HbSTMb gene and application thereof in improvement of hevea brasiliensis genetic transformation budding

Abstract

The invention belongs to the technical field of plant genetic engineering, particularly relates to an HbSTMb gene and cloning expression thereof, and further discloses application of the HbSTMb gene in improvement of hevea brasiliensis genetic transformation budding. Research finds that the HbSTMb gene of the rubber tree can effectively promote germination of positive embryos subjected to genetic transformation of the rubber tree, the capacity of further developing the positive embryos of the rubber tree into complete plants can be remarkably promoted by overexpression of the HbSTMb gene, and the positive embryos can easily generate bud points and then easily grow into the complete plants in a seedling emergence culture medium.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically an HbSTMb gene and its cloning and expression, and further discloses its application in improving the budding of rubber trees through genetic transformation. Background Technology

[0002] Natural rubber, originating in Brazil, has always been an important strategic material and reserve resource for my country. Currently, the breeding of new rubber tree varieties in my country still relies mainly on traditional hybridization breeding. However, due to its narrow genetic background, high heterozygosity, and long breeding cycle, the breeding process of Brazilian rubber trees is very slow, which seriously hinders the overall progress of rubber tree breeding. The use of transgenic technology can effectively improve breeding efficiency, shorten the breeding cycle, expand the genetic range of rubber trees, and accelerate the breeding process.

[0003] With the development of biotechnology, the establishment of efficient genetic transformation systems is fundamental to cultivating new rubber tree varieties using bio-breeding techniques such as transgenics and gene editing. Currently, ultrasound-assisted genetic transformation systems for rubber trees achieve a transformation efficiency of up to 1%, but suffer from a high rate of deformed positive embryos, making regeneration difficult. Therefore, simply increasing transformation efficiency to obtain more positive embryos and thus potentially more regenerated positive plants is unlikely to be successful.

[0004] Current research has confirmed that regulating the genes required for shoot tip development during genetic transformation can overcome the problem of positive embryo budding, thereby effectively improving crop regeneration efficiency, genetic transformation efficiency, and gene editing efficiency. These advances provide new ideas for solving the problem of positive embryo budding in rubber tree somatic embryogenesis and genetic transformation.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The first objective of this invention is to provide a rubber tree HbSTMb gene, which can effectively improve the budding efficiency of rubber tree genetic transformation.

[0007] A second objective of this invention is to provide a recombinant vector containing the HbSTMb gene;

[0008] The third objective of this invention is to provide the application of the above-mentioned rubber tree HbSTMb gene in improving the budding efficiency of rubber tree genetic transformation.

[0009] To address the aforementioned technical problems, this invention discloses a rubber tree HbSTMb gene, the CDS sequence of which is shown in SEQ ID NO.1.

[0010] The present invention also discloses a recombinant vector containing the HbSTMb gene;

[0011] Preferably, the recombinant vector comprises pCAMBIA1300-35S-HbSTMb-DsRed.

[0012] The present invention also discloses a method for constructing the recombinant vector, comprising the following steps:

[0013] (1) Design the following primers to amplify the CDS sequence of the HbSTMb gene to obtain the target fragment;

[0014] 3B-1059-F: 5'-CGGGATCCATGGAGGGTGGTTCCAATAGC-3';

[0015] 3S-1059-R: 5'-GCTCTAGATCAGAGAAGCGTGGGAGAGA-3';

[0016] (2) The plant expression vector pCAMBIA1300-35S-DsRed-Tnos was double-digested with BamHI and XbaI restriction endonucleases;

[0017] (3) The digested vector is ligated with the target fragment and transformed to obtain the desired recombinant vector.

[0018] The present invention also discloses a recombinant bacterium containing the HbSTMb gene or the recombinant vector.

[0019] The present invention also discloses a stable crop line obtained by stable genetic expression of the recombinant bacteria.

[0020] This invention also discloses the application of the HbSTMb gene, or the recombinant vector, or the recombinant bacteria, or the crop line in improving the budding efficiency of genetically transformed positive embryos in rubber trees.

[0021] This invention also discloses a method for improving the budding efficiency of positive embryos in the genetic transformation of rubber tree embryos, including the step of transforming the HbSTMb gene into the target rubber tree crop. Specifically, it includes constructing the HbSTMb gene into a plant expression vector to obtain a recombinant vector, and transforming the recombinant vector into the cotyledon embryos of rubber trees using the optimized Agrobacterium-mediated transformation method to improve the budding efficiency of positive embryos in the genetic transformation of rubber tree cotyledon embryos.

