Rubber tree transcription factor hbbes1, cloning method and application

Abstract

The application provides a rubber tree HbBES1 gene, and the nucleotide sequence of the rubber tree HbBES1 gene is shown as SEQ ID NO:1. The rubber tree HbBES1 gene is cloned from the rubber tree for the first time, the gene is subcellularly located in the nucleus, the expression amount in high-yield germplasm of the rubber tree is higher than that in low-yield germplasm, which indicates that HbBES1 is probably related to latex formation and is probably involved in the latex synthesis process of the rubber tree, further research shows that the gene can improve the survival ability of yeast under oxidative stress, which indicates the regulation function of the gene under abiotic stress. The application provides a new candidate gene for improving the stress resistance of yeast and plants and the like, and has important significance for in-depth study on genetic improvement and breeding of the rubber tree.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to a rubber tree transcription factor HbBES1, its cloning method, and its application. Background Technology

[0002] The Brazilian rubber tree is a perennial tree, a typical tropical rainforest plant, native to the equatorial climate zone of the Amazon River basin in Brazil. Natural rubber is the only renewable material among the world's four major industrial materials. Increasing rubber tree yield through genetic improvement is a fundamental measure to improve the self-sufficiency rate of natural rubber. This is also an important strategy for improving the economic benefits of rubber cultivation.

[0003] As rubber becomes increasingly widely used, its molecular biology research is also deepening and developing, with gene expression analysis gradually being applied to reveal the mechanisms of gene expression and regulation in rubber trees. Therefore, the localization, cloning, and function analysis of important trait genes in rubber trees are of great significance for the comprehensive development and utilization of rubber. Abiotic stress severely affects the normal growth and production of rubber trees, representing a critical issue that urgently needs to be addressed in the rubber industry.

[0004] Therefore, by mining rubber tree genes with stress resistance functions and further verifying their regulatory functions under abiotic stresses, it is of great significance for in-depth research on the genetic improvement and breeding of rubber trees. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a rubber tree transcription factor HbBES1, its cloning method, and its application.

[0006] The first aspect of the present invention is to provide a rubber tree HbBES1 gene, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0007] The second aspect of the present invention is to provide a rubber tree transcription factor HbBES1, which is a protein encoded by the rubber tree HbBES1 gene described in the first aspect of the present invention.

[0008] A third aspect of the present invention is to provide the use of the rubber tree HbBES1 gene as described in the first aspect of the present invention, or the rubber tree transcription factor HbBES1 as described in the second aspect of the present invention, in improving the ability of yeast to resist oxidative stress.

[0009] A fourth aspect of the present invention is to provide the use of the rubber tree HbBES1 gene as described in the first aspect of the present invention, or the rubber tree transcription factor HbBES1 as described in the second aspect of the present invention, in improving the ability of yeast to resist H2O2 stress.

[0010] A fifth aspect of the present invention is to provide a recombinant vector or host bacterium containing the coding region of the rubber tree HbBES1 gene as described in the first aspect of the present invention.

[0011] The original vector for the recombinant vector can be a vector commonly used in the field of gene recombination, such as a virus or plasmid. This invention does not limit this. In one specific embodiment of this invention, the original vector uses vector plasmids such as Blunt-Zero, 35S:GFP, or pYES2; however, it should be understood that other plasmids or viruses can also be used.

[0012] A sixth aspect of the present invention is to provide the use of the recombinant vector or host bacteria as described in the fifth aspect of the present invention in enhancing the ability of yeast to resist oxidative stress.

[0013] A seventh aspect of the present invention is to provide the use of the recombinant vector or host bacteria as described in the fifth aspect of the present invention in improving the ability of yeast to resist H2O2 stress.

[0014] The eighth aspect of the present invention is to provide a method for cloning the rubber tree HbBES1 gene described in the first aspect of the present invention: total RNA is extracted from rubber tree leaves, reverse transcribed into cDNA, and PCR amplification is performed using the first strand of cDNA as a template and HbBES1-F:5′-ATGACGTCAGACGGGGCC-3′ and HbBES1-R:5′-TTAACTCCGAGCCTTCCCATT-3′ as primers. The amplification product is then recovered to obtain the gene.

