-KO2 (KO) genetically stable in KT background, Figure 1 OsRPM-L1 overexpression lines OsRPM-L1-OE1 and OsRPM-L1-OE2 (OE) genetically stable in KT background. Figure 3

[0084] Full seed grains were selected and placed in conical bottles with tap water, and germinated in a 37°C dark incubator, with water changed daily. After 2 days, the white seeds were selected and placed in a 96-well seedling plate, and the 96-well seedling plate was placed on a float in Hoagland nutrient solution. After 21 days, the last two rice leaves with uniform growth and size were selected, punctured and inoculated with 5 μL of 5 x 10 5 ​CFU / mL of Zhong10-8-14 Magnaporthe oryzae spores, and the length of the lesion was observed and counted after 5 days.

[0085] The results are shown in Table 1 Figure 2 and Figure 4 The values represent the mean ± standard deviation (n = 11), and the significance was analyzed using Turkey HSD test (p < 0.05 represents significant difference). The statistics show that there is no significant difference in the length of the lesion between OsRPM-L1-KO1 and OsRPM-L1-KO2 plants in the KT background and KT plants after the inoculation by the stab method Figure 2 ).

[0086] The length of the lesion of OsRPM-L1-OE1 and OsRPM-L1-OE2 plants in the KT background is significantly reduced compared with KT plants, and the disease resistance of the plants is significantly enhanced Figure 4 ). It is indicated that OsRPM-L1 positively regulates the disease resistance of the plants, and the gene encoding OsRPM-L1 can be used as a target gene for the molecular breeding for improving the disease resistance of the plants.

[0087] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope of the present application defined by the claims.

Claims

1. Use of a rice protein OsRPM-L1 in any one of the following: (1) use in regulating rice resistance to Magnaporthe oryzae; (2) use in breeding transgenic rice with improved resistance to Magnaporthe oryzae; up-regulating expression of the rice protein OsRPM-L1 or its encoding gene in rice to improve the rice resistance to Magnaporthe oryzae; the amino acid sequence of the protein OsRPM-L1 is shown as SEQ ID NO.

2.

2. Use of an encoding gene of the rice protein OsRPM-L1 as claimed in claim 1 in any one of the following: (1) use in regulating rice resistance to Magnaporthe oryzae; (2) use in breeding transgenic rice with improved resistance to Magnaporthe oryzae; up-regulating expression of the encoding gene in rice to improve the rice resistance to Magnaporthe oryzae; the nucleotide sequence of the encoding gene is shown as SEQ ID NO.

1.

3. Use of a recombinant vector comprising the encoding gene as claimed in claim 2 in any one of the following: (1) use in regulating rice resistance to Magnaporthe oryzae; (2) use in breeding transgenic rice with improved resistance to Magnaporthe oryzae; up-regulating expression of the encoding gene in rice to improve the rice resistance to Magnaporthe oryzae.

4. Use of an engineered bacterium comprising the recombinant vector as claimed in claim 3 in any one of the following: (1) use in regulating rice resistance to Magnaporthe oryzae; (2) use in breeding transgenic rice with improved resistance to Magnaporthe oryzae; up-regulating expression of the encoding gene in rice to improve the rice resistance to Magnaporthe oryzae. comprising the step of up-regulating expression level of an encoding gene of a rice protein OsRPM-L1 in rice to improve the rice resistance to Magnaporthe oryzae; the nucleotide sequence of the encoding gene of the rice protein OsRPM-L1 is shown as SEQ ID NO.

1. comprising the following steps: over-expressing an encoding gene of a rice protein OsRPM-L1 in rice cells, then cultivating the rice cells, and regenerating rice from the rice cells to obtain the rice with improved resistance to Magnaporthe oryzae; the nucleotide sequence of the encoding gene of the rice protein OsRPM-L1 is shown as SEQ ID NO.

1. ​ ​ ​ ​ ​ ​ ​ 5. A method for improving resistance to rice blast in rice, characterized by, ​ ​ 6. A breeding method of rice having improved resistance to rice blast, characterized by, ​ ​ ​

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