A rubber tree protein receptor kinase HbBAK1 and its application

Abstract

The present invention relates to a kind of rubber tree protein receptor kinase HbBAK1 and its application. Specifically relate to a kind of protein receptor kinase gene HbBAK1 related to rubber tree drought resistance, not only verify the stress resistance of HbBAK1 gene with yeast, obtain HbBAK1 has drought resistance function in the drought environment mediated by polyethylene glycol ‑ 6000 (PEG ‑ 6000), and the research of the gene function is carried out on Arabidopsis thaliana transformed with HbBAK1 gene, verify that the tolerance of transgenic Arabidopsis thaliana to drought stress is improved. The gene can be used in the drought-resistant transgenic research of rubber tree, and the protein receptor kinase gene HbBAK1 comes from rubber tree, has less impact on the environment, and provides new gene resources for improving the drought resistance of rubber tree.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a rubber tree protein receptor kinase HbBAK1 and an application thereof. Background Art

[0002] The Brazilian rubber tree (Hevea brasiliensis Muell.Arg.) belongs to tropical trees, originating in the Amazon forest of South America. It is the only commercial source of natural rubber and occupies an important position in the development of the world economy. The high elasticity, plasticity, insulation, air tightness and wear resistance of natural rubber make it an important strategic material and widely used in national defense, transportation, medical treatment and other aspects. However, in recent years, droughts have occurred repeatedly in Guangxi, Hainan, Yunnan and Guangdong in my country's rubber planting areas, which have brought serious impacts and losses to the growth and production of rubber trees. Therefore, in-depth research on the drought resistance of rubber trees is carried out to find out genes related to the drought resistance of rubber trees, which is of great significance to the rational allocation of strains in my country's rubber planting areas, as well as to the utilization of water resources and the improvement of the ecological environment.

[0003] BAK1 is a receptor kinase that has been studied in depth. The extracellular domain is responsible for sensing signals, transmembrane and transmitting extracellular signals to the cell, and the intracellular kinase domain transmits signals to the downstream. BAK1 mainly participates in multiple signal pathways by binding to different receptor kinases to form co-receptors to regulate plant growth and development and abiotic stress responses. BAK1 participates in immune responses and can bind to the receptor kinase FLS2, then phosphorylate the cytoplasmic receptor kinase BIK1, and regulate the innate immune response mediated by the flagellar protein flg22. Knocking out the BAK1 gene reduces the plant's ability to bind to flg22, resulting in abnormalities in the initial response of flg22, indicating that BAK1 plays a positive regulatory function in the early signal transduction process of the corresponding pathogen molecular pattern and plays a negative regulatory role in cell death. Overexpression of the millet receptor protein kinase gene BAK1 can significantly inhibit cell death in transgenic Arabidopsis, and BAK1 plays an important role in the stimulation immune response that depends on R protein. The receptor-like protein RLP30 and the receptor-like kinase SOBIR1 and BAK1 jointly participate in the immune response mediated by tetrasomal trophic fungi. BAK1 forms a receptor-co-receptor complex with PSKR1 to sense the extracellular peptide PSK signal, and forms a complex with the transmembrane channel CNGC17 and AHA to regulate the elongation of the primary root of the seedling. BAK1 antagonizes ABI1, a negative regulator of the ABA signaling pathway. The water loss rate of the bak1 mutant is higher than that of the wild type, and it is insensitive to ABA-induced stomatal closure. This is mainly because BAK1 regulates the ABA-induced stomatal closure process of leaves by combining with ABI1 or SnRK2.6. Therefore, conducting drought resistance-related research on rubber trees and identifying the related protein receptor kinase HbBAK1 can provide key candidate genes for regulating rubber tree yield. Summary of the invention

[0004] In view of the above-mentioned defects in the prior art, the present invention proposes a rubber tree protein receptor kinase HbBAK1 and applications thereof to solve the problems raised by the above-mentioned background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The rubber tree protein receptor kinase HbBAK1 gene was cloned using the whole genome cDNA of rubber tree variety 'Reyan 73397' as a template.