[0022] Specifically, the method for improving the budding efficiency of positive embryos in the genetic transformation of rubber tree embryos further includes the step of transforming rubber tree cotyledon embryos with the plant expression vectors pCAMBIA1300-35S-DsRed-Tnos and pCAMBIA2301 via Agrobacterium-mediated transformation, so as to improve the genetic transformation efficiency of rubber tree cotyledon embryos.

[0023] Specifically, the method for improving the budding efficiency of genetically transformed positive embryos in rubber trees includes the following steps:

[0024] (1) Construct a recombinant bacterium containing the HbSTMb gene and culture it to obtain an infected bacterial solution. Use rubber tree cotyledon embryos as explants to infect the bacteria and obtain infected somatic embryos.

[0025] (2) The embryos were inoculated into the first culture medium HCK-12-A for the first culture;

[0026] (3) Continue to soak the embryos in sterile water containing 400-600 mg / L termethin (preferably 500 mg / L), and then inoculate them into the second culture medium HCK-12-T for a second culture;

[0027] (4) After cutting the embryo into pieces, transfer it to the third culture medium HCK-12-TH for third culture to obtain embryo blocks;

[0028] (5) Continue to transfer the embryo to the fourth culture medium HE-7-TH for the fourth culture, and subculture until embryos emerge.

[0029] Specifically, the method for improving the budding efficiency of genetically transformed positive embryos in rubber trees is as follows:

[0030] In step (2), the first culture medium includes HCK-12 basal culture medium, and acetylsuccinone with a final concentration of 50-150 μM is added.

[0031] In step (3), the second culture medium includes HCK-12 basal culture medium and termethin with a final concentration of 400-600 mg / L is added.

[0032] In step (4), the third culture medium includes HCK-12 basal culture medium, and termethin and hygromycin with a final concentration of 400-600 mg / L are added.

[0033] In step (5), the fourth culture medium includes HE-7 basal culture medium, and termethin and hygromycin with a final concentration of 400-600 mg / L are added;

[0034] in,

[0035] The HCK-12 basal medium comprises a modified MS medium supplemented with 1-2 mg / L kinetin, 1-2 mg / L naphthaleneacetic acid, 1-2 mg / L 2,4-dichlorophenoxyacetic acid, 0.2-0.4 g / L asparagine, 60-80 g / L sucrose, 50-80 ml / L coconut water, and 2.0-2.5 g / L agar.

[0036] The HE-7 basal medium comprises a modified MS medium supplemented with 0.5-1.5 mg / L 6-benzylaminopurine, 2.5-3.5 mg / L kinetin, 0.3-0.6 mg / L gibberellin, 0.05-0.08 mg / L 2,4-dichlorophenoxyacetic acid, 60-80 g / L sucrose, 50-80 ml / L coconut water, 1-2 g / L activated charcoal, and 2.0-2.5 g / L plant gel.

[0037] Preferably, the method for improving the budding efficiency of genetically transformed positive embryos in rubber trees is as follows:

[0038] In step (2), the first culture medium includes HCK-12 basal culture medium, and acetylsuccinone is added to a final concentration of 100 μM.

[0039] In step (3), the second culture medium includes HCK-12 basal culture medium, HCK-12 basal culture medium, and termethin with a final concentration of 500 mg / L is added.

[0040] In step (4), the third culture medium includes HCK-12 basal culture medium, and termethin and hygromycin are added to a final concentration of 500 mg / L;

[0041] In step (5), the fourth culture medium includes HE-7 basal culture medium, and termethin and hygromycin are added to a final concentration of 500 mg / L.

[0042] in,

[0043] The HCK-12 basal culture medium comprises the following components:

[0044] MS modified medium was prepared and supplemented with a final concentration of 1.5 mg / L kinetin, 1.5 mg / L naphthaleneacetic acid, 1.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.3 g / L asparagine, 70 g / L sucrose, 50 ml / L coconut water and 2.2 g / L agar.

[0045] The HE-7 basal medium consisted of a modified MS medium supplemented with 1 mg / L 6-benzylaminopurine, 3 mg / L kinetin, 0.5 mg / L gibberellin, 0.06 mg / L 2,4-dichlorophenoxyacetic acid, 70 g / L sucrose, 50 ml / L coconut water, 1 g / L activated charcoal, and 2.2 g / L plant gel.

