Protein BrHIS4 interacting with heat-resistance regulatory gene BrAK3 in Chinese cabbage and its application

Abstract

The present invention provides an interacting protein BrHIS4 of the BrAK3 gene of Chinese cabbage, which is significantly induced to express under high temperature stress. Overexpression of the BrHIS4 single gene or the BrAK3 and BrHIS4 double genes in yeast enhances the ability of yeast cells to withstand high temperatures. When the BrHIS4 single gene or the BrAK3 and BrHIS4 double genes of Chinese cabbage are silenced under high temperature, the degree of wilting of the silenced plants increases, the stability of the cell membrane decreases, and the antioxidant capacity weakens, indicating that BrHIS4 can enhance the high temperature resistance of Chinese cabbage alone or by interacting with BrAK3 to form a module. The results of the present invention accumulate basic theories for studying the mechanism of responding to high temperature signals, and also provide genetic resources for breeding new varieties of heat-resistant Chinese cabbage, which has good potential application value.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and in particular relates to a protein BrHIS4 interacting with a heat-resistant regulatory gene BrAK3 of flowering cabbage and an application thereof. Background Art

[0002] Chinese cabbage (Brassica campestris L.ssp.chinensis var.utilis Tsenet Lee), also known as Chinese cabbage, is a one- or two-year-old herbaceous plant of the genus Brassica in the family Cruciferae. It is a specialty of southern my country, and is derived from the long-term selection and cultivation of Chinese cabbage subspecies and Chinese cabbage subspecies. Chinese cabbage likes coolness, and the optimal growth temperature is 15-25℃. In high temperature weather above 30℃, the growth and development of Chinese cabbage will be affected, resulting in small and poor quality cabbage, and a significant decrease in single plant weight and commercial yield. In recent years, high temperatures in summer have lasted for a long time, especially in southern China, where extreme high temperatures exceeding 40℃ have frequently occurred. High temperature has become one of the important factors restricting the normal growth of Chinese cabbage in the region.

[0003] At present, there are few studies on the heat stress resistance of Chinese cabbage. For example, patent CN108934814B discloses a method for identifying the heat resistance of Chinese cabbage, which includes the following steps: setting different sowing periods from November to December of the current year to April of the next year, sowing the test varieties and control varieties at intervals; setting the Chinese cabbage of the same sowing period in a high temperature adverse environment; using the plant height, spread, number of leaves, main stalk height and main stalk diameter of the Chinese cabbage at the same sowing period as heat resistance indicators, and using the flowering percentage as the heat resistance index to evaluate the heat resistance of the Chinese cabbage; the control varieties include early-maturing varieties and late-maturing varieties with known heat resistance; when the flowering percentage of the test variety is similar to that of the early-maturing variety with known heat resistance, the heat resistance is strong; when the flowering percentage of the test variety is similar to that of the late-maturing variety with known heat resistance, the heat resistance is weak. Patent CN113348992A discloses a method for evaluating the heat resistance of Chinese cabbage, comprising the following steps: determining the optimal high temperature stress germination temperature of Chinese cabbage, wherein the optimal high temperature stress germination temperature is screened through the indicators: relative germination rate, relative germination potential, relative germination index and relative vitality index; obtaining the heat resistance evaluation index value of Chinese cabbage at the optimal high temperature stress germination temperature, wherein the heat resistance evaluation index value includes the relative germination rate, relative germination potential, relative germination index, relative vitality index, relative sprout length, relative sprout thickness, relative sprout fresh weight, relative sprout dry weight, relative root length, relative root thickness, relative root fresh weight and relative root dry weight of the sprouts; calculating the grading evaluation value, comprehensive grading value D and grading coefficient GC of each Chinese cabbage strain through the heat resistance evaluation index value; the method has the advantages of short identification cycle, high efficiency and low cost, and is suitable for large-scale evaluation and screening of Chinese cabbage strains.