[0007] A rubber tree protein receptor kinase HbBAK1, the length of the HbBAK1 gene is 1845bp, and the cDNA sequence is shown as SEQ ID NO:1.

[0008] Preferably, the HbBAK1 gene is obtained by amplifying using the primer pair shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0009] Preferably, the sequences of primers SEQ ID NO: 3 and SEQ ID NO: 4 are as follows:

[0010] HbBAK1-F: 5′-ATGGAGCGAAAGATGTGG-3′

[0011] HbBAK1-R: 5′-TCATCTAGGACCAGACAAC-3′

[0012] Preferably, the amino acid residues encoded by the HbBAK1 gene are 614aa, and the specific amino acid sequence is shown in SEQ ID NO:2.

[0013] Preferably, the rubber tree protein receptor kinase HbBAK1 is used in the preparation of drought-resistant transgenic plants.

[0014] Preferably, the plants include rubber and Arabidopsis thaliana.

[0015] Preferably, a method for constructing drought-resistant transgenic plants comprises the following steps:

[0016] (1) Extracting total RNA from rubber tree leaves and reversely transcribing to obtain the first-chain cDNA;

[0017] (2) Using the first strand of cDNA as a template and SEQ ID NO: 3 and SEQ ID NO: 4 as primers, the cDNA sequence of the protein receptor kinase HbBAK1 gene was obtained by PCR amplification reaction. The amplified product was subjected to 1.2% agarose gel electrophoresis to detect the target band. The target band was cut out under ultraviolet light and purified and recovered. The pEASY-Blunt Zero vector was connected and transformed into DH5α Escherichia coli competent cells for colony PCR detection, and the product was sent to Genetech for sequencing verification. The results showed that the HbBAK1 protein receptor kinase belongs to the plant receptor kinase SERK family.

[0018] (3) Using the cDNA sequence of the HbBAK1 gene as a template, HbBAK1-HindIII-F: actatagggaatattaagcttATGGAGCGAAAGATGTGGGC and HbBAK1-XhoI-R: cctctagatgcatgctcgagTCTAGGACCAGACAACTCATCTGG as primers, the amplified product was double-digested with HindIII and XhoI and then connected to the expression vector after the same restriction digestion, and the plasmid carrying the cDNA sequence of the HbBAK1 gene was transformed into the Agrobacterium GV3101 strain by electroporation;

[0019] (4) Use transgenic technology to transform Agrobacterium carrying the transformation plasmid into the target plant to obtain a transgenic stable genetic plant.

[0020] Preferably, the expression vector carrying the target gene can be introduced into plant cells by conventional biotechnology methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, etc.

[0021] Preferably, the pEASY-Blunt Zero vector is purchased from Beijing Quanshijin Biotechnology Co., Ltd.; and the DH5α Escherichia coli competent cells are purchased from Beijing Bomade Gene Technology Co., Ltd.

[0022] The amino acid sequences of rubber tree (HbBAK1) were compared with those of Arabidopsis thaliana (AtBAK1), durian (DzBAK1), cassava (MeBAK1), citrus (CsBAK1), poplar (PeBAK1), castor (RcBAK1), jatropha (JcBAK1), sweet cherry (PaBAK1), grape (VrBAK1) and rice (OsBAK1). The results showed that the average homology was as high as 72.63%.

[0023] The expression levels of HbBAK1 in tissues and in different germplasms were detected respectively, and the expression results showed that: leaf>flower>root>stem>latex>branch, PR107>Reyan 73397>Zhanshi 327-13.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The relevant function research of rubber tree protein receptor kinase gene HbBAK1 not only verified the stress resistance of HbBAK1 gene with yeast, obtained that HbBAK1 has drought resistance function in the drought environment mediated by polyethylene glycol-6000 (PEG-6000), but also studied the function of this gene by transforming HbBAK1 gene into Arabidopsis thaliana, verified that the tolerance of transgenic Arabidopsis thaliana to drought stress was improved. This gene can be used in the drought resistance transgenic research of rubber tree. This protein receptor kinase gene HbBAK1 comes from rubber tree and has little impact on the environment, providing new gene resources for improving the drought resistance of rubber tree. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The results of amino acid sequence homology comparison between rubber tree and Arabidopsis, durian, cassava, citrus, poplar, castor, jatropha, sweet cherry, grape and rice;

