Plant immunoregulation related CUA gene, orthologous gene thereof and application of CUA gene and orthologous gene thereof in breeding of disease-resistant molecules

Abstract

The invention discloses a CUA gene related to plant immunoregulation, an orthologous gene of the CUA gene and application of the CUA gene and the orthologous gene in breeding of disease-resistant molecules. The nucleotide sequence of the CUA gene comprises the nucleotide sequence of an AT5G52750 gene and / or the nucleotide sequence of an AT5G52760 gene, the nucleotide sequence of the AT5G52750 gene is shown as SEQ ID NO. 1, and the nucleotide sequence of the AT5G52760 gene is shown as SEQ ID NO. 2; the application of the CUA gene is that the CUA gene or an orthologous gene of the CUA gene is used for breeding disease-resistant molecules of plants. The technical scheme of the invention provides new gene resources and theoretical basis for plant disease-resistant molecular breeding.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, specifically to plant immune regulation-related CUA gene and its orthologous gene and their application in disease-resistant molecular breeding. Background Art

[0002] Arabidopsis thaliana has become the preferred model species for plant gene function research due to its relatively simple chromosome genome, easy indoor cultivation, short growth cycle and convenient genetic transformation. It is widely used in current plant functional genomics research. For the molecular breeding of disease resistance of economic crops, the discovery of Arabidopsis thaliana disease resistance genes and their functional research has important reference and reference value.

[0003] In Arabidopsis, there is a class of mutants whose immune system is continuously activated due to gene mutations. They are called "spontaneous immune mutants". These mutants are important resources for exploring immune regulatory genes.

[0004] With the widespread application of CRISPR technology in the field of crop molecular breeding, technicians can create disease-resistant materials by knocking out the "disease-susceptible genes" in plants. However, the known disease-susceptible gene resources in plants are relatively scarce. Therefore, there is an urgent need to discover new disease-susceptible genes to meet the needs of disease-resistant breeding. Summary of the invention

[0005] To this end, the technical problem to be solved by the present invention is to provide a CUA gene related to plant immune regulation and its orthologous gene and its application in disease-resistant molecular breeding, so as to provide new gene resources and theoretical basis for plant disease-resistant molecular breeding.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] A CUA gene related to plant immune regulation, wherein the nucleotide sequence of the CUA gene contains the nucleotide sequence of the AT5G52750 gene and / or the AT5G52760 gene, the nucleotide sequence of the AT5G52750 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the AT5G52760 gene is shown in SEQ ID NO.2, and the CUA gene is used to regulate the disease resistance of the plant.

[0008] The protein encoded by the CUA gene related to plant immune regulation is encoded by the AT5G52750 gene or the AT5G52760 gene. The amino acid sequence of the protein encoded by the AT5G52750 gene is shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by the AT5G52760 gene is shown in SEQ ID NO.4.

[0009] The orthologous gene of the CUA gene related to plant immune regulation is the orthologous gene of the CUA gene in economic crops. "Orthologous genes" are homologous genes present in different species, which are derived from a common ancestral gene and separated by a speciation event.

[0010] The above-mentioned orthologous gene, the economic crop includes at least one of turnip Brassica rapa, rapeseed Brassicanapus and radish Raphanus sativus; the amino acid sequence of the protein encoded by the orthologous gene in turnip is shown as SEQ ID NO.12, the amino acid sequence of the protein encoded by the orthologous gene in rapeseed is shown as SEQ ID NO.13, and the amino acid sequence of the protein encoded by the orthologous gene in radish is shown as SEQ ID NO.14.

[0011] Application of CUA gene, the application is: using the above CUA gene or the orthologous gene of the above CUA gene for molecular breeding of plant disease resistance. The homology of AT5G52750 gene and AT5G52760 gene reaches 78.01%, and experiments have shown that both have the ability to regulate Arabidopsis immunity. When the homology of a paralogous gene or a orthologous gene with AT5G52750 gene or AT5G52760 gene is greater than 78%, it can be reasonably expected that the paralogous gene or orthologous gene also has the ability to regulate plant immunity.

