CN109207485B

CN109207485B - Application of OsAPS1 gene in improving disease resistance of rice

Info

Publication number: CN109207485B
Application number: CN201811110938.3A
Authority: CN
Inventors: 袁猛; 惠述刚; 杨泽宇
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2018-09-22
Filing date: 2018-09-22
Publication date: 2020-08-25
Anticipated expiration: 2038-09-22
Also published as: CN109207485A

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of an OsAPS1 gene in improving rice disease resistance, wherein the OsAPS1 gene is cloned from rice, and the nucleotide sequence of the OsAPS1 gene is shown as SEQ ID NO. 1. The research on the biological functions of the OsAPS1 gene proves that the inhibition or knockout of the OsAPS1 gene in rice can enable the rice to show the capability of enhancing the resistance of the rice to various bacterial blight bacteria and bacterial streak bacteria, and the broad-spectrum disease resistance of the rice to the bacterial blight bacteria and the bacterial streak diseases is influenced by regulating the expression level of the OsAPS1 gene.

Description

Application of OsAPS1 gene in improving disease resistance of rice

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of an OsAPS1 gene in improving the disease resistance of rice, wherein the gene is a rice OsAPS1 gene, and the gene can be applied to cultivation of rice varieties with enhanced broad-spectrum resistance to bacterial blight and bacterial streak.

Background

Rice is one of the most important food crops of human beings, China is the first large producing country of rice, and the yield of rice is directly related to the survival of the population in China and even the world. The yield of rice is affected by many factors, and among them, diseases are one of the important factors affecting the yield of rice. The traditional disease control is mainly to spray chemical agents, and the long-term spraying of the chemical agents from the sustainable development viewpoint of agriculture is easy to pollute the environment on one hand, and on the other hand, the chemical agents can accelerate the variation of pathogenic bacteria to cause rice diseases. Therefore, the development of the disease-resistant gene of the rice and the improvement of the defense system of the rice have important significance for cultivating high-quality disease-resistant varieties.

The disease resistance response of plants is a complex process in which multiple genes are involved in regulation. Genes involved in the disease resistance response of plants are divided into two categories: major disease-resistant gene and disease-resistant related gene. At present, more than thirty major disease-resistant genes are cloned from plants, such as rice bacterial leaf blight-resistant genes Xa21, Xa1, Xa26, Xa5, Xa27, Xa13 and the like, and more rice blast-resistant genes such as Pib, Pi-ta, Pi9, Pi2 and the like. However, the resources of the main disease-resistant genes are limited, most of the main disease-resistant genes only have resistance to one or a few pathogenic races, the disease-resistant range is limited, and the cloning progress of the main disease-resistant genes is always slower than the pathogenic change of pathogens, so the application of the main disease-resistant genes in agricultural production is greatly limited.

All genes participating in disease resistance reaction except the main disease resistance gene in the plant are called disease resistance related genes, and encoding products of the genes are involved in synthesizing disease resistance signal molecules, signal transmission or defense reaction and the like in the plant body. At present, many disease resistance related genes have been cloned. More than ten disease-resistant related genes are identified from rice, such as OsWRKY45, OsGH3-8, OsEDR1, OsMAPK6, OsYLP4 and other genes, the genes encode different types of proteins, the proteins participate in the disease-resistant process of the rice to different pathogenic bacteria by regulating and controlling different reaction ways in the rice body, and most of the disease-resistant related genes have broad-spectrum disease resistance to different types of pathogenic bacteria.

The sulfur element is one of macroelements necessary for plant growth and is one of the components of some important compounds in organisms. Plays a role in the growth and development of plants, biotic and abiotic stress and the like, and is also one of important factors affecting crop quality. The importance of elemental sulfur to plant growth has been recognized as early as the 19 th century, and the application of sulfur-containing fertilizers can increase crop resistance to fungal pathogens.

ATP Sulfurylase (ATPs) is the first enzyme that functions in plant sulfate assimilation, and is a key enzyme in the sulfate assimilation pathway. The gene encoding ATPS has been cloned from plants such as Arabidopsis, potato and tobacco. In most plants there are two isogenic ATPSs, located in the cytoplasm and chloroplast, respectively. The chloroplast ATPS initiates sulfate reduction, while the cytosolic ATPS is believed to supply APS for subsequent sulfonation without participating in sulfate reduction.

The research of the invention finds that the OsAPS1 gene in rice is located in chloroplast, and the inhibition or knockout of the OsAPS1 gene can enhance the broad-spectrum disease resistance of the rice to bacterial blight and bacterial leaf streak. The invention has important significance for improving the resistance of the important grain crop rice to bacterial leaf blight and bacterial leaf streak and assimilating and absorbing sulfate ions by the rice.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, and finds that the OsAPS1 gene has an important regulation and control function on the bacterial blight and bacterial leaf streak resistance of rice through the research on the biological function of the OsAPS1 gene, and influences the broad-spectrum disease resistance of the rice to the bacterial blight and bacterial leaf streak by regulating the expression level of the OsAPS1 gene. Therefore, the invention has important significance for improving the resistance of the important grain crop rice to bacterial blight and bacterial streak.

The technical scheme of the invention is as follows:

1. the invention proves that the inhibition or knockout of OsAPS1 gene in rice can lead the rice to show the capability of enhancing the resistance to a plurality of different bacterial blight bacteria and bacterial streak disease bacteria. Through systematic research, the applicant finds that the OsAPS1 gene has an important regulation function in the aspects of resisting bacterial blight and bacterial leaf streak of rice.