[0004] The above existing technologies have achieved the purpose of preliminarily understanding, identifying and evaluating the heat resistance of Chinese cabbage germplasm resources, but there are relatively few studies on the molecular mechanism of Chinese cabbage's response to high temperature stress. For example, patent CN116732053B discloses the meiosis-related gene BrASY2, and patent CN117887683B discloses the aspartate kinase gene BrAK3. These two genes can effectively regulate the tolerance of Chinese cabbage to high temperature, laying a theoretical foundation for studying the mechanism of plant response to high temperature signals.

[0005] However, the functions of most heat-responsive genes in Chinese cabbage are still unclear. In order to cultivate new varieties of heat-resistant Chinese cabbage, technicians in this field need to further explore and research to find more heat-responsive genes in Chinese cabbage. Summary of the invention

[0006] In view of the deficiencies in the prior art and actual needs, the present invention provides a protein BrHIS4 that interacts with the heat-resistance regulatory gene BrAK3 of Chinese flowering cabbage.

[0007] Specifically, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a Chinese flowering cabbage heat-resistant gene BrHIS4, the nucleotide sequence of the heat-resistant gene is shown in SEQ ID NO.1.

[0009] In a second aspect, the present invention provides a protein encoded by the Chinese cabbage heat-resistant gene BrHIS4, whose amino acid sequence is shown in SEQ ID NO.2.

[0010] In a third aspect, the present invention provides an expression vector containing the Chinese flowering cabbage heat-resistant gene BrHIS4.

[0011] In a fourth aspect, the present invention provides a cell line containing the Chinese flowering cabbage heat-resistant gene BrHIS4.

[0012] In a fifth aspect, the present invention provides a host bacteria containing the Chinese cabbage heat-resistant gene BrHIS4.

[0013] In a sixth aspect, the present invention provides a method for constructing a heat-resistant Chinese cabbage silencing system, which uses a homologous recombination method to construct a 170 bp fragment of the CDS non-conserved segment of the BrHIS4 gene and its complementary fragment into a pK7GWIW vector, thereby obtaining a BrHIS4 gene silencing vector pK7GWIW-BrHIS4, and then the silencing vector pK7GWIW-BrHIS4 is transferred to Agrobacterium GV3101 to obtain a BrHIS4 gene silenced plant;

[0014] The 170 bp fragment of the CDS non-conservative segment of the BrHIS4 gene and its complementary fragment replaced the ccdB gene of the pK7GWIW plasmid. The specific position of the ccdB gene was between attR1 and attR2 after the 35S promoter.

[0015] Furthermore, the nucleotide sequence of the 170 bp long fragment of the CDS non-conserved segment of the BrHIS4 gene is shown in SEQ ID NO.4.

[0016] In a seventh aspect, the present invention provides the use of the Chinese cabbage heat-resistant gene BrHIS4 in regulating the heat resistance of Chinese cabbage.

[0017] Specifically, the heat resistance of Chinese cabbage is reduced by inhibiting or silencing the gene BrHIS4; or the heat resistance of Chinese cabbage is improved by overexpressing the gene BrHIS4.

[0018] In an eighth aspect, the present invention provides an application of the heat-resistant gene BrHIS4 of Chinese cabbage in regulating the heat resistance of Chinese cabbage. Specifically, the heat resistance of Chinese cabbage is reduced by simultaneously inhibiting or silencing the BrAK3 and BrHIS4 genes; or the heat resistance of Chinese cabbage is improved by simultaneously overexpressing the BrAK3 and BrHIS4 genes.

[0019] Among them, the nucleotide sequence of the BrAK3 gene is shown in SEQ ID NO.3.

