Plant immune regulation-related CUA genes and their orthologs and their application in disease resistance molecular breeding

Abstract

This invention discloses the CUA gene, related to plant immune regulation, and its orthologous genes, as well as their application in molecular breeding for disease resistance. The nucleotide sequence of the CUA gene includes the nucleotide sequences of the AT5G52750 gene and / or the AT5G52760 gene, as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The application of the CUA gene involves using the CUA gene or its orthologous genes in molecular breeding for plant disease resistance. This invention provides new gene resources and theoretical basis for molecular breeding for plant disease resistance.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology. Specifically, it relates to the CUA gene, which is related to plant immune regulation, and its orthologous genes, and their application in molecular breeding for disease resistance. Background Technology

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

[0003] In Arabidopsis thaliana, there exists a class of mutants whose gene mutations lead to the continuous activation of the immune system, known as "spontaneous immune mutants." These mutants are an important resource for discovering immune regulatory genes.

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

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

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] A CUA gene related to plant immune regulation, wherein the nucleotide sequence of the CUA gene includes the nucleotide sequences 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 plant disease resistance.

[0008] The protein encoded by the CUA gene related to plant immune regulation, wherein 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.

[0009] The orthologous gene of the CUA gene related to plant immune regulation, wherein the orthologous gene is the orthologous gene of the aforementioned CUA gene in economic crops. An "orthologous gene" is a homologous gene existing in different species, originating from a common ancestral gene and diverging through speciation events.

[0010] The aforementioned orthologous genes, the economic crops include at least one of Brassica rapa, Brassica napus, and Raphanus sativus; the amino acid sequence of the protein encoded by the orthologous gene in Brassica rapa is shown in SEQ ID NO.12, the amino acid sequence of the protein encoded by the orthologous gene in Brassica napus is shown in SEQ ID NO.13, and the amino acid sequence of the protein encoded by the orthologous gene in Raphanus sativus is shown in SEQ ID NO.14.

[0011] The application of the CUA gene refers to using the aforementioned CUA gene or its orthologous gene for molecular breeding of plant disease resistance. The AT5G52750 and AT5G52760 genes share 78.01% homology, and experiments have shown that both have the ability to regulate Arabidopsis immunity. When a paralogous gene or orthologous gene shares more than 78% homology with the AT5G52750 or AT5G52760 genes, it is reasonable to expect that the paralogous gene or orthologous gene also has the ability to regulate plant immunity.

[0012] The aforementioned application, specifically the disease-resistant molecular breeding, aims to enhance plant resistance to infections caused by *Golovinomyces* and / or *Pseudomonas* microorganisms. "Powdery mildew microorganisms" refers to the pathogens causing powdery mildew.

[0013] In the above applications, when conducting molecular breeding for plant disease resistance, recombinant vectors are constructed to transform plants, thereby reducing the expression levels of the AT5G52750 and / or AT5G52760 genes in the plants, thus improving plant disease resistance.

[0014] In the above application, the recombinant vector is a CRISPR / Cas9 gene knockout vector used to knock out the AT5G52750 gene and / or the AT5G52760 gene in plants, and the original vector of the recombinant vector is pHEE401E. The CRISPR / Cas9 gene knockout vector is the recombinant vector used in the CRISPR / Cas9 gene knockout system. The original vector of this recombinant vector is pHEE401E, and the recombinant vector contains an sgRNA sequence that specifically targets the AT5G52750 or AT5G52760 gene. This sgRNA sequence helps the relevant enzymes in the CRISPR / Cas9 gene knockout system to accurately locate the gene containing this "target sequence," thereby achieving gene knockout. If it is necessary to knock out both the AT5G52750 and AT5G52760 genes in the same plant, two CRISPR / Cas9 gene knockout vectors can be designed. One vector contains a specific sgRNA sequence targeting the AT5G52750 gene, and the other vector contains a specific sgRNA sequence targeting the AT5G52760 gene. After transforming the plant with these two CRISPR / Cas9 gene knockout vectors, the AT5G52750 and AT5G52760 genes can be knocked out, thereby reducing the expression levels of these two genes.

[0015] The above application involves a recombinant vector for specifically knocking out the AT5G52750 gene in plants, which is linked with an sgRNA sequence specifically targeting the AT5G52750 gene, as shown in SEQ ID NO.11.