[0012] In the above application, the disease-resistant molecular breeding is to improve the plant's resistance to infection by powdery mildew microorganisms of the genus Golovinomyces and / or infection by Pseudomonas microorganisms. "Powdery mildew microorganisms" are powdery mildew pathogens.

[0013] The above application, when conducting molecular breeding for plant disease resistance, constructs a recombinant vector to transform the plant to reduce the expression level of the AT5G52750 gene and / or the AT5G52760 gene in the plant, thereby achieving the purpose of improving the disease resistance of the plant.

[0014] In the above application, the recombinant vector is a CRISPR / Cas9 gene knockout vector for knocking out the AT5G52750 gene and / or the AT5G52760 gene in the plant, and the original vector of the recombinant vector is pHEE401E. The CRISPR / Cas9 gene knockout vector is a recombinant vector used in the CRISPR / Cas9 gene knockout system. The original vector of the recombinant vector is pHEE401E, and the recombinant vector contains a sgRNA sequence that can specifically target the AT5G52750 gene or the AT5G52760 gene, and the sgRNA sequence can help the relevant enzymes in the CRISPR / Cas9 gene knockout system to accurately locate the gene containing the "target sequence" to achieve gene knockout. If it is necessary to knock out both the AT5G52750 gene and the AT5G52760 gene in the same plant, two CRISPR / Cas9 gene knockout vectors can be designed, one of which contains an sgRNA sequence that specifically targets the AT5G52750 gene, and the other contains an sgRNA sequence that specifically targets the AT5G52760 gene. After using these two CRISPR / Cas9 gene knockout vectors to transform plants, the AT5G52750 gene and the AT5G52760 gene can be knocked out, thereby reducing the expression levels of the two genes.

[0015] In the above application, a recombinant vector for specifically knocking out the AT5G52750 gene in a plant is connected to a sgRNA sequence specifically targeting the AT5G52750 gene, and the sgRNA sequence is shown in SEQ ID NO.11.

[0016] In the above application, the plant is any one of Arabidopsis thaliana, turnip, radish and rapeseed.

[0017] The technical solution of the present invention achieves the following beneficial technical effects:

[0018] 1. The present invention discloses for the first time CUA genes related to plant immune regulation, particularly CUA82 and CUA83 genes, which play an important role in regulating plant resistance to infection by powdery mildew microorganisms of the genus Golovinomyces and infection by Pseudomonas microorganisms. This discovery provides new gene resources and theoretical basis for plant disease resistance molecular breeding. By reducing the expression of CUA genes in plants, plant resistance to powdery mildew and Pseudomonas can be significantly improved. This method provides a new way to solve the problem of the scarcity of susceptible gene resources in current plant disease resistance breeding.

[0019] 2. The present invention also provides a specific method for CUA gene silencing using the CRISPR / Cas9 system, providing a new paradigm for the application of genetic engineering technology in plant disease resistance breeding.

[0020] 3. The CUA gene and its orthologous genes disclosed in the present invention exist in a variety of economic crops and have wide applicability and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Growth phenotypes of Col-0, bon1-1, camta1 / 2 / 3, cpr1-2 and cbp60b-1 at 3 weeks of age in the examples of the present invention;

[0022] Figure 2 Venn diagram of up-regulated genes (log2≥1.0) in bon1-1, camta1 / 2 / 3, cpr1-2 and cbp60b-1 mutants in the examples of the present invention;

[0023] Figure 3 The results of measuring the expression of the immune marker gene PR1 of each mutant strain 24 hours after inoculation of Pseudomonas DC3000 in the embodiment of the present invention;

[0024] Figure 4 Photos of the disease phenotypes of the mutants 14 days after inoculation with powdery mildew pathogens in the examples of the present invention;