2. The invention demonstrates that OsAPS1 is localized to the chloroplast, and exerts biological functions in the chloroplast.

Biological function verification proves that the rice OsAPS1 gene provided by the invention has the following characteristics:

1. the nucleotide sequence of the OsAPS1 gene is shown as SEQ ID NO 1 in the sequence table.

The nucleotide sequence shown in SEQ ID NO. 1 consists of 3413 deoxyribonucleotides of the rice OsAPS1 gene and the upstream and downstream non-coding sequences thereof. 1, the deoxyribonucleotides from 1 st to 183 th in a sequence shown by SEQ ID NO. 1 are upstream non-coding sequences of an OsAPS1 gene; the deoxyribonucleotides from 184 th to 716 th are the first exon sequences of the OsAPS1 gene; the deoxyribonucleotides from 717 th to 1781 th are the first intron sequence of the OsAPS1 gene; the deoxyribonucleotides from 1782 to 2187 are the second exon sequence of the OsAPS1 gene; the deoxyribonucleotides from 2188 to 2332 are the second intron sequence of the OsAPS1 gene; the deoxyribonucleotides from 2333 th to 2593 th are the third exon sequences of the OsAPS1 gene; the deoxyribonucleotides from position 2594 to position 2769 are the third intron sequence of the OsAPS1 gene; the deoxyribonucleotides from position 2770 to position 2856 are the fourth exon sequences of the OsAPS1 gene; the deoxyribonucleotides from 2857 to 2954 are the fourth intron sequence of the OsAPS1 gene; the deoxyribonucleotides from 2955 th to 3095 th are the fifth exon sequences of the OsAPS1 gene; the deoxyribonucleotides from position 3096 to position 3413 are downstream non-coding sequences of the OsAPS1 gene.

2. The OsAPS1 gene sequence can be applied to crops, particularly rice breeding for disease resistance, transgenic lines and new gene varieties.

For a more detailed technical scheme, refer to the detailed description.

Compared with the prior art, the invention has the beneficial effects that:

1. the OsAPS1 gene negatively regulates the broad-spectrum disease resistance of rice to the bacterial blight bacteria and the bacterial streak disease bacteria.

2. The OsAPS1 protein is chloroplast-localized and functions in chloroplast organelles.

Drawings

SEQ ID NO. 1 of the sequence Listing is the nucleotide sequence of OsAPS1 gene. The sequence length is 3413 bp.

FIG. 1: the invention relates to a flow chart for identifying, separating and cloning a rice disease-resistant related gene OsAPS1 gene and verifying the function of the OsAPS1 gene.

FIG. 2: the expression mode of the OsAPS1 gene after the flower 11 in the rice variety is respectively inoculated with different pathogenic bacteria is detected by using a quantitative reverse transcription-PCR (quantitative reverse transcription-PCR, qRT-PCR) technology. Description of reference numerals: FIG. 2A is a graph showing the expression levels of OsAPS1 gene at different time points after inoculation of bacterial blight of rice; FIG. 2B is a graph showing the expression levels of OsAPS1 gene at different time points after inoculation of rice with bacterial leaf streak pathogens. The expression level of OsAPS1 gene in each sample was relative to the expression level of OsAPS1 gene in the sample of rice mid-flower 11 before treatment. Each data is mean (3 replicates) ± standard deviation.

FIG. 3 is a map of a genetic transformation vector pDS1301-P35S-OsAPS1 used in the present invention, and further includes a vector map of an initial transformation vector pYLCRISPR/Cas9-MH (KR029109) and a vector map of a subcellular localization vector pM999-P35S-OsAPS1, and the reference numerals indicate that A in FIG. 3 represents a vector map of pDS1301-P35S-OsAPS1, RB and LB represent right and left borders of T-DNA, respectively, GUS represents β -glucuronidase gene, Hpt represents hygromycin phosphotransferase gene, 35S represents 35S promoter of cauliflower mosaic virus, AdhI represents maize alcohol dehydrogenase 1 gene sequence, Waxy-a represents rice Waxy gene partial sequence, OCS represents polyadenylation signal sequence, MH B in FIG. 3 represents a vector map of pYLISPR/Cas 25-9-OsAPS₃₅: HPT represents hygromycin phosphotransferase gene expressed by 35S promoter of cauliflower mosaic virus, P_UbiRepresents a maize ubiquitin gene promoter, NLS represents a nuclear localization signal sequence, T_nosRepresents the Agrobacterium tumefaciens terminator sequence. FIG. 3C shows a vector map of pM999-p35S-OsAPS 1. Ampicillin indicates Ampicillin gene, CaMV35S indicates cauliflower mosaic virus 35S promoter, GFP indicates green fluorescent protein gene, and polyA indicates polyadenylation signal sequence.

FIG. 4: and (3) knocking out the design site and the genotype detection of the rice strain target site by the OsAPS1 gene. Description of reference numerals: fig. 4 a shows a diagram of the design sites for the U3 promoter-directed gRNA and the U6a promoter-directed gRNA; FIG. 4B is a graph showing the number and position of deoxyribonucleotides deleted in an OsAPS1 knock-out rice line as compared with the wild type; FIG. 4C shows that the PCR product of OsAPS1 knock-out rice line was significantly smaller than that of the wild type by agarose gel electrophoresis.