[0016] In the above applications, 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. This invention discloses for the first time CUA genes related to plant immune regulation, particularly CUA82 and CUA83, which play a crucial role in regulating plant resistance to infections by *Golovinomyces* and *Pseudomonas*. This discovery provides new gene resources and theoretical basis for molecular breeding of plant disease resistance. By reducing the expression level of CUA genes in plants, plant resistance to powdery mildew and *Pseudomonas* can be significantly improved. This method provides a new approach to address the current problem of scarce disease-susceptibility gene resources in plant disease resistance breeding.

[0019] 2. This invention also provides a specific method for silencing CUA genes 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 this invention exist in a variety of economic crops and have broad applicability and application prospects. Attached Figure Description

[0021] Figure 1 The growth phenotypes of Col-0, bon1-1, camta1 / 2 / 3, cpr1-2 and cbp60b-13 weeks of seedling age in the embodiments of the present invention;

[0022] Figure 2 Venn diagrams of upregulated genes (log2≥1.0) in the bon1-1, camta1 / 2 / 3, cpr1-2 and cbp60b-1 mutants in this embodiment of the invention;

[0023] Figure 3 The results of measuring the expression level of the immunomarker gene PR1 in each mutant strain 24 hours after inoculation with Pseudomonas DC3000 in this embodiment of the invention;

[0024] Figure 4 Photographs of the disease phenotypes of various mutants 14 days after inoculation with powdery mildew pathogens in this embodiment of the invention;

[0025] Figure 5 Nucleotide sequence alignment of CUA82 and CUA83 genes in this embodiment of the invention;

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

[0027] Figure 7 The expression levels of the CUA83 gene in the Col-0, cua83-1 mutants and CUA83-OE overexpression lines 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 this embodiment of the invention;

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

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

[0031] Figure 11 Sequence comparison of the CUA82 gene in this invention and its highly homologous gene in turnip;

[0032] Figure 12 Sequence comparison of the CUA82 gene in this invention and its highly homologous gene in rapeseed;

[0033] Figure 13 The sequence comparison of the CUA82 gene in this invention and its highly homologous gene in radish. Detailed Implementation

[0034] "Spontaneous immune mutants" are a type of mutant whose immune system is constitutively activated, and they often exhibit certain growth and developmental defects. Figure 1 This figure compares the growth of wild-type Arabidopsis thaliana and its spontaneously immunized mutants at 3 weeks of age. Col-0 represents wild-type Arabidopsis thaliana, while bon1-1, camta1 / 2 / 3, cpr1-2, and cbp60b-1 represent four different spontaneously immunized mutants (all mutants were obtained from the Arabidopsis SALK mutant library). The figure shows that the growth and development of the spontaneously immunized mutants are significantly worse than that of the wild-type.

[0035] To discover new immune regulatory genes, the inventors performed transcriptome sequencing on Col-0 and four spontaneously immunized mutants: bon1-1, camta1 / 2 / 3, cpr1-2, and cbp60b-1. They screened for 90 differentially expressed genes whose expression was upregulated in all four mutants (screening criteria: log2 ≥ 1.0, meaning the expression level of a certain gene differed by at least two-fold between the wild type and the mutant). Figure 2As shown in the figure. These genes were named CUA (Common up-regulated gene in autoimmunity) genes, and each gene was named sequentially in ascending order of gene number, i.e., these 90 differentially expressed genes were named CUA1 to CUA90. Among them, 36 genes (40%) are reported immune regulatory genes, and the functions of 54 genes in immune regulation have not been reported. Information on the 36 known CUA genes with immune regulatory functions is shown in Table 1.

[0036] Table 1

[0037]

[0038]

[0039] Fifty-four CUA genes, not yet reported to have immunomodulatory functions, were selected as candidate genes. CUA gene deletion mutants were collected for disease resistance screening. Some lines identified as homozygous mutants were inoculated with *Pseudomonas syringae* DC3000 and *Golovinomyces cichoracearum* UCSC1, respectively. Twenty-four hours after inoculation with *Pseudomonas syringae* DC3000, the expression level (relative expression level) of the immune marker gene PR1 in each mutant line was measured. Fourteen days after inoculation with *Golovinomyces cichoracearum* UCSC1, the resistance / susceptibility phenotype of each mutant line to powdery mildew (i.e., whether it showed resistance or susceptibility) was observed. The results of PR1 expression level measurement and the observation of resistance / susceptibility phenotypes were combined to screen and identify the disease resistance of each mutant line.