[0025] Figure 5 Comparison of the nucleotide sequences of the CUA82 gene and the CUA83 gene in the examples of the present invention;

[0026] Figure 6 The expression detection results of the CUA82 gene in the Col-0, cua82-1 mutants and the CUA82-OE overexpression strain in the embodiments of the present invention;

[0027] Figure 7 The expression level detection results of the CUA83 gene in the Col-0, cua83-1 mutant and CUA83-OE overexpression strains in the embodiments of the present invention;

[0028] Figure 8 Sequence comparison of the CUA82 gene between the cua82-2 / cua83-1 double mutant and the cua83-1 single mutant in the examples of the present invention;

[0029] Fig. 9Photos of the disease phenotypes of Col-0, cua82-1 mutant, CUA82-OE overexpression strain, cua83-1 mutant, CUA83-OE overexpression strain and cua82-2 / cua83-1 double mutant in the embodiments of the present invention two weeks after inoculation with powdery mildew spores;

[0030] Fig.10 The colony counts of Col-0, cua82-1 mutant, CUA82-OE overexpression strain, cua83-1 mutant, CUA83-OE overexpression strain and cua82-2 / cua83-1 double mutant after inoculation with DC3000 in the embodiments of the present invention;

[0031] Fig.11 Sequence comparison of the CUA82 gene of the present invention and its highly homologous genes in Brassica rapa;

[0032] Fig.12 Sequence comparison of the CUA82 gene in the present invention and its highly homologous genes in rapeseed;

[0033] Fig.13 Sequence comparison of the CUA82 gene in the present invention and its highly homologous genes in radish. DETAILED DESCRIPTION

[0034] "Autoimmune mutants" are a type of mutant in which the immune system is constitutively activated and often exhibit certain growth and development defects. Figure 1 This is a comparison of the growth conditions of wild-type Arabidopsis and Arabidopsis spontaneous immune mutants at 3 weeks of age, where Col-0 is wild-type Arabidopsis, and bon1-1, camta1 / 2 / 3, cpr1-2, and cbp60b-1 are four different spontaneous immune mutants (all mutants were purchased from the Arabidopsis SALK mutant library). As can be seen from the figure, the growth and development of the spontaneous immune mutants is significantly worse than that of the wild type.

[0035] In order to discover new immune regulatory genes, the inventors sequenced the transcriptomes of Col-0 and four spontaneous immune mutants, bon1-1, camta1 / 2 / 3, cpr1-2 and cbp60b-1, and screened 90 differentially expressed genes whose expression levels were upregulated in the four mutants (the screening criterion was log2 ≥ 1.0, that is, the difference in the expression level of a gene between the wild type and the mutant was at least 2 times). Figure 2As shown. These genes were named CUA (Common up-regulated gene inautoimmunity) genes, and each gene was named in order from small to large gene numbers, that is, these 90 differentially expressed genes were named CUA1 to CUA90. Among them, 36 genes (40%) were reported immune regulatory genes, and the functions of 54 genes in immune regulation have not been reported. The information of 36 CUA genes known to have immune regulatory functions is shown in Table 1.

[0036] Table 1

[0037]

[0038]

[0039] The above-mentioned 54 CUA genes that have not been reported to have immune regulation function were used as candidate genes, and CUA gene deletion mutants were collected for disease resistance screening. For some strains that have been identified as homozygous mutants, Pseudomonas syringae DC3000 and powdery mildew UCSC1 (Golovinomyces cichoracearumUCSC1) were inoculated respectively. After 24 hours of inoculation with Pseudomonas DC3000, the expression level (relative expression level) of the immune marker gene PR1 in each mutant strain was detected. After 14 days of inoculation with powdery mildew UCSC1, the resistance-susceptibility phenotype of each mutant strain to powdery mildew (i.e., whether it is resistant or susceptible) was observed. The measurement results of the expression level of PR1 and the observation results of the resistance-susceptibility phenotype were combined to screen and identify the disease resistance of each mutant strain.