FIG. 5: and identifying the expression quantity of the OsAPS1 gene in the rice strain with OsAPS1 gene inhibition expression. Description of reference numerals: FIG. 5A shows that qRT-PCR shows that the expression level of OsAPS1 gene inhibiting expression of rice pedigree 13 positive individual is significantly lower than that of wild type and separated negative; FIG. 5B shows that qRT-PCR shows that the expression level of OsAPS1 gene inhibiting expression of rice pedigree 17 positive individual is obviously lower than that of wild type and separated negative.

FIG. 6: OsAPS1 subcellular localization analysis. GFP served as an empty vector control. The results showed that the OsAPS1 protein is a chloroplast protein.

FIG. 7: the OsAPS1 gene inhibits and expresses the lesion length of a rice line and an OsAPS1 gene knockout rice line after the rice line is inoculated with the Xanthomonas albuginea PXO 347. Description of reference numerals: FIG. 7A shows that the length of disease spots of OsAPS1 gene suppression expression family 13 inoculated with P.albugineus PXO347 is obviously shortened compared with wild plants and transgenic negative plants; FIG. 7B shows that the length of disease spots of OsAPS1 gene suppression expression family 17 inoculated with P.albugineus PXO347 is obviously shortened compared with wild plants and transgenic negative plants; FIG. 7C shows that the length of disease spots of the OsAPS1 gene knockout rice line inoculated with P.albugineus PXO347 is significantly shortened compared with that of wild type plants and transgenic negative plants. The results show that the OsAPS1 gene suppression expression rice line and the OsAPS1 gene knockout rice line show a phenotype of enhanced disease resistance to the blight disease PXO 347.

FIG. 8: the OsAPS1 gene suppression expression rice strain has broad-spectrum disease resistance to different bacterial blight pathogens (PXO99, PXO61, PXO86, PXO112, PXO341, PXO347 and FuJ23) relative to a wild type.

FIG. 9: the OsAPS1 gene suppression expression rice strain has broad-spectrum disease resistance to different bacterial leaf spot pathogens (RH3, RS85, RS105, JSB224, HNB8-47 and GX01) compared with a wild type.

FIG. 10: the OsAPS1 gene inhibits the growth amount of the bacterial blight in the leaves of the rice strain inoculated with the bacterial blight PXO 347. Compared with wild type control, the amount of the bacterial blight disease in the rice line with OsAPS1 gene suppression expression is obviously reduced.

FIG. 11: the OsAPS1 gene inhibits the growth amount of bacterial leaf streak germ in the leaf after the rice strain is inoculated with the bacterial leaf streak germ RH 3. Compared with wild type control, the quantity of bacterial leaf streak germs in the OsAPS1 gene suppression expression rice strain is obviously reduced.

Detailed Description

The invention is further illustrated by the following specific examples. FIG. 1 depicts the procedures for identifying and isolating the cloned OsAPS1 gene and for verifying the function of the OsAPS1 gene. It should be noted that these examples are only for illustrating the present invention and should not be construed as limiting the scope of the claims of the present invention in any way.

The methods used in the following examples are conventional unless otherwise specified, and reference is made to the specific procedures: molecular Cloning A laboratory; ry Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor) or related products. The reagents or instruments used are not indicated by manufacturers, and are all conventional products which can be purchased through the market.

Example 1: analysis of expression patterns of OsAPS1 gene after rice is treated by bacterial blight and bacterial streak

In order to verify whether the OsAPS1 gene participates in the regulation of the disease resistance response of rice. The applicant respectively inoculates bacterial blight and bacterial streak disease in flower 11 (from the institute of crop science of Chinese academy of agricultural sciences, the same below) of a rice variety in booting stage, and analyzes the expression pattern of the OsAPS1 gene by adopting a qRT-PCR technology. The results show that the OsAPS1 gene can be inhibited and expressed by the bacterial blight bacteria and the bacterial streak germ. This result suggests that: the OsAPS1 gene may participate in regulating rice resistance reaction to bacterial blight disease and bacterial streak disease, and inhibiting or knocking out the expression of the OsAPS1 gene may improve the disease resistance of rice. The results are shown in FIG. 2.

Example 2: OsAPS1 gene expression inhibition and acquisition of knockout mutant material

(1) Construction of OsAPS1 gene suppression expression vector

This example is a general description of the construction of the pDS1301-p35S-OsAPS1 vector.

Using cDNA of flower 11 in middle quality as template, designing primers dsAPS1F (5'-AGACTAGTGGTACCGGGACCTGTATGATGCTGA-3') and dsAPS1R (5'-ACGAGCTCGGATCCCTTCCAATCAAACCCAAAT-3'), and amplifying by high fidelity DNA polymerase PCR to obtain partial cDNA fragment of OsAPS1 gene (nucleotide fragment is shown as 2511-2593 deoxyribonucleotide, 2770-2856 deoxyribonucleotide, and the second deoxyribonucleotide in sequence shown in SEQ ID NO:1Deoxyribonucleotides at positions 2955 to 3228, and 444 deoxyribonucleotides in total). The PCR amplification reaction was carried out with high fidelity enzyme, the PCR product was recovered by electrophoresis, the PCR product was digested with restriction enzymes BamHI and KpnI, the digested product was recovered by electrophoresis, and simultaneously the vector pDS1301 was digested with KpnI and BamHI overnight and recovered. Recovering the cleaved product from the vector fragment in a molar ratio of about 3: 1 (T4 Ligase)

) Selecting a single clone to culture, simultaneously carrying out PCR verification on the single clone, extracting plasmids from the clone verified to be correct, further carrying out sequencing verification for later use, carrying out enzyme digestion on a part of cDNA fragment of OsAPS1 gene obtained by PCR amplification and the recombinant vector which is connected into the first chain in the previous step by using restriction enzymes Sac I and Spe I, and connecting the recovered enzyme digestion product and the recombinant vector fragment which is connected into the first chain in a molar ratio of about 3: 1 (T4 Ligase)

) And (3) transforming the ligation product into escherichia coli DH5 α the next day, culturing at 37 ℃ overnight to obtain a single clone, selecting the single clone for culturing, simultaneously performing PCR verification on the single clone, verifying the correct clone to extract a plasmid, and further performing sequencing verification to obtain a plant transformation vector pDS1301-p35S-OsAPS1, wherein the map of the plant transformation vector is shown as a picture A in figure 3.