[0040] Meanwhile, Pseudomonas DC3000 and Pseudomonas ulmoides UCSC1 were inoculated into pad4-1 as susceptible control material (pad4-1 is an Arabidopsis thaliana mutant with PAD4 gene deletion, which has no disease resistance), and two pathogenic microorganisms were inoculated into cpr1-2 as resistant control material (cpr1-2 is an Arabidopsis thaliana mutant with CPR1 gene deletion, which has strong disease resistance).

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

[0042] Fourteen days after inoculation with the powdery mildew pathogen, both the cua82-1 and cua83-1 mutants exhibited a phenotype that was more resistant to powdery mildew than the Col-0 mutant. Figure 4 As shown in the figure, compared with the wild-type strain Col-0 and the susceptible strain pad4-1, the cua82-1 mutant and cua83-1 mutant have significantly fewer white lesions, and the number of white lesions on the cua82-1 mutant and cua83-1 mutant is similar to that of the resistant strain cpr1-2.

[0043] Furthermore, nucleotide sequence comparison revealed that CUA82 and CUA83 shared a sequence identity of up to 78.01% (e.g., Figure 5 As shown in the figure, this indicates that the two may be homologous genes.

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

[0045] The primer sequence used in the "three-primer method" for identification is:

[0046] CS852976_LP (for identification of CUA82-1): 5'-TTTCAAAATCAACTCAATGCATG-3';

[0047] CS852976_RP (for CUA82-1 identification): 5'-CAGGAAGAAAGCGTTTGTCAC-3';

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

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

[0050] BP (for identification of CUA82-1 and CUA83-1): 5'-ATTTTGCCGATTTCGGAAC-3'.

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

[0052] ATGCCTCCAATGAAAGCTGTGTTGCAATTGAGCATTCACGAGGAAAGAATCAGGAAGAAAGCGTTTGTCACCGTTTCTCGATGTCCAGGTGTTACTTCGATAACAATGGATGACAAAACCGGGAAAATGACAGTAGTTGGTGAAGTTGATGTACCGGTTATCGTGATGAAGCTAAGGAAGCTATGTAACACAGAGCTTGTTTCGGTTGAA GTTGTTAAACCACCTGAGAAAAAAGCCTGAACCAGAGAAACCGGCTCCACCTAAACCAGCTCCAGCTCCAGCTAAACCGGCTGAAATTGTTGCCTGGCCGGTTCAGATGAACAACCCTTACCAATACAATCCTGCCTATGCAAATTCTTACTATCAACCATATGGGAACTCTAGATTTGTAACGGACGAATCAAATTGTGTGATTATGTGA

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

[0054] ATGACCGCAAAGAACGCTGTGTTGCAATTGAGTATTCACGAGGAGAGAACCAGGAAGAAAGCGCTTGTCACCGTTTCTCGATTTTCAGGAGTTACTTCGATAACAATGGACAAAAGTGGGAAAATGACAATAGTTGGAGAAGTTGATGTACCAGCTGTCGTGATGAAGCTAAGGAAGCTATGTAACACAG AGATCGTTCAGTCGATGATGTTAAACCACCTGTTAAAAAGCCTGAACCTGAGAAACCAGCTGAATCTATTGCATATCCTGTTCCGATGAACTACGCGTACCAATTCAATCCTGCCTATGCAAATTCTTACTATCATCAACCATACGGAAATTGTAGAGTGGTAGACGAACCAAATTGTGTGATTATGTGA

[0055] The amino acid sequence (SEQ ID NO.3) of the protein encoded by the CUA82 gene (also known as 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 (also known as 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 two Arabidopsis thaliana lines, CUA82-OE and CUA83-OE, respectively, overexpressing the CUA82 and CUA83 genes. For the construction of the overexpressing Arabidopsis lines, Agrobacterium GV3101 carrying the recombinant overexpression vector was used to transform Arabidopsis Col-0 via pollen tube infection. The original vector for the recombinant overexpression vector was pMDC32. After transformation into Arabidopsis with the recombinant overexpression vector, positive transgenic lines were screened using hygromycin resistance plates, and the expression level of the target gene in the positive transgenic lines was further detected by RT-qPCR. The successfully overexpressed positive transgenic lines were self-crossed for two generations to obtain homozygous T3 lines.