[0020] The present invention provides an interacting protein BrHIS4 of the BrAK3 gene of Chinese cabbage, which is significantly induced to express under high temperature stress. Overexpression of the BrHIS4 single gene or the BrAK3 and BrHIS4 double genes in yeast enhances the ability of yeast cells to withstand high temperatures. When the BrHIS4 single gene or the BrAK3 and BrHIS4 double genes are silenced under high temperature, compared with the empty vector control plants and the BrAK3 single gene silenced plants, the BrHIS4 single gene or the BrAK3 and BrHIS4 double gene silenced plants have aggravated wilting, reduced cell membrane stability, and weakened antioxidant capacity. The above results indicate that BrHIS4 can enhance or reduce the high temperature resistance of Chinese cabbage alone or by interacting with BrAK3 to form a module. The results of the present invention accumulate basic theories for studying the mechanism of responding to high temperature signals, and also provide genetic resources for breeding new varieties of heat-resistant Chinese cabbage, which has good potential application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a transcriptional expression analysis diagram of the BrHIS4 gene in Example 2 in the heat-resistant material CX1-7 and the heat-sensitive material CX7-3.

[0022] Figure 2This is an analysis chart of the relative expression of the BrHIS4 gene in Example 2 in the heat-resistant material CX1-7 and the heat-sensitive material CX7-3.

[0023] Figure 3 This is a diagram of the analysis of the cis-acting elements of the BrHIS4 gene in Example 3.

[0024] Figure 4 It is the survival rate of yeast cells overexpressing BrHIS4 gene under high temperature stress in Example 4.

[0025] Figure 5 The expression of the BraAK3 gene in the silenced and control Chinese flowering cabbage leaves under high temperature stress in Example 5.

[0026] Figure 6 The expression of BrHIS4 gene in the silenced and control Chinese flowering cabbage leaves under high temperature stress in Example 5.

[0027] Figure 7 These are the results of measuring the water content in the leaves of the silent and control Chinese flowering cabbage under high temperature stress in Example 5.

[0028] Figure 8 These are the results of determining the MDA content in the leaves of the silenced and control Chinese flowering cabbage under high temperature stress in Example 5.

[0029] Fig. 9 These are the results of measuring the relative conductivity of the leaves of the silenced and control Chinese flowering cabbage under high temperature stress in Example 5.

[0030] Fig.10 These are the results of measuring the H2O2 content in the leaves of the silenced and control Chinese flowering cabbage under high temperature stress in Example 5.

[0031] Fig.11 These are the results of measuring the SOD antioxidant enzyme activity in the leaves of the silenced and control Chinese flowering cabbage under high temperature stress in Example 5.

[0032] Fig.12 This is the expression analysis of the key antioxidant enzyme gene BrMn-SOD in the silenced and control Chinese cabbage leaves under high temperature stress in Example 5.

[0033] Fig.13 This is the expression analysis of the heat-resistant marker gene BrHsfA2 in the silenced and control Chinese flowering cabbage leaves under high temperature stress in Example 5.

[0034] Fig.14 This is the expression analysis of the heat-resistant marker gene BrHsp90 in the silenced and control Chinese flowering cabbage leaves under high temperature stress in Example 5. DETAILED DESCRIPTION

[0035] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0036] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0037] When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs.

[0038] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product instructions shall be followed.

[0039] The examples involve the addition amounts, contents and concentrations of various substances, wherein the percentages described therein, unless otherwise specified, are all by mass percentages.

[0040] Unless otherwise specified, the experimental materials and reagents used in the following examples are all consumables and reagents that can be obtained from conventional commercial channels.

[0041] The Chinese cabbage materials used in the following examples were all provided by the Leafy Vegetable Research Laboratory of the Vegetable Research Institute of Hainan Academy of Agricultural Sciences.

[0042] The Chinese cabbage heat-resistant material CX1-7 and heat-sensitive material CX7-3 (CX1-7 and CX7-3 have been disclosed in patent publication document CN113348992A and are currently stored in the Vegetable Research Institute of Hainan Academy of Agricultural Sciences) involved in the following examples have all been identified under high temperature stress at the seedling stage and natural high temperature in the field.

[0043] The plant phenotypes involved in the following examples, such as MDA (malondialdehyde) content, leaf electrolyte permeability (relative conductivity), H2O2 content, cell membrane stability, SOD antioxidant enzyme activity, etc. are all common knowledge in the plant field, and their detection methods are all conventional detection methods in the field, so they are not described in detail below.