[0046] Specifically, the method for improving the budding efficiency of genetically transformed positive embryos in rubber trees is as follows:

[0047] In step (1), the dyeing process includes: overall brushing with a nanobrush + ultrasonic treatment for 15-25 seconds + vacuuming for 2-4 minutes.

[0048] In step (2), the second culture step takes 2-4 days;

[0049] In step (3), the third culture step is dark culture, and the culture time is 24-30 h;

[0050] In step (4), the third culture step is dark culture, and the culture time is 20-30 days;

[0051] In step (5), the fourth culture step is dark culture, and the culture time is 20-25 days.

[0052] This invention has found that the HbSTMb gene in rubber trees can effectively promote the budding of positive embryos from genetic transformation in rubber trees. Overexpression of the HbSTMb gene can significantly promote the ability of positive embryos in rubber trees to further develop into complete plants. Positive embryos can not only produce buds, but also easily grow into complete plants in the seedling culture medium.

[0053] The present invention utilizes the HbSTMb gene in the genetic transformation of rubber trees to promote the budding and regeneration ability of positive embryos, and has broad application prospects in gene function research and new variety creation using transgenic and gene-editing biological breeding technologies of rubber trees.

[0054] This invention optimizes the infection system and, through transient expression detection, obtains a more efficient infection method than the existing rubber tree cotyledon embryo infection system, achieving a significant difference in transient conversion efficiency compared to the control group. Attached Figure Description

[0055] Figure 1 RNA-seq levels of the HbSTMb gene in rubber tree anther callus at different stages (0-70 d) during embryogenesis (0-70 d) in 'Reyan 73397'; statistical data are expressed as mean ± standard deviation, and the results are from three independent replicates.

[0056] Figure 2 The results show the expression of the HbSTMb gene in rubber tree at different stages of cotyledon embryo formation in the 'Reyan 73397' variety. Statistical data are expressed as mean ± standard deviation. The experimental results are from three independent replicates.

[0057] Figure 3 A schematic diagram of the pCAMBIA1300-35S-DsRed-Tnos and pCAMBIA2301 carriers;

[0058] Figure 4 A schematic diagram of the structure of the HbSTMb gene overexpression vector pCAMBIA1300-35S-HbSTMb-DsRed;

[0059] Figure 5 The dyeing effects under different dyeing methods in Example 3;

[0060] Figure 6 The results are as follows: PCR detection of resistant embryos of the "Reyan 73397" transgenic gene; where 1: DL2000 Marker; 2-4: water, negative control, and positive control; 5-25: PCR detection of resistant embryos of HbSTMb (A) and DsRed (B) transgenic genes;

[0061] Figure 7 Emergence of T0-positive embryos of HbSTMb transgenic in emergence medium

[0062] Figure 8 The results show the effect of HbSTMb gene overexpression on the budding of positive embryos. The red arrow indicates that positive embryos can directly grow into buds (A). The results show that HbSTMb gene overexpression can promote the budding and seedling formation of positive embryos of rubber tree “Reyan 73397”; T0 positive embryos continue to be cut and proliferated (B).

[0063] Figure 9 Results of field transplantation of the transgenic rubber tree line HbSTMb; among them, A: direct emergence of HbSTMb T0 generation; B: direct emergence of HbSTMb T1 generation; C: bud-grafted emergence of HbSTMb T1 generation; D: bud-grafted emergence of DsRed empty vector T0.

[0064] Figure 10 The results are PCR detection results of the HbSTMb transgenic rubber tree line after transplanting and survival; where 1: DL2000 Marker; 2-4: water, negative control, and positive control; 5-16: PCR detection of HbSTMb (A) and DsRed (B) transgenic lines after transplanting and survival. Detailed Implementation

[0065] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0066] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0067] To enable those skilled in the art to better understand the technical content of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0068] Example 1

[0069] This embodiment analyzes the function of HbSTMb gene expression in improving the budding of positive embryos in the genetic transformation efficiency of rubber tree embryos.

[0070] In this embodiment, a gene associated with the height of somatic embryo budding was screened, and the CDS sequence of HbSTMb is as follows (SEQ ID NO.1).

[0071] SEQ ID NO.1:

[0072]

[0073] In this embodiment, the expression characteristics of the HbSTMb gene during the embryonic development and seedling formation process of rubber trees were verified.