[0061] The recombinant overexpression vector was constructed using the Gateway method, employing the introductory vector pDONR207 and the Gateway kit (for the BP reaction). TM BP Clonase TM and Gateway for LR reaction TM LR Clonase TM All primers were purchased from Thermo Fisher Scientific. The primers used for amplifying the target genes (CUA82 and CUA83 genes, respectively) 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 was ligated to the entry vector pDONR207 via a backpropagation (BP) reaction, and then further transferred to the overexpression vector (final vector) pMDC32 via a backpropagation (LR) reaction, thus completing the construction of the recombinant overexpression vector. The procedures for the BP and LR reactions were performed according to the instructions of the respective kits. After the recombinant overexpression vector was constructed, it needed to be sequenced. The correctly sequenced recombinant overexpression vectors were then transformed into Agrobacterium GV3101, and further transformed into Arabidopsis thaliana using Agrobacterium GV3101 carrying the recombinant overexpression vector.

[0067] In this embodiment, qPCR was used to detect the expression level of the CUA82 gene in Arabidopsis thaliana. First, the extracted Arabidopsis RNA was reverse transcribed, and then qPCR was performed using the reverse transcription product (cDNA) as a template. The kit used for reverse transcription was [kit name missing]. The Plus All-in-one 1st Strand cDNA Synthesis SuperMi x (gDNAPurge) (catalog number E047) kit used for qPCR is: SYBR qPCR SuperMix Plus (item number E096).

[0068] The primers used in qPCR are:

[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 was ACTIN. The reaction system for qPCR is shown in Table 2.

[0076] Table 2

[0077]

[0078]

[0079] The reaction procedure was performed according to the kit's instructions.

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

[0081] Considering the potential functional redundancy between the CUA82 and CUA83 genes, the inventors created the cua82-2 / cua83-1 double mutant by knocking out the CUA82 gene in the cua83-1 single mutant background where the CUA83 gene has completely lost function. The partial sequence alignment of the CUA82 gene between this double mutant and the cua83-1 single mutant is as follows: Figure 8 As shown.

[0082] Using the cua83-1 single mutant obtained from the Arabidopsis SALK mutant library as material, the CUA82 gene was specifically knocked out using a CRISPR / Cas9 system. The vector used was a recombinant vector containing an sgRNA sequence. The original vector of this recombinant vector was the pHEE401E vector, and the sgRNA sequence (SEQ ID NO. 11) inserted into the pHEE401E vector for specifically targeting the CUA82 gene was: GCATTCACGAGGAAAGAATC. The knockout was performed using standard CRISPR / Cas9 knockout procedures. Successful knockout (defined as the absence of expression of both the CUA82 and CUA83 genes in the knockout strain) yielded the cua82-2 / cua83-1 double mutant.

[0083] Furthermore, spores of powdery mildew UCSC1 were inoculated into cua82-1 mutant, cua83-1 mutant, cua82-2 / cua83-1 double mutant, CUA82-OE strain, and CUA83-OE strain, all aged 4 weeks. Simultaneously, wild-type Arabidopsis thaliana Col-0 was inoculated with spores of powdery mildew UCSC1 as a control. The disease resistance phenotype of these Arabidopsis thaliana strains was observed after two weeks. The phenotypic observation results are as follows: Figure 9As shown, both the cua82-1 and cua83-1 single mutants exhibited more localized necrotic spots on their leaves than Col-0, accompanied by less powdery mildew growth. The appearance of necrotic spots is due to the activation of a hypersensitive response in the plant's immune response. This is a strategy employed by plants to prevent further pathogen proliferation by inducing spontaneous necrosis of localized invading cells; in other words, more necrotic spots indicate stronger plant resistance. The cua82-2 / cua83-1 strains showed more localized necrotic spots and less powdery mildew growth than the single mutants, suggesting partial functional redundancy in the CUA82 and CUA83 genes. Conversely, the CUA82-OE and CUA83-OE overexpression lines showed almost no visible localized necrotic spots on their leaves, and compared to Col-0, the CUA82-OE and CUA83-OE overexpression lines exhibited more powdery mildew growth. These results indicate that the CUA82 and CUA83 genes negatively regulate powdery mildew resistance in Arabidopsis thaliana.

[0084] CUA82-1 mutant, CUA83-1 mutant, CUA82-2 / CUA83-1 double mutant, CUA82-OE strain, and CUA83-OE strain, all with a seedling age of 3 weeks, were inoculated with Pseudomonas DC3000. Figure 10 The figure shows the colony counts on day 0 and day 3 after inoculation with *Pseudomonas* DC3000. Different letters indicate significant differences between genotypes (P < 0.01, one-way ANOVA). As shown in the figure, three days after inoculation, the cua82-1 and cua83-1 single mutants exhibited significantly more resistant phenotypes than Col-0, and the cua82-2 / cua83-1 double mutant showed a more significant resistant phenotype than the single mutants. Conversely, the CUA82-OE and CUA83-OE overexpression lines showed significantly susceptible phenotypes.