(2) Construction of OsAPS1 Gene knockout vector

This example is a general description of the construction of pYLRISPR/Cas 9-MH-pUBIQUITION-OsAPS1 vector.

Primers APS1U3F (5'-GGCAGTGGTGGTGGTGGAGGAGGGTTTTAGAGCTAG-3') and APS1U3R (5'-CCTCCTCCACCACCACCACTGCCACGGATCATCTGC-3') (i.e., deoxyribonucleotides from position 137 to position 155 in the sequence shown in SEQ ID NO:1 of the sequence Listing), APS1U6aF (5'-GCCGCGAGCCTGATCGAGCCCGAGTTTTAGAGCTAG-3') and APS1U6aR (5'-TCGGGCTCGATCAGGCTCGCGGCAGCCAAGCCAGCAC-3') (deoxyribonucleotides from position 344 to position 362 in the sequence shown in SEQ ID NO: 1) were designed at the sites as shown in Panel A of FIG. 4. Performing two rounds of nested PCR, performing 2 reactions in the first round of PCR, performing PCR by respectively using U-F (5'-CTCCGTTTTACCTGTGGAATCG-3')/APS 1U3R and APS1U3F/gRNA-R (5'-CGGAGGAAAATTCCATCCAC-3') and pYLgRNA-OsU3 vector (KR029104) as DNA templates to obtain products (i) and (ii), and performing PCR by using U-F/APS1U6aR and APS1U6 aF/NA-R and pYLgRNA-OsU6a vector (KR029105) as templates to obtain products (R) and (iv); the second round is overlapped PCR, primers Up-T1 (5'-ACCGGTAAGGCGCGCCGTAGTGCTCGACTAGTGGAATCGGCAGCAAAGG-3') and gR-T1 (5'-CAGGGAGCGGATAACAATTTCACACAGGCACATCCACTCCAAGCTCTTG-3') are used as templates to obtain PCR products (v), and primers Up-T2 (5'-GTGCCTGTGTGAAATTGTTATCCGCTCCCTGGAATCGGCAGCAAAGG-3') and gR-T2 (5'-CCACGCATACGATTTAGGTGACACTATAGCGCATCCACTCCAAGCTCTTG-3') are used as templates to obtain PCR products (g). The pYLCRISPR/Cas9-MH vector was digested with BsaI (see Panel B in FIG. 3); cutting PCR products of a fifth enzyme, a sixth enzyme and BsaI enzyme, namely pYLCRISPR/Cas9-MH vector according to the volume ratio of 2: 2: 1, reacted with pEASY-Uni Seamless Cloning and Assembly Kit (available from Beijing Kokai Tokyo Kogyo Co., Ltd.) at 50 ℃ for 15 minutes, transformed into E.coli DH 5. alpha. and cultured overnight at 37 ℃ to obtain a monoclonal. And selecting a single clone for culturing, simultaneously carrying out PCR verification on the single clone, extracting a plasmid from the clone with correct verification, and carrying out further sequencing verification to obtain a plant transformation vector pYLCRISPR/Cas9-MH-OsAPS 1.

(3) Obtaining and identifying OsAPS1 gene suppression expression rice strain

The applicant introduces a pDS1301-p35S-OsAPS1 vector containing a partial sequence of OsAPS1 gene driven by a strong promoter 35S (AJ007626) into a rice variety Zhonghua 11 by an agrobacterium-mediated genetic transformation method to obtain a plurality of independent transgenic families, selects a family 13 and a family 17, and detects the expression level of the OsAPS1 gene by utilizing a qRT-PCR technology.

The rice leaf blades of the rice at the booting stage were taken from the fields of China, Hubei and Wuhan and total RNA was extracted according to the TransZol (available from Beijing Quanjin Biotechnology Co., Ltd.) using the instructions. 1-5. mu.g of total RNA was treated with DNaseI (Invitrogen, USA) for 15 minutes to removeAfter removing genomic DNA contamination, reverse transcription was performed using oligo (dT)15 oligo primers and M-MLV reverse transcriptase (Promega corporation, USA). Reagent kit adopting real-time quantitative PCR analysis

Green PCR MasterMix (Bao bioengineering, Inc.) was performed according to the kit instructions of the company, and Real-Time quantitative PCR reaction was performed on ABI7500Real-Time PCR system (Applied Biosystems, USA) instrument. And (4) measuring the expression quantity of the endogenous actin gene of the rice and homogenizing the RNA content of the sample. OsAPS1 gene specific PCR primers in qRT-PCR analysis are APS1realF (5'-AGTTTGCCAGACGCAATGCT-3') and APS1realR (5'-AAGGCGTTTGCGTGTATCAGT-3'), and actin gene PCR primers are actin F (5'-TGCTATGTACGTCGCCATCCAG-3') and actin R (5'-AATGAGTAACCACGCTCCGTCA-3').