[0074] In this embodiment, the expression of the HbSTMb gene in the anther callus tissue of “Reyan 73397” at different stages of somatic embryogenesis (0-42 days) and somatic embryo development (42-70 days) was detected. Materials at 0 days, 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, 56 days, 63 days and 70 days were immediately frozen in liquid nitrogen.

[0075] RNA was extracted using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441), and the concentration and purity of the extracted RNA were detected using a Nanodrop 2000. Sequencing libraries were then constructed using the extracted RNA, and RNA-seq and data analysis were performed. Clean reads were aligned to reference transcripts from rubber trees, and the number of reads aligned to each transcript was counted and converted to FPKM (Fragments Per Kilobase Per Million Bases) to obtain the transcript expression level. Each experimental design had three biological replicates. The transcript expression level results showed that the HbSTMb gene had the highest expression in the 70-day somatic embryo during the entire somatic embryonic development process, with lower or no expression at other stages. The 70-day somatic embryo has developed into a complete embryoid structure, meaning that HbSTMb gene expression increases with the formation of the dicotyledonous somatic embryo (see attached figure). Figure 1 Preliminary results confirm that the HbSTMb gene is involved in the embryonic budding process.

[0076] In this embodiment, the different stages of embryonic development of "Reyan 73397" were further examined. Mature cotyledon embryos that met the emergence criteria were cultured in the emergence medium for 1, 3, 5, 7 and 10 days, and then immediately frozen in liquid nitrogen. RNA was extracted using the RNAprepPure polysaccharide and polyphenol plant total RNA extraction kit (Tiangen, DP441). The concentration and purity of the extracted RNA were detected using Nanodrop 2000, and the RNA concentration was uniformly adjusted to 1 μg. Subsequently, cDNA was reverse transcribed using ToloScript All-in-one RT EasyMix for qPCR (TOLOBIO, 22107). The 20 μL reverse transcription system includes: 4 μL of 5×All-in-one RT Buffer, 1 μL of All-in-one Enzyme Mix, and RNase-free ddH2O, which are then added to a final volume of 20 μL. The mixture is gently pipetted 8-10 times until fully mixed, then briefly centrifuged to collect the contents at the bottom of the tube. The mixture is reacted at 50°C for 15 minutes, followed by a further reaction at 85°C for 5 seconds. The resulting product is the reverse transcription cDNA stock solution, which is diluted 20 times for use as the working solution.

[0077] The cDNA working solution of the above materials was used for quantitative PCR analysis using the 2 × Q3 SYBR qPCR MasterMix (Universal) (TOLOBIO, 22204) kit. The reaction mixture was prepared in the qPCR tube as follows: 10 μL of 2 × Q3 SYBR qPCR Master Mix (Universal); 0.4 μL each of the left and right primers; 9.2 μL of cDNA and ddH2O. The reaction program was 95℃ pre-denaturation for 30 seconds, 95℃ for 10 seconds, and 60℃ for 30 seconds, for 40 cycles. Melting curves were obtained using the default program of the Bio-Rad quantitative PCR instrument to determine the expression levels of the target gene at different emergence times. Each experiment was designed with three biological replicates. (See attached...) Figure 2 The results showed that the HbSTMb gene gradually increased from day 3 to day 10 during the germination stage, further confirming that the HbSTMb gene is involved in the embryonic budding process.

[0078] Example 2

[0079] This embodiment involves the construction of an overexpression vector based on the HbSTMb gene and its transformation into rubber tree embryos.

[0080] In this embodiment, in order to further study the function of HbSTMb in the embryogenesis and development of rubber trees, an expression vector of 35S-HbSTMb-DsRed was constructed and overexpressed in the dicotyledonous embryos of rubber trees through Agrobacterium-mediated stable transformation.

[0081] The CDS sequence of the HbSTMb gene was amplified using the primers shown below:

[0082] 3B-1059-F: 5'-CGGGATCCATGGAGGGTGGTTCCAATAGC-3';

[0083] 3S-1059-R: 5'-GCTCTAGATCAGAGAAGCGTGGGAGAGA-3'.