[0044] The information on the heat-resistance regulatory gene BrAK3 of Chinese cabbage and the construction of its gene silencing system involved in the following examples has been disclosed in patent CN117887683B.

[0045] Example 1: Source of BrHIS4 gene

[0046] In the early stage, the BrAK3 protein was used as bait, and the yeast two-hybrid technique was used to screen and identify the target genes of the BrAK3 gene regulating the high temperature stress response of Chinese cabbage. The genes of the interacting proteins obtained by the screening were cloned, and the screened target genes were verified by yeast two-hybrid rotation and bimolecular fluorescence complementation technology. Finally, it was determined that BrHIS4 might be the interacting protein of BrAK3. Subsequently, BrHIS4 was used as the object for subsequent functional studies.

[0047] The full-length CDS sequence of the BrHIS4 gene used is 813 bp in length, and the specific sequence (SEQ ID NO.1) is:

[0048] ATGATACAGGTTTTGAGGTACACATCAGAGAATGTCTTTGTACCACTAACCGTTGGAGGTGGTATTAGAGATTTTACAGATGCTACCGGCAGGTACTATTCTAGTTTGGAAGTTGCTGCTGAGTATTTCAGATCCGGTGCTGATAAGATCTCCATAGGAAGTGACGCTGTTTATGCTGCGGAAGAGTTTATAAAATCAGGGGCGAAGACAGGAAAGAGTAGTTTAGAACAAATATCCAGAGTTTATGGAAACCAGGCAGTGGTTGTAAGTATCGATCCTCGTAGAGTTTATGTGAACGATCCTGATGATGTGCCATACAAAGTCATCAAAGTAACTAACCCAGGTCCGAATGGGGAAGAATACGCATGGTATCAGTGCACGGTAAGTGGAGGACGAGAAGGTCGGCCTATTGGAGCGTATGAGCTTGCAAAAGCGGTTGAAGAGTTAGGTGCTGGTGAAATTCTACTGAACTGCATAGACTGTGATGGTCAAGGAAAAGGATTTGACACAGACTTGGTGAAGCTCATATCTGATTCTGTAGGCATACCGGTTATCGCAAGCAGTGGAGCAGGCACTCCTGAGCACTTTTCTGAGGTGTTTGAGACGACTAATGCATCTGCTGCTCTTGCTGCCGGCATTTTCCCACCGGAAAGAGGTACCCATCCAGTCTGTGAAAGAGCACTTGCTAGAGAAGAGCATAGAAGTCAGGATGTAAGAAGTTCCGTTTCTGTTTCAAGACTTCACGTGTTACAATTATTCATGTTACACTTCCAACAAGATCGATTCTTATTTTTCAATAAGTTATTATTATAA

[0049] The amino acid sequence of the encoded protein (SEQ ID NO.2) is specifically as follows:

[0050] MIQVLRYTSENVFVPLTVGGGIRDFTDATGRYYSSLEVAAEYFRSGADKISIGSDAVYAAEEFIKSGAKTGKSSLEQISRVYGNQAVVVSIDPRRVYVNDPDDVPYKVIKVTNPGPNGEEYAWYQCTVSGGREGR PIGAYELAKAVEELGAGEILLNCIDCDGQGKGFDTDLVKLISDSVGIPVIASSGAGTPEHFSEVFETTNASAALAAGIFPPERGTHPVCERALAREEHRSQDVRSSVSVSRLHVLQLFMLHFQQDRFLFFNKLLL

[0051] Example 2: Transcriptional expression and relative expression analysis results of BrHIS4 gene under high temperature stress

[0052] The high temperature stress treatment temperature used in this embodiment is 37°C, and the treatment time is 6h (for specific treatment, refer to "(Pang Qiangqiang et al. Transcriptome analysis and transcription factor screening of Chinese cabbage leaves in response to high temperature stress [J / OL]. Molecular Plant Breeding: 1-23[2023-05-24].)").