[0040] At the same time, pad4-1 was inoculated with Pseudomonas DC3000 and Echinops UCSC1 respectively as a susceptible material control (pad4-1 is an Arabidopsis mutant lacking the PAD4 gene and has no disease resistance), and cpr1-2 was inoculated with the two pathogenic microorganisms respectively as a resistant material control (cpr1-2 is an Arabidopsis mutant lacking the CPR1 gene and has strong disease resistance).

[0041] The expression level measurement results of PR1 in some mutant strains are as follows Figure 3As shown, only the measurement results of the strains with significantly increased expression of PR1 are shown in the figure. The vertical axis in the figure represents the expression of the PR1 gene relative to the ACTIN1 gene as an internal reference, and 24hpi Pst DC3000 represents 24 hours after inoculation of Pseudomonas DC3000. As can be seen from the figure, 24 hours after infection with Pseudomonas DC3000, the expression of the immune marker gene PR1 in the cua82-1 mutant (mutant with cua82 gene deletion, "cua82" indicates that the deleted gene is CUA82 gene, and "1" indicates the strain number) and cua83-1 mutant (mutant with cua83 gene deletion) are significantly higher than Col-0, indicating that a stronger immune response occurred in cua82-1 and cua83-1 compared with Col-0.

[0042] 14 days after inoculation with powdery mildew pathogens, both cua82-1 and cua83-1 mutants showed a phenotype that was more resistant to powdery mildew than Col-0, as shown in Figure 2. Figure 4 As shown in the figure, compared with the wild-type strain Col-0 and the susceptible strain pad4-1, the number of white lesions on the cua82-1 mutant and cua83-1 mutant was significantly less, and the number of white lesions on the cua82-1 mutant and cua83-1 mutant was close to that of the resistant strain cpr1-2.

[0043] In addition, nucleotide sequence comparison revealed that the sequence identity between CUA82 and CUA83 was as high as 78.01% (e.g. Figure 5 This suggests that the two genes may be homologous to each other.

[0044] The above-mentioned cua82-1 (CS852976) and cua83-1 (SALK_013684) mutants were purchased from the Arabidopsis SALK mutant library and were identified as homozygous by the "three-primer method" of LP+BP and LP+RP.

[0045] Primer sequences used in the “three-primer method” identification:

[0046] CS852976_LP (for identification of cua82-1): 5′-TTTCAAAATCAACTCAATGCATG-3′;

[0047] CS852976_RP (for identification of cua82-1): 5′-CAGGAAGAAAGCGTTTGTCAC-3′;

[0048] SALK_013684-LP (for identification of cua83-1): 5′-CTCCACGTCACCCTAGACTTG-3′;

[0049] SALK_013684-RP (for identification of cua83-1): 5'-TTCTGGAGTAGTTTCTCGAGCC-3';

[0050] BP (common to cua82-1 and cua83-1): 5'-ATTTTGCCGATTTCGGAAC-3'.

[0051] The coding sequence (SEQ ID NO.1) of the CUA82 gene (i.e., the AT5G52750 gene) is as follows:

[0052] ATGCCTCCAATGAAAGCTGTGTTGCAATTGAGCATTCACGAGGAAAGAATCAGGAAGAAAGCGTTTGTCACCGTTTCTCGATGTCCAGGTGTTACTTCGATAACAATGGATGACAAAACCGGGAAAATGACAGTAGTTGGTGAAGTTGATGTACCGGTTATCGTGATGAAGCTAAGGAAGCTATGTAACACAGAGCTTGTTTCGGTTGAA GTTGTTAAACCACCTGAGAAAAAAGCCTGAACCAGAGAAACCGGCTCCACCTAAACCAGCTCCAGCTCCAGCTAAACCGGCTGAAATTGTTGCCTGGCCGGTTCAGATGAACAACCCTTACCAATACAATCCTGCCTATGCAAATTCTTACTATCAACCATATGGGAACTCTAGATTTGTAACGGACGAATCAAATTGTGTGATTATGTGA