The qRT-PCR results show that the expression level of OsAPS1 gene in positive individuals in transgenic families is significantly lower than that in negative individuals and wild-type controls, and the results are shown in FIG. 5.

(4) Obtaining and identifying OsAPS1 gene knockout rice strain

Applicants introduced ppYLCRISPR/Cas9-MH-pUBIQUITION-OsAPS1, which contains a strong promoter UBIQUITIN (JX947345) driving the OsAPS1 gene sequence, into flower 11 of rice variety by Agrobacterium-mediated genetic transformation. Obtaining a plurality of independent transgenic families, selecting a family 1 and a family 2, carrying out PCR amplification on the family 1 and the family 2 of wild type and OsAPS1 gene knockout rice strains by using primers 53230CgF (5'-AAGACCGATGGCATGACAGAGTA-3') and 53230CgR (5'-TCCACTCATGTAATTAGTAAT-3'), and identifying the gene knockout condition. The OsAPS1 gene knockout rice line has 218 deoxyribonucleotides deleted between target sites in

family

1 and 208 deoxyribonucleotides deleted between target sites in family 2. The results are shown in panel B of FIG. 4. Agarose gel electrophoresis of PCR products shows that large-fragment deoxyribonucleotide deletions exist in the family 1 and the family 2 of the OsAPS1 gene knockout rice line at the genome level. The results are shown in panel C of FIG. 4.

Example 3: subcellular localization analysis of OsAPS1 protein

(1) Construction of OsAPS1 gene subcellular localization vector

This example is a general description of the construction of the pM999-p35S-OsAPS1 vector.

Using cDNA of Zhonghua 11 as a template, designing primers APS1PM999F (5'-AAGAATTCATGGCGATGCAGGCCGCCTT-3') and APS1PM999R (5'-AAGGTACCGGCCGCAACCGCCTCGC-3'), and amplifying the OsAPS1 gene (184 th to 716 th deoxyribonucleotides, 1782 th to 2187 th deoxyribonucleotides, 2333 th to 2593 th deoxyribonucleotides, 2770 th to 2856 th deoxyribonucleotides, 2955 th to 3095 th deoxyribonucleotides in a sequence shown by SEQ ID NO: 1) by using high-fidelity DNA polymerase PCR. Recovering PCR products through electrophoresis, carrying out enzyme digestion by using EcoRI and KpnI, and recovering an OsAPS1 gene fragment; meanwhile, the vector pM999 (map: FIG. 3C) was digested with EcoRI and KpnI overnight and recovered. Recovering the OsAPS1 fragment and the vector fragment in a molar ratio of about 3: 1 (T4 Ligase)

) And selecting the monoclone for culture, simultaneously carrying out PCR verification on the monoclone, verifying the correct clone to extract a plasmid, and further carrying out sequencing verification to obtain the subcellular localization vector pM999-p35S-OsAPS 1.

(2) Transient expression of pM999-p35S-OsAPS1 in rice protoplast, and positioning of OsAPS1 by fluorescence

Taking rice seedlings which are aseptically cultured for 10-15 days, rapidly cutting leaf sheaths into small sections with the diameter of less than 1mm by using a sharp blade, and balancing with 0.6M mannitol buffer solution for 10 minutes. The mannitol buffer solution is removed, and the mixture is subjected to enzymolysis by using an enzymolysis buffer solution (0.6M mannitol, 10mM MES, cellulase (1.5%), pectinase (0.75%), 0.1% bovine serum albumin, 1mM calcium chloride and beta-mercaptoethanol) and is subjected to a light-shielding reaction for 4 to 5 hours. Then, protoplast cells were collected with W5 buffer (154mM sodium chloride, 125 mM calcium chloride, 5mM potassium chloride, 2mM MES (pH 5.7)). The protoplast cells were resuspended in MMG buffer (0.6M mannitol, 15mM magnesium chloride, 4mM MES (pH5.7)), pM999-p35S-OsAPS1 vector and PEG4000 solution were added, mixed by immediately gentle inversion, and left at room temperature for 10-15 minutes. The transformation was then stopped by the addition of W5 buffer. The supernatant was discarded by centrifugation, and WI buffer (0.6M mannitol, 4mM MES (pH5.7), 4mM potassium chloride) was added thereto, followed by dark culture at 28 ℃ for 12 hours.

Fluorescence was observed with a confocal fluorescence microscope. Referring to FIG. 6, the above results indicate that the OsAPS1 protein is localized in chloroplast organelles.

Example 4: correlation analysis and functional verification of OsAPS1 gene suppression expression rice line and OsAPS1 gene knockout rice line

(1) OsAPS1 gene suppression expression rice line and OsAPS1 gene knockout rice line resistance bacterial blight phenotype analysis at booting stage

In summer fields of China, Hubei and Wuhan, the inoculation test of the bacterial blight of the white leaf blight is carried out on OsAPS1 gene suppression expression rice strains, knockout rice strains and wild type controls. The results show that the OsAPS1 gene suppression expression rice strain and the OsAPS1 gene knockout rice strain are inoculated with the pathogenic microspecies PXO347 of the blight bacteria at the booting stage, and compared with the wild type (non-transgenic, the same below), the OsAPS1 gene suppression expression rice strain 13 and 17 positive single strains and the OsAPS1 gene knockout rice strain OsAPS1-1 and OsAPS1-2 are obviously shorter in the length of the disease than the negative single strain and the wild type control (p is less than 0.01). See fig. 7. The results show that the OsAPS1 gene suppression expression rice line and the OsAPS1 gene knockout rice line can enhance the resistance of rice to small pathogenic species PXO347 of the bacterial blight.