[0085] In this embodiment, the cua82-1 and cua83-1 mutants are T-DNA insertion mutants that result in the loss of function of the corresponding genes. In other embodiments, when it is necessary to regulate the disease resistance of Arabidopsis thaliana, especially resistance to powdery mildew and Pseudomonas infections, a recombinant vector for CRISPR / Cas9 gene knockout (i.e., a CRISPR / Cas9 gene knockout vector) can be designed based on the sequences of the CUA82 and CUA83 genes provided in this embodiment and transformed into Arabidopsis thaliana to knock out the CUA82 or CUA83 gene, or knock out both CUA82 and CUA83 genes, thereby reducing the expression level of the corresponding genes in Arabidopsis thaliana and further enhancing its disease resistance. Alternatively, RNAi can be used to reduce the expression level of the CUA82 and CUA83 genes in Arabidopsis thaliana, i.e., constructing a recombinant vector for RNAi to transform Arabidopsis thaliana plants.

[0086] The results above show that the CUA82 and CUA83 genes share 78.01% homology, and both have the function of regulating plant immunity. Both CUA82 and CUA83 genes are derived from Arabidopsis thaliana, and theoretically, their orthologous genes also have the function of regulating plant immunity.

[0087] The aforementioned CUA82 and CUA83 genes contain proteins with highly similar amino acid sequences in economic crops (such as turnip *Brassica rapa*, rapeseed *Brassicanapus*, and radish *Raphanus sativus*). The genes encoding these proteins are orthologous genes of CUA82 and CUA83. Figures 11-13 The figures show a comparison of the amino acid sequence encoded by the CUA82 gene with the amino acid sequences of related proteins in the economic crops turnip, rapeseed, and radish. The orthologs (encoded proteins) of the CUA82 gene in these economic crops are involved in regulating plant immune processes.

[0088] The amino acid sequence of a homologous protein from turnip is shown in SEQ ID NO.12:

[0089] MTAKKAVLQLSVHDEKIRKKAFVTVSRSQGVTSITMDDKTGKMTVVGEVDTPVLVMKLRKLCNAEIVSVEVVKPPEKKPEPAKPAPAKPDTTKPAEIVAFPVTHMNYPYQYHSSYANSHYQPYGNSRVVVEEPNTCVLM

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

[0091] MTAKKAVLQLSVHDERIRKKAFVTVSRSQGVTSITMDDKTGKMTVVGEVDTPVLVMKLRKLCNAEIVSVEVVKPPEKKPEPAKPAPAKPDTAKPAEIVAFPVTHMNYPYQYHSSFANSHYQPYGNSRVVVEEPNTCVLM

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

[0093] MTAKKAVLQLSVHEERIRKKAFVTVSRSPGVSSITMDDKTGKMTVVGEVDVPVLVMKLRKLCNAEIVSVEVVKPPEKKPEPAKPDPAKPAEIVAYPVTHMNYSYQYHSSYANSHYQPFGNSRVVVEEPNTCVLM

[0094] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. CUA The application of genes is characterized by, The CUA The gene is at least one of the AT5G52750 gene and the AT5G52760 gene; the sequence of the AT5G52750 gene is shown in SEQ ID NO.1; the sequence of the AT5G52760 gene is shown in SEQ ID NO.2; the application is: to use the... CUA Genes used to enhance plant resistance to powdery mildew Golovinomyces Microbial infection and / or Pseudomonas Pseudomonas This involves molecular breeding for resistance to microbial infection; the plant in question is Arabidopsis thaliana. When conducting molecular breeding for plant disease resistance, recombinant vectors are constructed to transform plants, thereby reducing the expression levels of the AT5G52750 and / or AT5G52760 genes in the plants and thus improving plant disease resistance.

2. The application according to claim 1, 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 the plant, and the original vector of the recombinant vector is pHEE401E.

3. The application according to claim 2, characterized in that, The recombinant vector for specifically knocking out the AT5G52750 gene in plants contains an sgRNA sequence that specifically targets the AT5G52750 gene, as shown in SEQ ID NO. 11.

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