[0053] The coding sequence of the CUA83 gene (i.e., the AT5G52760 gene) (SEQ ID NO. 2) is as follows:

[0054] ATGACCGCAAAGAACGCTGTGTTGCAATTGAGTATTCACGAGGAGAGAACCAGGAAGAAAGCGCTTGTCACCGTTTCTCGATTTTCAGGAGTTACTTCGATAACAATGGACAAAAGTGGGAAAATGACAATAGTTGGAGAAGTTGATGTACCAGCTGTCGTGATGAAGCTAAGGAAGCTATGTAACACAG AGATCGTTCAGTCGATGATGTTAAACCACCTGTTAAAAAAGCCTGAACCTGAGAAACCAGCTGAATCTATTGCATATCCTGTTCCGATGAACTACGCGTACCAATTCAATCCTGCCTATGCAAATTCTTACTATCATCAACCATACGGAAATTGTAGAGTGGTAGACGAACCAAATTGTGTGATTATGTGA

[0055] The amino acid sequence (SEQ ID NO. 3) of the protein encoded by the CUA82 gene (i.e., the AT5G52750 gene) is as follows:

[0056] MPPMKAVLQLSIHEERIRKKAFVTVSRCPGVTSITMDDKTGKMTVVGEVDVPVIVMKLRKLCNTELVSVEVVKPPEKKPEPEKPAPPKPAPAPAKPAEIVAWPVQMNNPYQYNPAYANSYYQPYGNSRFVTDESNCVIM

[0057] The amino acid sequence (SEQ ID NO. 4) of the protein encoded by the CUA83 gene (i.e., the AT5G52760 gene) is as follows:

[0058] MTAKNAVLQLSIHEERTRKKALVTVSRFSGVTSITMDKSGKMTIVGEVDVPAVVMKLRKLCNTEIVSVDDVKPPVKKPEPEKPAESIAYPVPMNYAYQFNPAYANSYYHQPYGNCRVVDEPNCVIM

[0059] Based on the above results, the inventors preliminarily determined that CUA82 and CUA83 are involved in plant immune regulation (i.e., regulating plant disease resistance).

[0060] To further verify the functions of CUA82 and CUA83 in immune regulation, the inventors constructed the Arabidopsis strain CUA82-OE overexpressing the CUA82 gene and the Arabidopsis strain CUA83-OE overexpressing the CUA83 gene, respectively. When constructing the overexpression Arabidopsis strain, Agrobacterium GV3101 carrying the recombinant overexpression vector was used to transform Arabidopsis Col-0 by pollen tube infection. The original vector of the recombinant overexpression vector was pMDC32. After the recombinant overexpression vector was transformed into Arabidopsis, the positive transgenic strain was screened out using a hygromycin resistance plate, and the expression level of the target gene in the positive transgenic strain was further detected by RT-qPCR. For the positive transgenic strain that successfully achieved overexpression, it was self-pollinated for two consecutive generations to obtain a T3 generation homozygous strain.

[0061] The recombinant overexpression vector was constructed by the Gateway method, using the entry vector pDONR207 and a kit (Gateway for BP reaction). TM BP Clonase TM and Gateway for LR reactions TM LR Clonase TM ) were purchased from Thermo Fisher Scientific. When amplifying the target genes (CUA82 gene and CUA83 gene respectively), the primers used were:

[0062] CDS_AT5G52750_F(CUA82-OE):5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCATGCCTCCAATGAAAGCTGTG-3'

[0063] CDS_AT5G52750_R(CUA82-OE):5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCACATAATCACACAATTTGATTC-3'

[0064] CDS_AT5G52760_F(CUA83-OE):5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCATGACCGCAAAGAACGCTGT-3'

[0065] CDS_AT5G52760_R(CUA83-OE):5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCACATAATCACACAATTTGG-3'

[0066] The amplified target gene is connected to the entry vector pDONR207 through the BP reaction, and further transferred to the overexpression vector (final vector) pMDC32 through the LR reaction, and the recombinant overexpression vector is constructed. The operation method during the BP reaction and the LR reaction is in accordance with the instructions of the corresponding kit. After the recombinant overexpression vector is constructed, it needs to be sequenced, and the recombinant overexpression vector with correct sequencing is selected to transform Agrobacterium GV3101, and the Agrobacterium GV3101 carrying the recombinant overexpression vector is further used to transform Arabidopsis.