[0015] A ninth aspect of the present invention is to provide a primer pair, said primer pair being HbBES1-F: 5′-ATGACGTCAGACGGGGCC-3′, HbBES1-R: 5′-TTAACTCCGAGCCTTCCCATT-3′; or the primer pair is qHbBES1-F: 5′-CCAAGTCCACTATCTTCATCATTTCC-3′, qHbBES1-R: 5′-GCCATGGATTGTTTGGCAAT-3′; or the primer pair is HbBES1-KpnI-F: acgggggacgagctcggtaccATGACGTCAGACGGGGCC, HbBES1-BamHI-R: catgtcgactctagaggatccACTCCGAGCCTTCCCATTTC; the primer pair is HbBES1-HindIII-F: actatagggaatattaagcttATGACGTCAGACGGGGCC, HbBES1-XhoI-R: cctctagatgcatgctcgagACTCCGAGCCTTCCCATTTC.

[0016] This invention marks the first cloning of the HbBES1 gene from rubber trees. This gene is subcellularly located in the cell nucleus, and its expression level is higher in high-yielding rubber tree germplasm than in low-yielding germplasm, indicating that HbBES1 may be related to latex formation and may participate in the latex synthesis process of rubber trees. Further research shows that this gene can improve the survival ability of yeast under oxidative stress, demonstrating its regulatory function under abiotic stress. This invention provides a new candidate gene for research on improving the stress resistance of yeast and plants, and is of great significance for in-depth research on the genetic improvement and breeding of rubber trees. Attached Figure Description

[0017] Figure 1 This is the nucleotide sequence of the HbBES1 gene from the rubber tree.

[0018] Figure 2 This study shows the expression of the HbBES1 gene in high-yielding and low-yielding rubber tree germplasms.

[0019] Figure 3 Subcellular localization results of the HbBES1 gene in rubber trees.

[0020] Figure 4 The value is the OD600 of the transgenic yeast under H2O2 stress.

[0021] Figure 5 The growth status of recombinant plasmid yeast under oxidative stress. Detailed Implementation

[0022] The present invention will be further described below with reference to the accompanying drawings and specific embodiments to better understand the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0023] Example 1: Gene Cloning of HbBES1

[0024] Leaves of the rubber tree cultivar 'CATAS73397' were used as tissue. Total RNA was extracted from the leaves using the FastPure Plant Total RNA Isolation Kit (Novizan, Nanjing, China). The first strand of cDNA was obtained by reverse transcription using the HiScript II 1st Strand cDNA Synthesis Kit (Novizan, Nanjing, China). Using this as a template, HbBES1 was amplified to its full length using primers HbBES1-F: 5′-ATGACGTCAGACGGGGCC-3′ and HbBES1-R: 5′-TTAACTCCGAGCCTTCCCATT-3′, combined with Phanta Max Master Mix (Novizan, Nanjing, China) high-fidelity amplification enzyme.

[0025] The PCR amplification reaction system is as follows: Phanta Max Master Mix - 25 μL, cDNA - 2 μL, F - 2 μL, R - 2 μL, and finally add ddH2O to 50 μL.

[0026] The amplification reaction program was set as follows: 98 °C pre-denaturation for 3 min, 98 °C denaturation for 15 s, 60 °C annealing for 15 s, 72 °C extension for 150 s, with a cycle number of 35, and a final extension at 72 °C for 10 min.

[0027] After the amplification reaction, the target band was detected by electrophoresis on a 1.5% agarose gel. The target band was purified and recovered using the FastPure Gel DNA Extraction Mini (Novizan, Nanjing, China) purification kit. The recovered product was ligated into the intermediate cloning vector Blunt-Zero (Novizan, Nanjing, China) and transformed into competent E. coli DH5α cells (Novizan, Nanjing, China). After colony PCR detection, positive clones were selected and sent to Qingke Biotechnology Co., Ltd. for sequencing verification. Single clones with correct sequences were preserved in glycerol brine and plasmids were extracted and labeled with the HbMAX2-Blunt plasmid. The obtained CDS sequence of the rubber tree HbBES1 gene is shown in SEQ ID NO:1 and... Figure 1 As shown, the sequence length is 939 bp.