[0053] pass Figure 1 and Figure 2 It can be found that before high temperature stress (0h), the transcriptional expression level of BrHIS4 gene in the heat-sensitive material CX7-3 and the heat-resistant material CX1-7 was low; after 6h of high temperature stress, the transcriptional expression level of BrHIS4 gene in CX1-7 increased rapidly, which was 34.07 times that before high temperature stress, while the expression level in CX7-3 was only 1.48 times that before high temperature stress; the expression level of BrHIS4 gene in CX1-7 after 6h of high temperature stress was 31.85 times that in CX7-3. The results of relative expression analysis were similar to those of transcriptional expression analysis. The above results show that BrHIS4 gene in Chinese cabbage is involved in the response of Chinese cabbage to high temperature stress.

[0054] Example 3: Analysis of cis-acting elements of BrHIS4 gene

[0055] The online tool PlantCARE (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) was used to analyze the cis-acting elements of the BrHIS4 gene. The results are as follows: Figure 3 As shown in Figure 2, it can be found that the BrHIS4 gene contains five high temperature stress response elements, CAAT-box.

[0056] Example 4: High temperature tolerance of yeast overexpressing the BrHIS4 gene

[0057] The CDS of BrHIS4 and BrAK3 were cloned into the yeast expression vector pYES-Dest52 using Gateway recombination technology to obtain recombinant vectors pYES-BrHIS4, pYES-BrAK3 and pYES-BrHIS4 / BrAK3. The recombinant vector and the empty vector pYES-Dest52 were respectively transferred into the competent cells of the yeast G19 strain, and the transformants were screened on the leucine-deficient medium (SD / Leu-). A sterile water suspension of yeast cells was prepared, treated at 50°C for 0, 25, 35, and 40 minutes, and then transferred to 30°C for recovery for 10 minutes. 100 μL of the bacterial suspension was spread and inoculated on a SD / Leu- medium plate, and a bacterial suspension that had not been treated with high temperature was spread as a control. After culturing at 30°C for 48 hours, the number of colonies was counted and the survival rate was calculated. The statistical results are as follows Figure 4 The results showed that the survival rate of yeast cells overexpressing BrHIS4 single gene or BrHIS4 and BrAK3 double genes was significantly higher than that of the control at 25min, 35min and 40min of stress, indicating that overexpression of this gene enhanced the ability of yeast cells to withstand high temperatures.

[0058] Example 5: Analysis of BrHIS4 and BrAK3 synergistically regulating high temperature resistance in Chinese cabbage

[0059] CN117887683B has disclosed the relevant information of the heat-resistant regulatory gene BrAK3 of Chinese cabbage, and its nucleotide sequence (SEQ ID NO.3) is as follows:

[0060] ATGCGGCCAGCTAGCGAAGGGAACATTCCTGTAAGGGTTAAGAACTCTTACAATCCACTGCACCAGGAACTCTCATCACTAGATCAAGAGACATGAGCAAGGCTGTACTGACCAGCATCGTTCTGAAACGTAATGTTACCATGTTGGACATCACTAGCAACCGTATGCT CGGTCAATATGGTTTCCTTTCCAAGGTGCTCTCCACATTTGAAAAGATGGGCATATCTGTAGATGTTGTTGCAACCAGCGAAGTTAGCGTTTCCTTGACATTGGATCCTTCAAAGTTCTGCAGCAAAGAGTTAATTCAACAGGAGATTAATCACATGGTAGAGGAACTGG AGAAGATTGCTTCGGTAAACCTGCTCAGCACAGATCAATTATCTCACTCATTGGAAATTTTCAGAGATCATCATCTTTCATATTAGAGAAGGGCTTCCGAGTTCTTAGAACCAATGGAATCAATGTCCAGATGATCTCTCAGGGTGCATCTAAGGTAAACATCTCGCTG ATAGTGAATGATGATGAGGCAGAGCATTGTGTGAAGGCTCTCCATTCAGCCTTCTTCGAGACAGGAACCTCCAAAGCTGTTCCTCAGATAGGGCGTCTAGCTACTACACCTCTGCCTTGTCGTGAAAACCTCTTGGTGCAACCTGACCTGCGAGATCTACAAGTAGTTTGA

