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CN-122012519-A - HbSTMb gene and application thereof in improving genetic transformation budding of rubber tree

CN122012519ACN 122012519 ACN122012519 ACN 122012519ACN-122012519-A

Abstract

The invention belongs to the technical field of plant genetic engineering, in particular to HbSTMb genes and clone expression thereof, and further discloses application thereof in improving genetic transformation budding of rubber trees. According to the research of the invention, the gene HbSTMb of the rubber tree can effectively promote the germination of positive embryos of the genetic transformation of the rubber tree, and the capability of further developing the positive embryos of the rubber tree into complete plants can be obviously promoted by utilizing the over-expression HbSTMb gene, so that the positive embryos can easily generate bud points and further can easily grow into complete plants in a seedling emergence culture medium.

Inventors

  • LI JI
  • YANG HONGLI
  • LI YANSHUANG
  • LIU HAOBIN
  • JIANG MIN
  • WU RIZHI
  • Fu Qinghuan
  • HUANG TIANDAI

Assignees

  • 中国热带农业科学院橡胶研究所

Dates

Publication Date
20260512
Application Date
20251117

Claims (10)

  1. 1. The rubber tree HbSTMb gene is characterized in that the CDS sequence of the HbSTMb gene is shown in SEQ ID NO. 1.
  2. 2. A recombinant vector comprising the HbSTMb gene of claim 1; preferably, the recombinant vector comprises pCAMBIA1300-35S-HbSTMb-DsRed.
  3. 3. The method for constructing a recombinant vector according to claim 2, comprising the steps of: (1) Designing the following primers to amplify the CDS sequence of HbSTMb gene in claim 1 to obtain target fragment; 3B-1059-F:5’-CGGGATCCATGGAGGGTGGTTCCAATAGC-3’; 3S-1059-R:5’-GCTCTAGATCAGAGAAGCGTGGGAGAGA-3’; (2) Double digestion is carried out on a plant expression vector pCAMBIA1300-35S-DsRed-Tnos by utilizing BamHI and XbaI restriction enzymes; (3) And (3) connecting and converting the vector after enzyme digestion with the target fragment to obtain the required recombinant vector.
  4. 4. A recombinant bacterium comprising the HbSTMb gene of claim 1 or the recombinant vector of claim 2.
  5. 5. A stable crop line obtained by stable genetic expression of the recombinant bacterium of claim 4.
  6. 6. Use of HbSTMb gene according to claim 1, recombinant vector according to claim 2, recombinant strain according to claim 4, or crop strain according to claim 5 for increasing the efficiency of budding of a genetic transformation positive embryo of a rubber tree.
  7. 7. A method for improving the germination efficiency of a genetic transformation positive embryo of a rubber tree embryo, comprising the step of transforming the HbSTMb gene of claim 1 into a desired rubber tree crop.
  8. 8. The method for improving the germination efficiency of a genetic transformation positive embryo of a rubber tree embryo according to claim 7, comprising the steps of: (1) Constructing recombinant bacteria containing HbSTMb genes, culturing to obtain infectious bacteria liquid, and infecting with rubber tree cotyledon embryo as explant to obtain transfected somatic embryo; (2) Inoculating the somatic embryo into a first culture medium HCK-12-A for first culture; (3) Continuously soaking the somatic embryo with sterilized water containing timentin, and then inoculating the somatic embryo into a second culture medium HCK-12-T for second culture; (4) Cutting the somatic embryo into blocks, and transferring the blocks into a third culture medium HCK-12-T-H for third culture to obtain briquettes; (5) And transferring the briquettes into a fourth culture medium HE-7-T-H for fourth culture, and carrying out subculture until the embryos are obtained.
  9. 9. The method for improving the germination efficiency of genetic transformation positive embryos of rubber tree embryos according to claim 8, wherein the method comprises the following steps: in the step (2), the first culture medium comprises HCK-12 basal medium, and acetosyringone with the final concentration of 50-150 mu M is added; in the step (3), the second culture medium comprises HCK-12 basal medium, and the termestin with the final concentration of 400-600 mg/L is added; In the step (4), the third culture medium comprises HCK-12 basal medium, and the termestin with the final concentration of 400-600 mg/L and the hygromycin with the final concentration of 5-10 mg/L are added; In the step (5), the fourth culture medium comprises HE-7 basal medium, and the final concentration of the termestin is 400-600 mg/L and the hygromycin is 5-10 mg/L are added; Wherein, the The HCK-12 basal medium comprises an improved MS medium, and is added with kinetin with the final concentration of 1-2 mg/L, 1-2 mg/L naphthylacetic acid, 1-2 mg/L2, 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 an improved MS medium, and is added with 6-benzyl amino purine, kinetin with the final concentration of 0.5-1.5 mg/L, gibberellin with the final concentration of 2.5-3.5 mg/L, gibberellin with the final concentration of 0.3-0.6 mg/L, sucrose with the final concentration of 0.05-0.08 mg/L, dichlorophenoxyacetic acid with the final concentration of 4-dichlorophenoxyacetic acid, sucrose with the final concentration of 60-80 g/L, coconut water with the final concentration of 50-80 ml/L, activated carbon with the final concentration of 1-2 g/L and plant gel with the final concentration of 2.0-2.5 g/L.
  10. 10. The method for improving the germination efficiency of a genetic transformation positive embryo of a rubber tree embryo according to claim 8 or 9, wherein the method comprises the following steps: in the step (1), the dip dyeing step comprises the steps of brushing and dyeing the whole nano brush, carrying out ultrasonic treatment for 15-25 seconds and vacuumizing for 2-4 minutes; In the step (2), the time of the second culturing step is 2-4 days; In the step (3), the third culturing step is dark culturing, and the culturing time is 24-30 h; In the step (4), the third culturing step is dark culturing for 20-30 days; in the step (5), the fourth culturing step is dark culturing for 20-25 days.