[0084] The plant expression vector pCAMBIA1300-35S-DsRed-Tnos (structure as shown) was expressed using two restriction endonucleases, BamHI and XbaI. Figure 3 The vector plasmid and the amplified fragment were double-digested with enzymes. The digested vector and the amplified fragment were then recovered and ligated using T4 DNA ligase (Takara, 2011A). 10 μl of the ligation product was added to 100 μl of DH5α competent cells (TOLOBIO, CC96102). After incubating on ice for 30 min, the cells were heat-shocked at 42°C for 90 s, followed by an ice incubation for 2 min. The cells were then cultured in antibiotic-free LB medium at 37°C on a shaker at 200 rpm for 1 h. Finally, the cells were plated onto LB solid medium containing 50 μg / mL kanamycin and cultured overnight.

[0085] Transformants were picked and cultured in LB broth containing 50 μg / mL kanamycin at 37℃ with shaking at 220 rpm for 12 h. Plasmids were extracted and sequenced for verification, ultimately yielding the HbSTMb expression vector pCAMBIA1300-35S-HbSTMb-DsRed, the structure of which is shown in the attached figure. Figure 4 As shown.

[0086] The constructed pCAMBIA1300-35S-HbSTMb-DsRed, expression vector, and the aforementioned empty vector pCAMBIA1300-35S-DsRed-Tnos were used. After plasmid extraction, 10 μl of the plasmid was added to 100 μl of EHA105 Agrobacterium competent cells (Weidi Biotechnology, AC1010S). The mixture was incubated on ice for 5 min, in liquid nitrogen for 5 min, in a 37℃ water bath for 5 min, and on ice for 5 min. Then, 700 μl of LB liquid medium was added, and the mixture was incubated at 28℃ and 220 rpm for 2-3 h. After centrifugation at 6000 rpm for 5 min, the bacteria were harvested. Approximately 100 μl of the supernatant was collected, and the bacterial clumps were gently resuspended by pipetting and spread onto LB solid medium containing 50 mg / L kanamycin and 10 mg / L rifampin. The clumps were then incubated upside down at 28℃ for 2-3 days. Single clones were selected for PCR verification. The correctly detected single clones were added to an equal volume of 50% glycerol (50 ml glycerol + 50 ml glycerol). Sterilize with ddH2O at 121℃ for 20 min, and store in an ultra-low temperature freezer at -80℃ for subsequent experiments.

[0087] Example 3

[0088] This embodiment optimizes the infection system based on a genetic transformation system using rubber tree embryos as explants.

[0089] As the research object, the plant expression vectors pCAMBIA1300-35S-DsRed-Tnos and pCAMBIA2301 were used to transform rubber tree cotyledon embryos via Agrobacterium-mediated transformation in order to improve the genetic transformation efficiency of rubber tree cotyledon embryos.

[0090] In this embodiment, the specific operations for converting rubber tree cotyledon embryos are as follows:

[0091] Agrobacterium containing the recombinant vector was cultured. Agrobacterium was scraped off from the culture dish with a pipette tip and placed in a resuspension solution. The solution was aspirated and shaken until no particles were visible. This solution was then used as the infection solution and placed on a shaker at 28°C and 200 rpm for 4 hours before infection. Rubber tree cotyledon embryos were used as explants. Thirteen infection methods were designed and tested, as shown in Table 1. DsRed and GUS were used as selection marker genes for transformation. Three embryoids were treated in each experiment, and the experiment was repeated three times.

[0092] Table 1

[0093]

[0094] After inoculation, blot the bacterial solution from the cotyledon embryos with sterile triangular paper, then blow-dry for about 15 minutes before placing the cotyledon embryos on HCK-12-A medium and culturing for 72 hours. Rinse the embryos with sterile water until the water is clear, then soak them in sterile water containing 500 mg / L termethin for 10 minutes. Blot dry with sterile triangular paper and place them on HCK-12-T medium lined with sterile filter paper for dark incubation for at least 24 hours. Cut the embryos into 3 mm × 3 mm pieces and transfer them to HCK-12-TH medium for dark incubation for 20-30 days. Transfer the dark-cultured embryo pieces to HE-7-TH embryo culture medium and incubate in the dark for 20-25 days, subculturing several times until embryos emerge.

[0095] In this embodiment, the resuspension is a solution containing 100 µM acetylsuccinone.

[0096] In this embodiment, the culture medium formulation involved in optimizing the infiltration method is as follows:

[0097] HCK-12 culture medium formulation:

[0098] The modified MS medium was prepared by adding 1.5 mg / L kinetin, 1.5 mg / L naphthaleneacetic acid, 1.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.3 g / L asparagine, 70 g / L sucrose, 50 ml / L coconut water and 2.2 g / L agar, pH 5.8.