[0067] In this embodiment, when detecting the expression level of CUA82 gene in Arabidopsis thaliana, the qPCR method is used to first reverse transcribe the extracted Arabidopsis thaliana RNA, and then perform qPCR using the reverse transcription product (cDNA) as a template. The kit used for reverse transcription is Plus All-in-one 1st Strand cDNA Synthesis SuperMi x (gDNAPurge) (Cat. No. E047), the kit used for qPCR is: SYBR qPCR SuperMix Plus (Cat. No. E096).

[0068] The primers used in qPCR were:

[0069] AT5G52760-qPCR-F (SEQ ID NO.5): 5'-AGTCGATGATGTTAAAACCACCT-3'

[0070] AT5G52760-qPCR-R (SEQ ID NO.6): 5'-GAATTGGTACGCGTAGTTCATC-3'

[0071] AT5G52750-qPCR-F (SEQ ID NO.7): 5'-GAAACCGGCTCCACCTAAAC-3'

[0072] AT5G52750-qPCR-R(SEQ ID NO.8):5'-CATAATCACACAATTTGATTCG-3'

[0073] ACTIN-qPCR-F(SEQ ID NO.9):5'-GACCAGCTCTTCCATCGAGAA-3'

[0074] ACTIN-qPCR-R (SEQ ID NO.10): 5'-CAAACGAGGGCTGGAACAAG-3'

[0075] The internal reference gene is ACTIN. The reaction system for qPCR is shown in Table 2.

[0076] Table 2

[0077]

[0078]

[0079] The reaction procedure was carried out according to the instructions of the kit.

[0080] The expression levels of CUA82 gene in cua82-1 and CUA82-OE are shown in Figure 6 As shown in Figure 2, the expression levels of the CUA83 gene in cua83-1 and CUA83-OE strains are shown in Figure 2. Figure 7 shown.

[0081] Considering that there may be functional redundancy between CUA82 and CUA83, the inventors knocked out the CUA82 gene in the cua83-1 single mutant background in which the CUA83 gene function was completely lost to create the cua82-2 / cua83-1 double mutant. The partial sequence comparison of the CUA82 gene between the double mutant and the cua83-1 single mutant showed that the double mutant had a CUA82 gene with a CUA83 gene with ... Figure 8 shown.

[0082] The cua83-1 single mutant purchased from the Arabidopsis SALK mutant library was used as a material. When the CUA82 gene was specifically knocked out, the CRISPR / Cas9 system was used. The vector used was a recombinant vector containing an sgRNA sequence. The original vector of the recombinant vector was a pHEE401E vector. The sgRNA sequence (SEQ ID NO.11) for specific targeting of the CUA82 gene inserted (contained) on the pHEE401E vector was: GCATTCACGAGGAAAGAATC. The conventional operation method of CRISPR / Cas9 knockout was used for knockout. After successful knockout (the CUA82 gene and the CUA83 gene in the knockout strain were not expressed, that is, the knockout was successful), the cua82-2 / cua83-1 double mutant was obtained.