Description

HbSTMb gene and application thereof in improving genetic transformation budding of rubber tree Technical Field The invention belongs to the technical field of plant genetic engineering, in particular to HbSTMb genes and clone expression thereof, and further discloses application thereof in improving genetic transformation budding of rubber trees. Background Natural rubber is native in Brazil and is an important strategic material and reserve resource in China all the time. At present, the breeding of new rubber tree varieties in China is still mainly based on traditional cross breeding. However, the Brazil rubber tree breeding process is very slow due to the characteristics of narrow genetic background, high heterozygosity, long breeding period and the like, which seriously hinders the rubber tree breeding process. The transgenic technology can effectively improve the breeding efficiency, shorten the breeding period, expand the genetic range of the rubber tree and accelerate the breeding process. Along with the development of biotechnology, the establishment of a high-efficiency genetic transformation system is the basis for cultivating new varieties of rubber trees by utilizing biological breeding technologies such as transgenosis, gene editing and the like. At present, the transformation efficiency can reach 1% by utilizing an ultrasonic-assisted mediated genetic transformation system of the rubber tree, but the problem that the positive embryo deformity rate is high and the plant is difficult to regenerate exists. Therefore, by improving the transformation efficiency, more positive embryos are obtained, and positive regenerated plants are possibly difficult to succeed. At present, research has proved that the problem of positive plant budding can be overcome by regulating and controlling genes required by stem tip development in genetic transformation, so that the regeneration efficiency, genetic transformation efficiency and gene editing efficiency of crops are effectively improved, and the progress provides a new thought for solving the problems of embryogenesis of rubber trees and positive embryo budding in genetic transformation. The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art. Disclosure of Invention The first object of the present invention is to provide a gene HbSTMb of rubber tree, said HbSTMb gene being effective for improving the germination efficiency of genetic transformation of rubber tree; a second object of the present invention is to provide a recombinant vector containing the HbSTMb gene; The third object of the present invention is to provide an application of the above-mentioned rubber tree HbSTMb gene in improving the germination efficiency of genetic transformation of rubber tree. In order to solve the technical problems, the invention discloses a rubber tree HbSTMb gene, wherein the CDS sequence of the HbSTMb gene is shown as SEQ ID NO. 1. The invention also discloses a recombinant vector, which comprises the HbSTMb gene; preferably, the recombinant vector comprises pCAMBIA1300-35S-HbSTMb-DsRed. The invention also discloses a construction method of the recombinant vector, which comprises the following steps: (1) Designing the following primers to amplify the CDS sequence of the HbSTMb gene to obtain a target fragment; 3B-1059-F:5’-CGGGATCCATGGAGGGTGGTTCCAATAGC-3’; 3S-1059-R:5’-GCTCTAGATCAGAGAAGCGTGGGAGAGA-3’; (2) Double digestion is carried out on a plant expression vector pCAMBIA1300-35S-DsRed-Tnos by utilizing BamHI and XbaI restriction enzymes; (3) And (3) connecting and converting the vector after enzyme digestion with the target fragment to obtain the required recombinant vector. The invention also discloses a recombinant bacterium which contains the HbSTMb gene or the recombinant vector. The invention also discloses a stable crop strain obtained by stable genetic expression of the recombinant bacteria. The invention also discloses application of HbSTMb gene, recombinant vector, recombinant strain or crop strain in improving the germination efficiency of the genetic transformation positive embryo of the rubber tree. The invention also discloses a method for improving the germination efficiency of the genetic transformation positive embryo of the rubber tree embryo, which comprises the steps of transforming the HbSTMb gene into a target rubber tree crop, specifically comprises the steps of constructing the HbSTMb gene onto a plant expression vector to obtain a recombinant vector, and transforming the recombinant vector into the cotyledon embryo of the rubber tree by the optimized agrobacterium-mediated method so as to improve the germination efficiency of the genetic transformation positive embryo of the cotyledon embryo of t