[0061] In order to further explore the regulatory relationship of BrHIS4 on high temperature resistance of Chinese cabbage, we replaced the ccdB gene of the pK7GWIW plasmid (purchased from Shanghai Yuanmu Biotechnology Co., Ltd.) with a 170bp fragment (CDS644-813bp) of the non-conserved region of the BrHIS4 gene and its complementary fragment through gene recombination exchange. The specific location of the ccdB gene is between attR1 and attR2 behind the 35S promoter, thus obtaining the BrHIS4 gene silencing vector (pK7GWIW-BrHIS4).

[0062] The length of the non-conserved segment of the BrHIS4 gene used in the present invention is 170 bp.

[0063] The specific nucleotide sequence (SEQ ID NO. 4) is: 5'-CACCGGAAAGAGGTACCCATCCAGTCTGTGAAAGAGCACTTGCTAGAGAAG AGCATAGAAGTCAGGATGTAAGAAGTTCCGTTTCTGTTTCAAGACTTCACGTGT TACAATTATTCATGTTACACTTCCAACAAGATCGATTCTTATTTTTCAATAAGTT ATTATTATAA-3'.

[0064] The gene silencing vector pK7GWIW-BrHIS4 was transformed into Agrobacterium GV3101. After cultivation, shaking, and resuspension, an equal amount of static bacterial solution was drawn with a 1 mL syringe and injected into the pinholes on the back of the leaves of the empty vector and BrAK3-silenced Chinese cabbage plants (the front of the Chinese cabbage leaves were blocked by hand). The injected Chinese cabbage seedlings were cultured in the dark for 12 hours and then cultured at 22°C for 3 days. Plants with consistent growth were selected and treated with 37°C for 6 hours, and the control was 25°C. The phenotypic changes of the plants before and after the treatment were recorded by taking pictures, and the leaves of the Chinese cabbage seedlings before and after high temperature stress were collected for physiological indexes and related gene expression analysis.

[0065] The identification of BrHIS4 gene silenced plants and gene expression analysis were performed as follows: RNA was extracted using the TRIzol kit (Invitrogen TRIzol), cDNA synthesis and purification were completed using the SuperScript IV First-Strand cDNA Synthesis System Kit (Invitrogen), and related gene expression analysis was performed. The cDNA product was diluted 3 times and used as a template for PCR amplification for qRT-PCR amplification. The reagents used for qRT-PCR amplification were Premix Ex TaqTM kit (TAKARA), real-time fluorescence quantitative PCR instrument is BioRad CFX96 TM (BioRad, USA).

[0066] The qRT-PCR amplification reaction system was 5 μL 2×SYBR Premix Ex TaqTM, 0.5 μL forward primer (10 μmol / L), 0.5 μL reverse primer (10 μmol / L), 4 μL cDNA template (diluted 3 times), and 10 μL sterile distilled water.

[0067] qRT-PCR reaction program: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s, 60℃ retardation for 20s, 72℃ extension for 20s, 40 cycles. Each reaction was repeated 3 times.

[0068] Gene expression analysis was performed using 2-△△Ct The calculation and drawing were performed by the method, with BrCKⅠ gene as the internal reference gene (Pang Qiangqiang, et al. Internal reference gene of high temperature stress in Chinese cabbage and its screening method and application [P]. Hainan Province: CN115927296A.).