[0083] Furthermore, the spores of the powdery mildew UCSC1 were inoculated into the 4-week-old cua82-1 mutant, cua83-1 mutant, cua82-2 / cua83-1 double mutant, CUA82-OE strain, and CUA83-OE strain, and the wild-type Arabidopsis thaliana Col-0 was inoculated with the powdery mildew UCSC1 spores as a control. After two weeks, the disease resistance phenotypes of these Arabidopsis thaliana were observed. The phenotypic observation results are shown in Figure 2. Fig. 9As shown in the results, the leaves of cua82-1 and cua83-1 single mutants showed more local necrotic spots than Col-0, accompanied by less powdery mildew growth. The reason for the appearance of necrotic spots is that the hypersensitive response in the plant immune response is activated. This is a strategy for plants to prevent the further reproduction and expansion of pathogens by stimulating spontaneous necrosis of local invaded cells. In other words, the more necrotic spots, the stronger the disease resistance of the plant. However, cua82-2 / cua83-1 had more local necrotic spots than the single mutants and less powdery mildew growth, indicating that there was partial functional redundancy between the CUA82 and CUA83 genes. In contrast, the leaves of the CUA82-OE and CUA83-OE overexpression lines had almost no local necrotic spots visible to the naked eye. Compared with Col-0, the CUA82-OE and CUA83-OE overexpression lines showed more powdery mildew growth. The above results indicate that the CUA82 and CUA83 genes negatively regulate the powdery mildew resistance of Arabidopsis.

[0084] The cua82-1 mutant, cua83-1 mutant, cua82-2 / cua83-1 double mutant, CUA82-OE strain, and CUA83-OE strain, all of which were 3 weeks old, were inoculated with Pseudomonas sp. DC3000. Fig.10 The colony counts on the 0th day and 3 days after inoculation with Pseudomonas DC3000 are shown. Different letters indicate significant differences between genotypes (P<0.01, one-way ANOVA). As shown in the figure, 3 days after inoculation, cua82-1 and cua83-1 single mutants showed a significantly disease-resistant phenotype compared with Col-0, and the disease-resistant phenotype of the cua82-2 / cua83-1 double mutant was more significant than that of the single mutant. In contrast, the CUA82-OE and CUA83-OE overexpression strains showed a significantly susceptible phenotype.

[0085] The cua82-1 mutant and cua83-1 mutant in the present embodiment are mutants that T-DNA insertion causes the corresponding gene function to lose. In some other embodiments, when it is necessary to regulate the disease resistance of Arabidopsis thaliana, especially to regulate the disease resistance to powdery mildew microorganisms and pseudomonas microorganism infection, the sequence of the CUA82 gene and the CUA83 gene provided in the present embodiment can be used to design a recombinant vector (i.e., CRISPR / Cas9 gene knockout vector) for CRISPR / Cas9 gene knockout and transform Arabidopsis thaliana, to knock out the CUA82 gene or CUA83 gene in Arabidopsis thaliana, or to knock out the CUA82 gene and the CUA83 gene, so that the expression of the corresponding gene in Arabidopsis thaliana is reduced, and the disease resistance of Arabidopsis thaliana is further enhanced. Alternatively, the expression of the CUA82 gene and the CUA83 gene in Arabidopsis thaliana can be reduced by the RNAi method, i.e., a recombinant vector for RNAi can be constructed to transform Arabidopsis thaliana plants.

[0086] According to the above results, the homology between CUA82 and CUA83 reached 78.01%, and both have the function of regulating plant immunity. Both CUA82 and CUA83 genes are from Arabidopsis, and the orthologous genes of these two genes also have the function of regulating plant immunity in theory.

[0087] The CUA82 gene and CUA83 gene have proteins with highly similar amino acid sequences in economic crops (such as Brassica rapa, Brassicanapus and Raphanus sativus), and the genes encoding these proteins are orthologous genes of CUA82 and CUA83. Figure 11 to Figure 13 The amino acid sequence of CUA82 gene is compared with the amino acid sequence of its related proteins in economic crops such as turnip, rape and radish. The orthologous genes (proteins encoded) of CUA82 gene in these economic crops are involved in regulating the immune process of plants.