(2) Broad-spectrum resistance analysis of OsAPS1 gene inhibition expression rice strain on different bacterial blight bacteria

In China, Hubei and Wuhan summer fields, the inoculation experiment of the bacterial blight of the white leaf blight is carried out on OsAPS1 gene suppression expression rice strains and wild type controls. The results show that the OsAPS1 gene suppression expression rice strain lines are inoculated with different pathogenic microspecies of the bacterial blight of the leaf blight (PXO99, PXO61, PXO86, PXO112, PXO341, PXO347 and FuJ23, all the bacterial blight of the leaf blight are given by the International Rice research institute), and compared with the wild type, the pathogenic length of the positive individual strains of the OsAPS1 gene suppression expression rice family 13 and family 17 is remarkably shortened (p is less than 0.01). See fig. 8. The results show that the inhibition of expression of the OsAPS1 gene enhances the broad-spectrum disease resistance of rice to different bacterial blight germs.

(3) Broad-spectrum resistance analysis of OsAPS1 gene suppression expression rice line to different bacterial leaf streak

In China, Hubei and Wuhan summer, the OsAPS1 gene suppression expression rice strain and wild type control are subjected to bacterial leaf streak germ inoculation experiment. The results show that the OsAPS1 gene suppression expression rice strain is inoculated with different pathogenic strains of bacterial streak pathogens (RH3, RS85, RS105, JSB224, HNB8-47 and GX01, all bacterial streak bacterial strains are given by professor of Cheng Yongyou of Shanghai university of transportation), and compared with the wild type, the incidence length of the positive individual strains of the OsAPS1 gene suppression expression rice family 1 and family 10 is obviously shortened (p is less than 0.01). See fig. 9. The results show that the inhibition expression of the OsAPS1 gene enhances the broad-spectrum disease resistance of rice to different bacterial leaf streak pathogens.

(4) Analysis of quantity of bacterial blight in OsAPS1 gene suppression expression rice line

In China, Hubei and Wuhan summer fields, OsAPS1 gene suppression expression rice strain lines are inoculated with small pathogenic strains PXO347 of the bacterial blight in booting stage, and then the growth conditions of the bacterial blight in leaves are analyzed. In the booting stage, an OsAPS1 gene suppression expression rice line and a wild type control are respectively inoculated with small pathogenic species PXO347 of the bacterial blight in the field (donated by the International Rice research institute), leaf materials (leaves of 6cm below an inoculation cut) are taken at different times after inoculation, and three leaves are taken from the same material (representing the repetition of three experiments). The leaf material was treated according to the reported method and analyzed for the number of bacteria grown. The main analysis steps are as follows: disinfecting the surface of the leaf blade by using 75% alcohol for 1 minute, airing, placing the leaf blade into a mortar, adding 1ml of sterilized distilled water, grinding the leaf blade into homogenate, then diluting the homogenate by doubling the sterilized water into different concentration echelons, repeatedly coating three PSA culture dishes (200 g of potatoes, 20g of agar, 20g of cane sugar and constant volume of deionized water to 1000ml) in each concentration gradient, and counting bacterial colonies after growing for 2-3 days in the dark at the temperature of 22-25 ℃. The bacterial growth curve is plotted as LOG value of the number of colonies of the bacterial blight fungus per leaf. The growth analysis of the bacterial blight of the white leaf shows that after the bacterial blight pathogenic microspecies PXO347 are inoculated, the quantity of the bacterial blight of the white leaf in the leaves of the rice line of OsAPS1 gene suppression expression is obviously lower than that of the wild type control. See fig. 10.

(5) Analysis of bacterial leaf streak disease number in OsAPS1 gene suppression expression rice strain

In China, Hubei and Wuhan summer, OsAPS1 gene suppression expression rice strain is inoculated with a bacterial streak germ pathogenic microspecies RH3 (donated by professor of Cheng Yong of Shanghai university) in the tillering stage and then the growth condition of the bacterial streak germ in leaves is analyzed. In a tillering stage, an OsAPS1 gene suppression expression rice line and a wild type control are respectively inoculated with a pathogenic microspecium RH3 of bacterial leaf streak pathogens in the field, leaf blade materials (6 cm leaf blades above and below an inoculated part) are taken at different time after inoculation, and three leaf blades (representing the repetition of three experiments) are taken from the same material. The leaf material was treated according to the reported method and analyzed for the number of bacteria grown. The main analysis steps are as follows: disinfecting the surface of the leaf with 75% alcohol for 1 minute, drying, placing in a mortar, adding 1ml of sterilized distilled water, grinding into homogenate, diluting with double sterilized water to obtain different concentration gradients, treating the leaf material according to the reported method, and analyzing the growth amount of bacterial streak pathogens. The main analysis steps are as follows: disinfecting the surface of the leaf blade by using 75% alcohol for 1 minute, airing, placing the leaf blade in a mortar, adding 1ml of sterilized distilled water, grinding the leaf blade into homogenate, then diluting the homogenate by doubling the sterilized water into different concentration gradients, repeatedly coating three PSA culture dishes for each concentration gradient, and counting the bacterial colony number of the bacterial streak germs after the bacterial colony grows for 2 to 3 days at the temperature of 22 to 25 ℃ in the dark. Bacterial growth curves were plotted as LOG number of bacterial colonies per leaf. Growth analysis of bacterial leaf streak pathogens shows that after inoculation of pathogenic microspecies RH3 of bacterial leaf streak pathogens, the quantity of bacterial leaf streak pathogens in leaves of OsAPS1 gene suppression expression rice strains is obviously lower than that of wild type controls. See fig. 11.