[0099] HE-7 medium formulation: Modified MS medium supplemented with 1 mg / L 6-benzylaminopurine, 3 mg / L kinetin, 0.5 mg / L gibberellin, 0.06 mg / L 2,4-dichlorophenoxyacetic acid, 70 g / L sucrose, 50 ml / L coconut water, 1 g / L activated charcoal, and 2.2 g / L plant gel, pH 5.8.

[0100] The HCK-12-A medium is prepared by adding 1 mL of 100 μM acetylsalicylic acid to 1 L of HCK-12.

[0101] The HCK-12-T medium is prepared by adding 1 mL of 500 mg / mL termethin to 1 L of HCK-12.

[0102] The HCK-12-TH medium is prepared by adding 1 mL of 500 mg / ml termethin and 200 μl of 50 mg / ml hygromycin to 1 L of HCK-12.

[0103] The HE-7-TH medium is prepared by adding 1 mL of 500 mg / ml termethin and 200 μl of 50 mg / ml hygromycin to 1 L of HE-7 medium.

[0104] In this embodiment, the dyeing results of the leaf blank under the above-mentioned optimized scheme are shown in the appendix. Figure 5 As shown.

[0105] It is evident that, under the same conditions, cotyledon embryos of similar size and thickness, and in good condition, exhibited different infection efficiencies under 13 different infection methods. When cotyledon embryos infected with Agrobacterium tumefaciens EHA105 were subjected to a combination of three infection methods—microblogging (whole-body), ultrasound for 20 seconds, and vacuum infiltration for 3 minutes—an average of up to 58% of the embryo mass area was infected (e.g., ...). Figure 5 In the case of cotyledonous embryos infected with Agrobacterium tumefaciens (A), when three infection methods were combined—micro-brushing of the entire embryo, ultrasonication for 20 seconds, and vacuum infiltration for 3 minutes—an average of up to 62% of the embryo mass area was infected (e.g., ...). Figure 5 (B)

[0106] In summary, this embodiment used 13 different conventional infection methods for experiments. The final statistical data and SPSS data analysis of each group revealed that the treatment group using "micro-brushing + ultrasonic treatment for 20 seconds + vacuum immersion for 3 minutes" had the highest infection efficiency and was significantly different from the other treatment groups.

[0107] Therefore, in the following embodiments of the present invention, the "micro-brushing the whole + ultrasonic 20 s + vacuum impregnation 3 min" impregnation method is selected as the optimal impregnation method for rubber tree somatic embryos obtained in this experimental study.

[0108] Example 4

[0109] In this embodiment, the recombinant vector / recombinant bacteria constructed in Example 2 were used to conduct an infiltration experiment according to the optimized method obtained in Example 3.

[0110] Agrobacterium containing the recombinant vector was cultured. Agrobacterium was scraped off the culture dish with a pipette tip and placed in a suspension. The mixture was aspirated and shaken until no particles were visible. The resulting infecting bacterial solution was placed on a shaker at 28°C and 200 rpm for 4 hours.

[0111] In this embodiment, the infection operation of the rubber tree cotyledon embryo is as follows:

[0112] 1) Use a nano brush to gently brush the surface of the cotyledon embryo 3-4 times in a clean bench. Then place the brushed embryo in a 150 ml Erlenmeyer flask, pour in the inoculation solution, and ultrasonically inoculate for 20 seconds. Then vacuum for 3 minutes.

[0113] 2) Discard the bacterial inoculum and use sterile forceps to place the cotyledon embryos onto a sterile culture dish (with a few sheets of sterile filter paper). After blotting the bacterial inoculum off the cotyledon embryos with sterile triangular paper, blow dry for about 15 minutes and then place the cotyledon embryos onto HCK-12-A medium and culture for a total of 72 hours.

[0114] 3) Rinse the embryos with sterile water until the water is clear, then soak them in sterile water containing 500 mg / L termethin for 10 min, blot dry with sterile triangular paper, and then place them on HCK-12-T medium lined with a sterile filter paper and incubate in the dark for more than 24 h.

[0115] 4) Cut the somatic embryos into 3 mm × 3 mm pieces, transfer them into HCK-12-TH medium, and incubate in the dark for 20-30 days;

[0116] 5) Transfer the dark-cultured embryos to the embryo-producing medium HE-7-TH, culture in the dark for 20-25 days, and subculture several times until embryos emerge.