[0027] Figure 2 is the expression level of HbBAK1 in different tissues;

[0028] Figure 3 is the expression level of HbBAK1 in different germplasms;

[0029] Figure 4 The growth difference between the recombinant plasmid HbBAK1-pYES2-INVSC1 and the yeast empty vector pYES2-INVSC1 under liquid culture conditions;

[0030] Figure 5 The growth difference between the recombinant plasmid HbBAK1-pYES2-INVSC1 and the yeast empty vector pYES2-INVSC1 under solid culture conditions. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments, but is not intended to limit the scope of the present invention. Unless otherwise specified, the embodiments are all based on conventional test conditions or the conditions recommended by the manufacturer's instructions.

[0032] RNAprep Pure polysaccharide and polyphenol plant total RNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.;

[0033] RevertAid Reverse Transcriptase kit was purchased from Thermo Fisher Scientific (China) Co., Ltd.;

[0034] Phanta Max Super-Fidelity DNA Polymerase was purchased from Nanjing Novozyme Biotechnology Co., Ltd.;

[0035] Example 1 Cloning of Hevea brasiliensis protein receptor kinase HbBAK1 gene

[0036] 1. The rubber tree protein receptor kinase HbBAK1 gene was cloned using the whole genome cDNA of the rubber tree variety 'Reyan 73397' as a template. The total RNA of rubber tree leaves was extracted using the RNAprep Pure polysaccharide and polyphenol plant total RNA extraction kit, and the first chain of cDNA was obtained by reverse transcription using the RevertAid Reverse Transcriptase kit.

[0037] 2. Use cDNA as template, HbBAK1-F and HbBAK1-R as primers, and Phanta Max Super-Fidelity DNA Polymerase as amplification enzyme for PCR amplification. The method is as follows:

[0038] Primer sequence: HbBAK1-F: 5′-ATGGAGCGAAAGATGTGG-3′

[0039] HbBAK1-R: 5′-TCATCTAGGACCAGACAAC-3′

[0040] PCR amplification reaction system: cDNA-1μL, primer F, R 2μL each, 2×Phanta Max Buffer-25μL, Phanta Max Super-Fidelity DNA Polymerase-1μL, add ddH2O to 50μL.

[0041] PCR amplification reaction program: pre-deformation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 2 min, for a total of 35 cycles; extension at 72°C for 5 min.

[0042] 3. The amplified product was subjected to 1.2% agarose gel electrophoresis to detect the target band. The target band was cut out under ultraviolet light and purified and recovered. The pEASY-Blunt Zero vector was connected and transformed into DH5α Escherichia coli competent cells. Colony PCR detection was performed and the product was sent to Genetron Biotech for sequencing verification.

[0043] 4. The results showed that HbBAK1 protein receptor kinase belongs to the plant receptor kinase SERK family member, its cDNA sequence length is 1845bp, and the encoded amino acid sequence is 614aa.

[0044] 5. The amino acid sequences of rubber tree (HbBAK1) were compared with those of Arabidopsis thaliana (AtBAK1), durian (DzBAK1), cassava (MeBAK1), citrus (CsBAK1), poplar (PeBAK1), castor (RcBAK1), jatropha (JcBAK1), sweet cherry (PaBAK1), grape (VrBAK1) and rice (OsBAK1). The average homology was as high as 72.63% ( Figure 1 ).