[0028] Example 2: Expression analysis of HbBES1 in rubber tree germplasms with differential latex yield

[0029] Based on the HbBES1 sequence, full-field quantitative PCR primers qHbBES1-F: 5′-CCAAGTCCACTATCTTCATCATTTCC-3′ and qHbBES1-R: 5′-GCCATGGATTGTTTGGCAAT-3′ were designed. The difference in HbBES1 expression levels between high- and low-yielding rubber tree germplasms 'CATAS73397' and 'PR107' was detected using quantitative PCR. Total RNA was extracted from latex samples of both germplasms and reverse transcribed into cDNA. Quantitative PCR was performed using Taq Pro Universal SYBR qPCR Master Mix (Novizan, Nanjing, China). The reaction mixture was as follows: Taq Pro Universal SYBR qPCR Master Mix - 10 μL, qHbBES1-F - 0.4 μL, qHbBES1-R - 0.4 μL, 1 ng / μL cDNA - 1 μL, ddH2O - 8.2 μL. The instrument used for this quantitative PCR was a CFX96 Touch real-time system (Bio-Rayet, California, USA). The reaction procedure was as follows: First, pre-denaturation at 95 ℃ for 30 s; second, denaturation at 95 ℃ for 10 s, followed by annealing at 60 ℃ for 30 s, for 40 cycles. Finally, the instrument's default melting curve was used for data acquisition. Each experiment was performed in triplicate, using HbActin (Genbank: HQ260674.1) as the reference gene. The final results were analyzed using 2... -ΔΔCt The relative expression levels were calculated using the following methods. qHbActin-F: 5′-GATGTGGATATCAGGAAGGA-3′, qHbActin-R: 5′-CATACTGCTTGGAGCAAGA-3′. Quantitative fluorescence analysis showed that the gene expression level of HbBES1 in the high-yielding germplasm 'CATAS73397' was higher than that in the low-yielding germplasm 'PR107'. Figure 2 This suggests that HbBES1 may be related to latex formation and may participate in the latex synthesis process of rubber trees.

[0030] Example 3: Subcellular localization of HbBES1

[0031] Using the HbBES1-Blunt plasmid as a template and 35S:GFP as a vector, homologous recombination primers HbBES1-KpnI-F: acgggggacgagctcggtaccATGACGTCAGACGGGGCC and HbBES1-BamHI-R: catgtcgactctagaggatccACTCCGAGCCTTCCCATTTC were designed and high-fidelity PCR amplification was performed, following the same procedures as in Example 1. The amplified and recovered products were then homologously recombinated with 35S:GFP by double digestion with KpnI and BamHI to obtain the HbBES1-GFP recombinant plasmid, which was then transformed into Agrobacterium tumefaciens GV3101 (Weidi, Shanghai, China). After inoculation on antibiotic-resistant plates and growth for 2 days, bacterial cells were scraped and resuspended in 10 Mm MgCl2 and 120 μM acetylsyleugenol, adjusting the OD600 to approximately 0.8. Healthy *Nicotiana benthamiana* plants, approximately one month old, were selected. Resuspended HbBES1-GFP and GFP empty vectors were injected into the lower epidermal cells of the tobacco plant. After 12 hours of treatment in the dark, the cells were transferred to normal light for 3 days of culture. Subcellular localization of HbBES1 was observed under a laser confocal microscope (Zeiss, Baden-Württemberg, Germany). The results showed that HbBES1 was localized in the cell nucleus (…). Figure 3 ).

[0032] Example 4: Verification of the stress resistance of the HbBES1 gene

[0033] Using the HbBES1-Blunt plasmid as a template and pYES2 as a vector, homologous recombination primers HbBES1-HindIII-F (actatagggaatattaagcttATGACGTCAGACGGGGCC) and HbBES1-XhoI-R (cctctagatgcatgctcgagACTCCGAGCCTTCCCATTTC) were designed and amplified by PCR, following the same method as in Example 1. The amplified product was then digested with pYES2 using HindIII and XhoI for homologous recombination to obtain the HbBES1-pYES2 recombinant plasmid. The recombinant plasmid HbBES1-pYES2 and the empty vector plasmid pYES2 were transformed into competent yeast cells INVSC1. Single yeast clones were picked and colony PCR was performed to verify the success of the transformation. Two methods were used in this experiment to verify the stress resistance of yeast transformed with HbBES1.