[0117] In this embodiment, the culture medium formulation involved in the method of infecting rubber tree cotyledon embryos is as follows:

[0118] The resuspension was prepared by removing CaCl2 from a modified MS medium, adding 0.3 g / L asparagine and 20 g / L sucrose, pH 5.2, and adding a 100 µM acetylsylgenone solution before use.

[0119] HCK-12 medium formulation: Modified MS medium supplemented with 1.5 mg / L kinetin, 1.5 mg / L naphthaleneacetic acid, 1.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.3 g / L asparagine, 70 g / L sucrose, 50 ml / L coconut water and 2.2 g / L agar, pH 5.8;

[0120] HE-7 medium formulation: Modified MS medium supplemented with 1 mg / L 6-benzylaminopurine, 3 mg / L kinetin, 0.5 mg / L gibberellin, 0.06 mg / L 2,4-dichlorophenoxyacetic acid, 70 g / L sucrose, 50 ml / L coconut water, 1 g / L activated charcoal, and 2.2 g / L plant gel, pH 5.8;

[0121] HCK-12-A medium is prepared by adding 1 mL of 100 μM acetylsalicylic acid to 1 L of HCK-12 medium;

[0122] HCK-12-T medium is prepared by adding 1 mL of 500 mg / mL termethin to 1 L of HCK-12.

[0123] HCK-12-TH medium is prepared by adding 1 mL of 500 mg / ml termethin and 200 μl of 50 mg / ml hygromycin to 1 L of HCK-12 medium;

[0124] HE-7-TH medium is prepared by adding 1 mL of 500 mg / ml termethin and 200 μl of 50 mg / ml hygromycin to 1 L of HE-7 medium.

[0125] Example 5

[0126] This embodiment verifies that the HbSTMb gene improves the budding ability of positive embryos in rubber trees.

[0127] Using the inoculation method described in Example 4 above, the number of transgenic positive embryoid buds formed by the pCAMBIA1300-35S-HbSTMb-DsRed expression vector and the empty vector pCAMBIA1300-35S-DsRed-Tnos was statistically analyzed based on the number of buds formed. The results are shown in Table 2 below. The results indicate that HbSTMb can significantly improve the budding efficiency of positive embryos in "Reyan 73397".

[0128] Table 2

[0129]

[0130] As shown in Table 2, 130 and 240 cotyledon embryoids were infected with the pCAMBIA1300-35S-HbSTMb-DsRed expression vector and the pCAMBIA1300-35S-DsRed-Tnos control empty vector, respectively. The resulting T0-resistant embryoids were 80 and 345, respectively. PCR verification confirmed 40 and 151 T0-positive embryos, respectively. Some of the resistant embryo detection results are attached. Figure 6 Among them, HbSTMb with buds in the T0 generation can directly produce 5 seedlings (as shown in the attached image). Figure 7 The results shown and the appendix Figure 8 (A) The control vector cannot directly germinate in the T0 generation.

[0131] Positive embryoids of HbSTMb and the control vector were proliferated via secondary embryogenesis (see attached). Figure 8 (B) 140 and 72 transgenic T1 generation resistant embryos of HbSTMb and control vector were obtained, respectively. PCR detection showed that 22 positive embryos of HbSTMb and 1 positive embryo of control vector were obtained. Among them, 4 HbSTMb T1 generation plants had buds and could be directly planted, while 3 plants that did not grow tall or did not grow leaves were planted by budding.

[0132] In summary, although the control vector was superior to the HbSTMb gene in T0 generation resistant and positive embryos, HbSTMb was significantly superior to the control vector in terms of the number of buds.

[0133] In this embodiment, transplanted and surviving plants (as shown in the attached image) are taken. Figure 9 DNA was extracted from leaves, and PCR testing confirmed that the plants were positive (results are attached). Figure 10 ).

[0134] In summary, this invention is based on the fact that the HbSTMb gene of rubber tree can effectively promote the budding of positive embryos from genetic transformation of rubber tree. Overexpression of the HbSTMb gene can significantly promote the ability of positive embryos of rubber tree to further develop into complete plants. Positive embryos can not only produce buds, but also easily grow into complete plants in the seedling culture medium.

[0135] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A rubber tree HbSTMb gene, characterized in that, The CDS sequence of the HbSTMb gene is shown in SEQ ID NO.