[0045] Example 2 Analysis of the expression of rubber tree protein receptor kinase HbBAK1 in different tissues and germplasms

[0046] Fluorescence quantitative PCR was used to detect the expression of HbBAK1 in different tissues and germplasms of rubber trees. Six tissues of rubber tree variety 'Reyan 73397' were collected: root, stem, leaf, flower, branch, and gum, and rubber tree varieties 'Reyan 73397', 'PR107', and 'Zhanshi 327-13'. Total RNA of each sample was extracted and reverse transcribed to obtain cDNA.

[0047] qRT-PCR used the ChamQ SYBR Color qPCR Master Mix reaction system: 10 μl of ChamQ SYBR Color qPCR Master Mix, 0.4 μl of forward fluorescent quantitative primer, 0.4 μl of reverse fluorescent quantitative primer, 1 μl of diluted cDNA, and 8.2 μl of ddH2O, a total of 20 μl.

[0048] The qRT-PCR reaction procedure was as follows: 95°C pre-denaturation for 10 s; 95°C for 10 s, 60°C annealing for 30 s, for a total of 40 cycles; 95°C for 15 s, 60°C for 1 min, 95°C for 15 s. HbActin (Genbank: HQ260674.1) was used as the internal reference primer and repeated three times. The results were analyzed by 2 -ΔΔCt The relative expression was calculated by the method.

[0049] qHbActin-F: 5′-GATGTGGATATCAGGAAGGA-3′

[0050] qHbActin-R: 5′-CATACTGCTTGGAGCAAGA-3′

[0051] qHbBAK1-F: 5′-TTGCTTCGTCTACGTGGCTT-3′

[0052] qHbBAK1-R: 5′-TGTGACTCCTGACGCTCTCT-3′

[0053] qRT-PCR results showed that the expression level of HbBAK1 in tissues was leaf>flower>root>stem>latex>branch( Figure 2 ), the expression levels in different germplasms were PR107>Reyan 73397>Zhanshi 327-13 ( Figure 3 ).

[0054] Example 3 Yeast verification of stress resistance of HbBAK1 gene

[0055] Using HbBAK1-Peasy plasmid as template and recombination primer HbBAK1-HindIII-F:

[0056] actatagggaatattaagcttATGGAGCGAAAGATGTGGGC, HbBAK1-XhoI-R:

[0057] cctctagatgcatgctcgagTCTAGGACCAGACAACTCATCTGG was PCR amplified, and the amplification reaction procedure was the same as in Example 1. The amplified product was recombinantly ligated with the yeast expression vector pYES2 double-digested with HindIII and XhoI to obtain the HbBAK1-pYES2 recombinant plasmid.

[0058] The recombinant plasmid HbBAK1-pYES2 and the empty plasmid pYES2 were respectively transferred into the yeast competent INVSC1, and the yeast single clone was picked for PCR positive detection to confirm the successful transformation. First, the growth difference between the recombinant yeast HbBAK1-pYES2-INVSC1 and the yeast empty pYES2-INVSC1 under liquid induction culture conditions was compared. After picking the positive single colony and culturing it in liquid culture medium for 24 hours, the bacteria were collected by centrifugation and resuspended in liquid induction medium and liquid induction medium containing 15% PEG-6000 to OD 600 0.2, pipette 10ml of bacterial solution into a 50ml centrifuge tube and culture for 36h before measuring OD 600 The results showed that the OD values ​​of recombinant yeast and yeast empty vector in the medium without stress treatment were 600 There is no significant difference in the values ​​( Figure 4 Black column), OD of HbBAK1-pYES2-INVSC1 bacterial culture under drought treatment in liquid induction medium containing 15% PEG-6000 600 The value is significantly higher than the OD of yeast empty pYES2-INVSC1 bacterial solution 600 value( Figure 4 Gray column). This indicates that HbBAK1 has drought resistance function in a drought environment mediated by PEG-6000.

[0059] At the same time, the growth differences between the recombinant yeast HbBAK1-pYES2-INVSC1 and the yeast empty pYES2-INVSC1 under solid culture conditions were compared. The positive single clones were picked and cultured in liquid selection medium for 24 hours. After centrifugation, the cells were collected and resuspended in ddH2O and 15% PEG-6000 solution to OD 600 Take 1 ml and culture for 24 hours, and adjust the OD 600 The starting concentration was 2, and a 5-fold concentration gradient dilution was performed with this as the starting concentration. 3 μL of each gradient was taken for yeast spotting, and each treatment was repeated three times. After culturing for 2 days, the growth of yeast colonies under different treatment conditions was observed.