[0088] Among them, the amino acid sequence of a protein with homology in turnip is shown in SEQ ID NO.12:

[0089] MTAKKAVLQLSVHDEKIRKKAFVTVSRSQGVTSITMDDKTGKMTVVGEVDTPVLVMKLRKLCNAEIVSVEVVKPPEKKPEPAKPAPAKPDTTKPAEIVAFPVTHMNYPYQYHSSYANSHYQPYGNSRVVVEEPNTCVLM

[0090] The amino acid sequence of a protein with homology in rapeseed is shown in SEQ ID NO.13:

[0091] MTAKKAVLQLSVHDERIRKKAFVTVSRSQGVTSITMDDKTGKMTVVGEVDTPVLVMKLRKLCNAEIVSVEVVKPPEKKPEPAKPAPAKPDTAKPAEIVAFPVTHMNYPYQYHSSFANSHYQPYGNSRVVVEEPNTCVLM

[0092] The amino acid sequence of a protein with homology in radish is shown in SEQ ID NO.14:

[0093] MTAKKAVLQLSVHEERIRKKAFVTVSRSPGVSSITMDDKTGKMTVVGEVDVPVLVMKLRKLCNAEIVSVEVVKPPEKKPEPAKPDPAKPAEIVAYPVTHMNYSYQYHSSYANSHYQPFGNSRVVVEEPNTCVLM

[0094] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A CUA gene related to plant immune regulation, characterized in that: The nucleotide sequence of the CUA gene includes the nucleotide sequence of the AT5G52750 gene and / or the AT5G52760 gene, the nucleotide sequence of the AT5G52750 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the AT5G52760 gene is shown in SEQ ID NO.

2. The CUA gene is used to regulate the disease resistance of plants.

2. A protein encoded by the CUA gene related to plant immune regulation, characterized in that: The protein is encoded by the AT5G52750 gene or the AT5G52760 gene. The amino acid sequence of the protein encoded by the AT5G52750 gene is shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by the AT5G52760 gene is shown in SEQ ID NO.

4.

3. A direct homologous gene of the CUA gene related to plant immune regulation, characterized in that: The orthologous gene is the orthologous gene of the CUA gene in economic crops as claimed in claim 1.

4. The orthologous gene according to claim 3, characterized in that The economic crop comprises at least one of Brassicarapa, Brassica napus and Raphanus sativus; the amino acid sequence of the protein encoded by the orthologous gene in Brassica rapa is shown as SEQ ID NO.12, the amino acid sequence of the protein encoded by the orthologous gene in rapeseed is shown as SEQ ID NO.13, and the amino acid sequence of the protein encoded by the orthologous gene in radish is shown as SEQ ID NO.

14.

5. The application of CUA gene is characterized by: The application is: using the CUA gene as claimed in claim 1 or the orthologous gene of the CUA gene as claimed in claim 3 for molecular breeding of disease resistance in plants.

6. The use according to claim 5, characterized in that: The disease-resistant molecular breeding is to improve the resistance of plants to infection by powdery mildew microorganisms of the genus Golovinomyces and / or infection by microorganisms of the genus Pseudomonas.

7. The use according to claim 5 or 6, characterized in that: When conducting molecular breeding for plant disease resistance, a recombinant vector is constructed to transform the plant to reduce the expression level of the AT5G52750 gene and / or the AT5G52760 gene in the plant, thereby achieving the purpose of improving the disease resistance of the plant.

8. The use according to claim 7, characterized in that: The recombinant vector is a CRISPR / Cas9 gene knockout vector used to knock out the AT5G52750 gene and / or the AT5G52760 gene in a plant, and the original vector of the recombinant vector is pHEE401 E.

9. The use according to claim 8, characterized in that: The recombinant vector for specifically knocking out the AT5G52750 gene in a plant is connected to a sgRNA sequence that specifically targets the AT5G52750 gene, and the sgRNA sequence is shown in SEQ ID NO.

11.

10. The use according to claim 5, characterized in that: The plant is any one of Arabidopsis thaliana, turnip, radish and rapeseed.

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