SEQUENCE LISTING

<110> university of agriculture in Huazhong

Application of OsAPS1 gene in improving disease resistance of rice

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<141>2018-09-22

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<213> Rice (Oryza sativa)

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<220>

<221>3'UTR

<222>(3096)..(3413)

<223>

<220>

<221>exon

<222>(2955)..(3095)

<223>

<220>

<221>Intron

<222>(2857)..(2954)

<223>

<220>

<221>exon

<222>(2770)..(2856)

<223>

<220>

<221>Intron

<222>(2594)..(2769)

<223>

<220>

<221>exon

<222>(2333)..(2593)

<223>

<220>

<221>Intron

<222>(2188)..(2332)

<223>

<220>

<221>exon

<222>(1782)..(2187)

<223>

<220>

<221>Intron

<222>(717)..(1781)

<223>

<220>

<221>exton

<222>(184)..(716)

<223>

<220>

<221>5'UTR

<222>(1)..(183)

<223>

<400>1

cccgcaatcc catccaggtt catccgcctc ccgtcttctt tttttttaat tcatccgcaa 60

ccactgcgaa cttgcgaagg ccgcgggtac aagaaaagaa gagaaaaata aaaaggagcc 120

gcattataag taggtagtgg tggtggtgga ggaggaggag agcaaatcga ggtagccatg 180

gcgatgcagg ccgccttcgc cgcctcgatg ttcccgcagc tggcgcagcg gaggggaagc 240

gatcgcgcgg tggtggtggc gccgccggcg ccggcgccgg tgagggtggc gatgaggagc 300

ggtggggcgg cggcggcggc ggcgagaggg gtgaggtgca gggcgagcct gatcgagccc 360

gacgggggga ggctggtgga gctggtggtg ccggaggagg gagggcggcg ggaggcggcg 420

cggcgggagg cggcggcgct ggcgcaccgg gtgaggctgg ggcgggtgga gacggagtgg 480

ctgcacgtgc tgagcgaagg gtgggcgagc ccgctgcgag ggttcatgcg cgaggccgag 540

ttcctccaag cacttcattt caacgccatc cgcggcggag acggggccat ggtcaacatg 600

tccgtgccca tcgtcctccc cctcggtgac gcccagcgcc gcgccatcga ggcgtccggc 660

gcccgccgcg tcgcgctcgt cgacgccgcc gaccgccccc tcgccgtcct cagcgagtga 720

gacatccccc ctaccccacc tcttcttgtt tcccttgttt agttagttgc atttccctgt 780

tcagacgctt aattgcaatt atttggttac taggagtgct tgctagaaga gtaaaattac 840

taattacatg agtggatgga atgtagatat attgattgtt cgtgttggtt ggttgattct 900

cacagttgtg ggatgccttt ggacctccaa aagccgtaat ttttgtgctt gggttggaaa 960

ttggaattgt gggggttgta gcctgaatgg ttcagagaag ttcataggtg gtttcttcaa 1020

ttggaaacat aatatgcaga aatacagatt atgagtgttt ctgtgcagtt cccacatttc 1080

gtgagcttat ccctcacaaa cttgtgctct ggttatattt tagactttaa tgctagtaaa 1140

caatttgttt cacagcaacc tggtcaatga tatatcattc atggcggagg ttttatcttg 1200

ggcgttaaac taattttaag gttgtggcat tcctcttttg tcgattagac tacaatatcg 1260

tcatgcggat aacctttgtc atgctatatc aggaactcag gacatcttta tacatctcgg 1320

tcgttttcag gtcatatttt tttgagtcgt atatatataa tacgatgatc ggtaatacca 1380

ttggtagtcc ttataaatac atgttccgta gttcttttgc cgtgttctct aggtatttta 1440

gttttatcta tattaaactg acaagtgata ttatatttgt ttcgttgttt tttcgccgcg 1500

ttctatgggt attttagttt aagcaactga cacatgatat gatttacatc agaaagatta 1560

ttttttattc accatttgtt tataaggttt tgtacacatc cagctattgt aagatgttgt 1620

tatagaggtg acgtaacaac gtcaaccgtt tacatctgtg tctaggaact cctcaagtca 1680

tgtgtgttca aaacaatccg acttgattta gcatggtttt agttcttttt aattagttat 1740

accatggcat tccctgacat atttttcttc tttgttgtca g cat tga gat cta caa 1796

His Asp Leu Gln

1

gca taa caa aga aga aag gat tgc acg gac atg ggg aac aac tgc acc 1844

Ala Gln Arg Arg Lys Asp Cys Thr Asp Met Gly Asn Asn Cys Thr

5 10 15

tgg gct gcc tta tgt tga tga ggc gat tac aaa tgc tgg tga ttg gtt 1892

Trp Ala Ala Leu Cys Gly Asp Tyr Lys Cys Trp Leu Val

20 25 30

aat tgg tgg tga ctt gga ggt tat aga acc aat caa gta caa tga tgg 1940

Asn Trp Trp Leu Gly Gly Tyr Arg Thr Asn Gln Val Gln Trp

35 40 45

tct tga tca gta tcg ctt gtc tcc agc cca gct gcg tga aga gtt tgc 1988