[0060] The results showed that there was no difference in the growth of HbBAK1-pYES2-INVSC1 and pYES2-INVSC1 under no stress treatment. Under the drought condition simulated by 15% PEG-6000, the bacterial solution was diluted to 5 -2 Afterwards, the growth results of HbBAK1-pYES2-INVSC1 yeast colonies were significantly better than those of pYES2-INVSC1 yeast colonies ( Figure 5 ), the results are consistent with the OD of liquid induction culture conditions 600 The results were consistent with those of the previous study, indicating that HbBAK1 has drought resistance function in a PEG-6000-mediated drought environment.

[0061] Example 4 Obtaining Arabidopsis thaliana plants expressing the protein receptor kinase HbBAK1 gene and identifying their drought resistance

[0062] The cDNA sequence of the HbBAK1 gene prepared in Example 1 was used as a template for amplification, HbBAK1-HindIII-F: actatagggaatattaagcttATGGAGCGAAAGATGTGGGC and

[0063] HbBAK1-XhoI-R:cctctagatgcatgctcgagTCTAGGACCAGACAACTCATCTGG was used as primer. The amplified product was double-digested with HindIII and XhoI and then connected to the expression vector digested with the same enzymes. The plasmid carrying the cDNA sequence of the HbBAK1 gene was transformed into the Agrobacterium GV3101 strain by electroporation. The HbBAK1 gene was transformed into transgenic Arabidopsis plants by Agrobacterium-mediated method for overexpression. The transgenic positive plants were identified, and the T3 generation positive plants containing the target gene were screened and stress resistance analyzed. The results are shown in Table 1.

[0064] Table 1 Phenotypic comparison between Arabidopsis thaliana plants transfected with the protein receptor kinase HbBAK1 gene and the wild type

[0065]

[0066] As shown in Table 1, the rhizome length of transgenic Arabidopsis overexpressing the HbBAK1 gene is longer than that of the wild type, which is conducive to obtaining deep nutrients. In a water-deficient and dry environment, it mainly relies on the extension of the rhizome to absorb nutrients from the deep soil layer. At the same time, the leaf and stem width of the overexpressed Arabidopsis is also wider than that of the wild type, which is conducive to photosynthesis, increasing the transpiration area and improving water use efficiency. In general, the tolerance of transgenic Arabidopsis overexpressing the HbBAK1 gene to drought stress is significantly improved, providing a good basis for the application of the HbBAK1 gene in other plants. Compared with wild-type Arabidopsis plants, the HbBAK1 transgenic strain has a well-developed root system and significantly improved drought tolerance in the soil.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An application of a rubber tree protein receptor kinase HbBAK1 in preparing drought-resistant transgenic plants, characterized in that: The HbBAK1 gene cDNA sequence is shown in SEQ ID NO: 1; The HbBAK1 gene is obtained by amplifying the primer pair shown in SEQ ID NO: 3 and SEQ ID NO: 4; The method for constructing the drought-resistant transgenic plant comprises the following steps: (1) Extracting total RNA from rubber tree leaves and reversely transcribing to obtain the first-chain cDNA; (2) Using the first strand of cDNA as a template and SEQ ID NO: 3 and SEQ ID NO: 4 as primers, a PCR amplification reaction was performed to obtain the cDNA sequence of the protein receptor kinase HbBAK1 gene; (3) using the electroporation method to transform the plasmid carrying the cDNA sequence of the HbBAK1 gene into Agrobacterium; (4) Using transgenic technology to transform the target plant with Agrobacterium carrying the transformation plasmid to obtain a transgenic stable genetic plant; The drought-resistant transgenic plants are rubber and Arabidopsis thaliana.

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