[0034] (1) After selecting recombinant plasmid-positive colonies and empty vector colonies, and culturing them in liquid induction medium for 1 day, the colonies were centrifuged at 5000 rpm and the cells were collected. The cells were resuspended in liquid induction medium and liquid induction medium containing 3 mM H2O2 until the OD600 reached 0.2. 5 mL of the bacterial solution was transferred to 50 mL sterile centrifuge tubes and cultured for 2 days. The OD600 value of each tube was then measured. Each treatment was repeated three times. The experimental groups are as follows:

[0035] pYES2 group: yeast transformed with pYES2 empty vector plasmid was cultured in normal liquid induction medium;

[0036] HbBES1p-YES2 group: Yeast transformed with HbBES1-pYES2 recombinant plasmid was cultured in normal liquid induction medium;

[0037] pYES-H2O2 group: yeast transformed with pYES2 empty vector plasmid was cultured in liquid induction medium containing 3 mM H2O2;

[0038] HbBES1-H2O2 group: Yeast transformed with HbBES1-pYES2 recombinant plasmid was cultured in liquid induction medium containing 3 mM H2O2.

[0039] The results showed that there was no significant difference in OD600 values ​​between recombinant plasmid yeast and empty vector yeast under no-stress conditions. However, under oxidative stress conditions formed in liquid induction medium containing 3 mM H2O2, the OD600 value of recombinant plasmid yeast was significantly higher than that of empty vector yeast. Figure 4 (and Table 1), indicating that HbBES1 has a resistance function to oxidative stress.

[0040] Table 1

[0041] deal with Repeat 1 Repeat 2 Repeat 3 average value pYES2 0.884 0.837 0.866 0.86 HbBES1p-YES2 0.916 0.928 0.893 0.91 <![CDATA[pYES-H2O2]]> 0.615 0.625 0.618 0.62 <![CDATA[HbBES1-H2O2]]> 0.744 0.784 0.772 0.77

[0042] Recombinant plasmid-positive colonies and empty vector colonies were cultured in liquid selective medium for 1 day, followed by centrifugation to collect the bacterial cells. The cells were then resuspended in sterile ddH₂O and 3 mM H₂O₂ solution until the OD₆₀ was 0.5. 1 mL of each bacterial culture was aspirated and cultured for 1 day, adjusting the OD₆₀ to 1. A five-fold serial dilution was performed using this initial bacterial culture as the original concentration. 5 μL of each dilution was spotted onto a yeast single-cell culture plate, and each treatment was repeated three times. The experimental groups are as follows:

[0043] pYES2 group: yeast transformed with pYES2 empty vector plasmid was cultured in normal liquid selective medium and liquid selective medium containing 3 mM H2O2, respectively.

[0044] HbBES1-pYES2 group: Yeast transformed with HbBES1-pYES2 recombinant plasmid was cultured in normal liquid selective medium and liquid selective medium containing 3 mM H2O2, respectively.

[0045] After 2 days of cultivation, the growth of yeast colonies under different treatment conditions was observed. The results showed that under no stress conditions, there was no difference in growth between the recombinant plasmid yeast and the empty plasmid yeast. However, under simulated oxidative stress conditions of 3 mM H2O2, after dilution to a certain concentration, the growth of the HbBES1-pYES2 recombinant yeast was superior to that of the pYES2 empty yeast. Figure 5 This result is consistent with the OD600 value under liquid-induced conditions, indicating that HbBES1 has antioxidant function in oxidative stress environments.

[0046] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to this utility model are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. The application of the rubber tree HbBES1 gene or rubber tree transcription factor HbBES1 in improving the ability of yeast to resist oxidative stress, wherein the nucleotide sequence of the rubber tree HbBES1 gene is shown in SEQ ID NO:1, and the rubber tree transcription factor HbBES1 is the protein encoded by the rubber tree HbBES1 gene.

2. Application of the rubber tree HbBES1 gene or rubber tree transcription factor HbBES1 in improving the ability of yeast to resist H2O2 stress; the nucleotide sequence of the rubber tree HbBES1 gene is shown in SEQ ID NO:1, and the rubber tree transcription factor HbBES1 is the protein encoded by the rubber tree HbBES1 gene.

3. The application of a recombinant vector or host bacterium containing the coding region of the rubber tree HbBES1 gene in improving the ability of yeast to resist oxidative stress, wherein the nucleotide sequence of the rubber tree HbBES1 gene is shown in SEQ ID NO:

1.

4. The application of a recombinant vector or host bacterium containing the coding region of the rubber tree HbBES1 gene in improving the ability of yeast to resist H2O2 stress, wherein the nucleotide sequence of the rubber tree HbBES1 gene is shown in SEQ ID NO:1.

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