Ser Ser Val Ser Leu Val Ser Ser Pro Ala Ala Arg Val Cys

50 5560

cag acg caa tgc tga tgc ggt att cgc gtt tca gct tcg caa tcc tgt 2036

Gln Thr Gln Cys Cys Gly Ile Arg Val Ser Ala Ser Gln Ser Cys

65 70 75

aca caa tgg aca tgc ttt gct cat gac tga tac acg caa acg cct tct 2084

Thr Gln Trp Thr Cys Phe Ala His Asp Tyr Thr Gln Thr Pro Ser

80 85 90

tga gat ggg tta caa aaa tcc tgt tct tct gct tca tcc att ggg agg 2132

Asp Gly Leu Gln Lys Ser Cys Ser Ser Ala Ser Ser Ile Gly Arg

95 100 105

att cac aaa agc aga tga tgt gcc tct tag ttg gag aat gaa gca gca 2180

Ile His Lys Ser Arg Cys Ala Ser Leu Glu Asn Glu Ala Ala

110 115

tga aaa g gtacgcttta agagtagcaa tcgtactgtc aaatcttttt agcaaatgca 2237

Lys

120

tgttggtgcc ttgttgttga tgaaaccatt tgatttaatt acactactgt aaggcccaat 2297

tcatgcacaa ttttgatgcg tgtttatttt ggtag gt tct tga gga agg tgt 2349

Gly Ser Gly Arg Cys

125

cct caa ccc aga atc aac tgt cgt tgc aat ctt ccc ctc tcc aat gca 2397

Pro Gln Pro ArgIle Asn Cys Arg Cys Asn Leu Pro Leu Ser Asn Ala

130 135 140

tta tgc agg gcc aac tga agt gca atg gca tgc taa ggc tcg tat aaa 2445

Leu Cys Arg Ala Asn Ser Ala Met Ala Cys Gly Ser Tyr Lys

145 150 155

tgc tgg tgc aaa ctt cta cat agt tgg aag aga tcc tgc tgg tat ggg 2493

Cys Trp Cys Lys Leu Leu His Ser Trp Lys Arg Ser Cys Trp Tyr Gly

160 165 170

cca ccc aac tga aaa gag gga cct gta tga tgc tga tca tgg gaa aaa 2541

Pro Pro Asn Lys Glu Gly Pro Val Cys Ser Trp Glu Lys

175 180

ggt gtt gag cat ggc tcc tgg gct tga gaa gct caa tat cct tcc ttt 2589

Gly Val Glu His Gly Ser Trp Ala Glu Ala Gln Tyr Pro Ser Phe

185 190 195

caa g gtatgtcatc ctaccagacc agatatcaca ttttttggat gcactagatt 2643

Gln

200

gtgtagttcg ctagcttact gtccatcttg tacatttgaa tttggatgtc aagcaacata 2703

ttttagtttt cagataaacc ttgttgtatt ctttttcttg gcatgactcc tgatttgttt 2763

tatcag gt ggc tgc ata tga cac aaa gca aaa gaa aat gga ctt ttt 2810

Gly Gly Cys Ile His Lys Ala Lys Glu Asn Gly Leu Phe

205 210

cga tcc atc aag gaa aga tga ttt cct gtt cat ctc tgg cac aaa g 2856

Arg Ser Ile Lys Glu Arg Phe Pro Val His Leu Trp His Lys

215 220 225

gtaacatcca tttcaatcct gttttggtac accagttaat tttgagcacc aatgtttttt 2916

tccagcccag tcctaacaac attgcctgtg ttatatag at gcg tac tct tgc caa 2971

Asp Ala Tyr Ser Cys Gln

230

gaa ctg cca gag ccc ccc tga cgg att cat gtg ccc ggg tgg ctg gaa 3019

Glu Leu Pro Glu Pro Pro Arg Ile His Val Pro Gly Trp Leu Glu

235 240 245

ggt cct tgt tga ata cta cga cag ctt gac gcc atc agc gga cag cag 3067

Gly Pro Cys Ile Leu Arg Gln Leu Asp Ala Ile Ser Gly Gln Gln

250 255 260

caa act gcg cga ggc ggt tgc ggc cta g aaagctgaaa ctacctcaaa 3115

Gln Thr Ala Arg Gly Gly Cys Gly Leu

265 270

taagcaagaa cattgcatct tgcaactgta aaatggttct ttcagagaga gaaccaatgg 3175

tatatgtgta tactactggt cgcaaggcct aaacatttgg gtttgattgg aagcgcattc 3235

tggtgtgatt tgtatgtgcc ctatcatctg gtaatgctac attggaagct catcatgttg 3295

ttggtgttaa taaggatgag cctttgtgtg ctctgctaga tatgtacata tacaatgtgg 3355

tggtatttat atagtgcttg gtgtgtttgc tattatagaa gagcaggttt catcttct 3413

Claims

1. The application of OsAPS1 gene derived from rice in enhancing the resistance of rice to bacterial blight and bacterial leaf streak, and is characterized in that the nucleotide sequence of the OsAPS1 gene is shown as SEQ ID NO:1 is shown.

2. The use of claim 1, further comprising transforming rice with the OsAPS1 gene on a suppression expression vector or a knock-out vector to obtain transgenic lines with enhanced resistance of rice to bacterial blight and bacterial leaf streak.