[0069] The primers for BrCKⅠ gene expression amplification are:

[0070] F:5'-GTCTAACTTCCTCGCCACCT-3',

[0071] R:5'-GATTCGACGACAAGCCAGAC-3';

[0072] The primers for amplifying the expression of BrHIS4 gene are:

[0073] F:5'-TGGGGAAGAATACGCATGGT-3',

[0074] R:5'-CTCCACTGCTTGCGATAACC-3';

[0075] The primers for amplifying the expression of BrAK3 gene are:

[0076] F:5'-GGCAGATGGGTTCAGATTCG-3',

[0077] R:5'-AAGAGGCGTGCTCATACTCA-3';

[0078] The primers for BrHsfA2 expression amplification are:

[0079] F:5'-GACCATTGCTCCAAGACACC-3',

[0080] R:5'-GGAAGAGACGGTGACCTACG-3';

[0081] The primers for amplifying the expression of BrHsp90 gene are:

[0082] F:5'-CTGCAACAGTCTCACCCTCT-3',

[0083] R:5'-CTGAGAGCAAGTGGTGGAAC-3';

[0084] The primers for BrMn-SOD expression amplification are:

[0085] F:5'-AGATGAGTGCTGAAGGTGCT-3';

[0086] R:5'-CTGATTGGCGGTTGTGTCAA-3'.

[0087] The physiological indexes were determined by the following methods: relative conductivity was determined by conductivity meter method, hydrogen peroxide content (H2O2) was determined by spectrophotometer method, malondialdehyde content (MDA) was determined by thiobarbituric acid method, and superoxide dismutase activity (SOD) was determined by nitroblue tetrazolium method. All the above indexes were determined three times.

[0088] Genetic test results such as Figure 5 and Figure 6 The expression levels of BrAK3 and BrHIS4 genes in the leaves of gene-silenced Chinese cabbage were significantly lower than those in the control plants, indicating that the BrHIS4 single gene and BrAK3 and BrHIS4 double gene silencing systems were successfully constructed.

[0089] The empty vector control plants, BrAK3 silenced plants, BrHIS4 silenced plants, and BrAK3 and BrHIS4 double gene silenced plants were treated at 25℃ and 37℃ for 6h, respectively. The results showed that under high temperature stress, compared with the empty vector control plants and BrAK3 silenced plants, after silencing BrHIS4, the wilting degree of BrHIS4 single gene, BrAK3 and BrHIS4 double gene silenced plants increased, and the water content of the plants was significantly reduced ( Figure 7 ).

[0090] Under high temperature stress, compared with the empty vector control plants and BrAK3 silenced plants, the high temperature resistance of BrHIS4 single gene, BrAK3 and BrHIS4 double gene silenced plants was significantly reduced, as reflected in: MDA content, leaf electrolyte permeability and H2O2 content increased significantly ( Figure 8-10 ), cell membrane stability decreased; SOD antioxidant enzyme activity decreased ( Fig.11 ), the expression of the key antioxidant enzyme gene BrMn-SOD decreased ( Fig.12 ), the antioxidant capacity was weakened; the expression of heat resistance marker genes BrHsfA2 and BrHsp90 was inhibited ( Fig.13 and Fig.14 ). Therefore, we concluded that BrHIS4 can regulate the high temperature resistance of Chinese cabbage alone or through interaction with BrAK3.

[0091] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. Application of the heat-resistant gene BrHIS4 of Chinese cabbage in regulating the heat resistance of Chinese cabbage, characterized in that: The heat resistance of Chinese flowering cabbage is reduced by inhibiting or silencing the BrHIS4 gene, and the nucleotide sequence of the heat resistance gene BrHIS4 of Chinese flowering cabbage is shown in SEQ ID NO.

1.

2. Application of the heat-resistant gene BrHIS4 of Chinese cabbage and the heat-resistant regulatory gene BrAK3 of Chinese cabbage in regulating the heat resistance of Chinese cabbage, characterized in that: The heat tolerance of Chinese cabbage was reduced by simultaneously inhibiting or silencing the BrHIS4 and BrAK3 genes; The nucleotide sequence of the Chinese flowering cabbage heat-resistant gene BrHIS4 is shown in SEQ ID NO.1, and the nucleotide sequence of the BrAK3 gene is shown in SEQ ID NO.3.

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