1.

2. A recombinant vector, characterized in that, The recombinant vector contains the HbSTMb gene as described in claim 1; Preferably, the recombinant vector comprises pCAMBIA1300-35S-HbSTMb-DsRed.

3. A method for constructing the recombinant vector as described in claim 2, characterized in that, Includes the following steps: (1) Design the following primers to amplify the CDS sequence of the HbSTMb gene described in claim 1 to obtain the target fragment; 3B-1059-F: 5'-CGGGATCCATGGAGGGTGGTTCCAATAGC-3'; 3S-1059-R: 5'-GCTCTAGATCAGAGAAGCGTGGGAGAGA-3'; (2) The plant expression vector pCAMBIA1300-35S-DsRed-Tnos was double-digested with BamHI and XbaI restriction endonucleases; (3) The digested vector is ligated with the target fragment and transformed to obtain the desired recombinant vector.

4. A recombinant bacterium, characterized in that, The recombinant bacteria contain the HbSTMb gene of claim 1 or the recombinant vector of claim 2.

5. A stable crop line obtained by stable genetic expression of the recombinant bacteria described in claim 4.

6. The application of the HbSTMb gene of claim 1, or the recombinant vector of claim 2, or the recombinant bacteria of claim 4, or the crop line of claim 5 in improving the budding efficiency of genetically transformed positive embryos in rubber trees.

7. A method for improving the budding efficiency of genetically transformed positive embryos in rubber trees, characterized in that, The method includes the step of converting the HbSTMb gene of claim 1 into the target rubber tree crop.

8. The method for improving the budding efficiency of positive embryos in the genetic transformation of rubber trees according to claim 7, characterized in that, Includes the following steps: (1) Construct a recombinant bacterium containing the HbSTMb gene and culture it to obtain an infected bacterial solution. Use rubber tree cotyledon embryos as explants to infect the bacteria and obtain infected somatic embryos. (2) The embryos were inoculated into the first culture medium HCK-12-A for the first culture; (3) Continue to soak the embryos in sterile water containing termethin, and then inoculate them into the second culture medium HCK-12-T for a second culture; (4) After cutting the embryo into pieces, transfer it to the third culture medium HCK-12-TH for third culture to obtain embryo blocks; (5) Continue to transfer the embryo to the fourth culture medium HE-7-TH for the fourth culture, and subculture until embryos emerge.

9. The method for improving the budding efficiency of positive embryos in the genetic transformation of rubber trees according to claim 8, characterized in that: In step (2), the first culture medium includes HCK-12 basal culture medium, and acetylsuccinone with a final concentration of 50-150 μM is added. In step (3), the second culture medium includes HCK-12 basal culture medium and termethin with a final concentration of 400-600 mg / L is added. In step (4), the third culture medium includes HCK-12 basal culture medium, and termethin and hygromycin with a final concentration of 400-600 mg / L are added; In step (5), the fourth culture medium includes HE-7 basal culture medium, and termethin and hygromycin are added to a final concentration of 400-600 mg / L. in, The HCK-12 basal medium comprises a modified MS medium supplemented with 1-2 mg / L kinetin, 1-2 mg / L naphthaleneacetic acid, 1-2 mg / L 2,4-dichlorophenoxyacetic acid, 0.2-0.4 g / L asparagine, 60-80 g / L sucrose, 50-80 ml / L coconut water, and 2.0-2.5 g / L agar. The HE-7 basal medium comprises a modified MS medium supplemented with 0.5-1.5 mg / L 6-benzylaminopurine, 2.5-3.5 mg / L kinetin, 0.3-0.6 mg / L gibberellin, 0.05-0.08 mg / L 2,4-dichlorophenoxyacetic acid, 60-80 g / L sucrose, 50-80 ml / L coconut water, 1-2 g / L activated charcoal, and 2.0-2.5 g / L plant gel.

10. The method for improving the budding efficiency of genetically transformed positive embryos in rubber trees according to claim 8 or 9, characterized in that: In step (1), the dyeing process includes: overall brushing with a nanobrush + ultrasonic treatment for 15-25 seconds + vacuuming for 2-4 minutes. In step (2), the second culture step takes 2-4 days; In step (3), the third culture step is dark culture, and the culture time is 24-30 h; In step (4), the third culture step is dark culture, and the culture time is 20-30 days; In step (5), the fourth culture step is dark culture, and the culture time is 20-25 days.

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