A method for obtaining transgenic plants of rubber tree with a lactiferous duct-specific promoter
A rubber tree, specific technology, applied in the field of plant genetic engineering, can solve the problem of transforming rubber tree with milk duct-specific expression vectors that have not yet been seen
Active Publication Date: 2020-06-23
RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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[0005] Although the studies on the promoter PHEV2.1 and HbHMGR1 genes have been reported separately, there is no research on using PHEV2.1 to construct the lactiferous duct-specific expression vector of HbHMGR1 to transform Hevea
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[0209] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. The application principle of the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.
[0210] A kind of method that the present invention obtains rubber tree transgenic plant with laticifer specific promoter, concrete steps are as follows:
[0211] Step 1. Cloning of the key rate-limiting enzyme gene HbHMGR1 in natural rubber biosynthesis
[0212] 1.1 Extraction of rubber tree total RNA and synthesis of cDNA first strand
[0213] Hevea latex total RNA was extracted using the Trizol method (Sambrook et al., 1996). The first strand of cDNA was synthesized according to the instructions of the Revert Aid TM cDNA First Strand Synthesis Kit, and stored at -20°C until use.
[0214] 1.2 HbHMGR1 gene cDNA chain synthesis
[0215] According to the HbHMGR1 gene of NCBI access...
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Abstract
The invention discloses a strong promoter PHEV2.1 specific for cloning rubber tree laticiferous ducts, which is connected with the key rate-limiting enzyme HbHMGR1 gene of the natural rubber biosynthesis pathway to construct a plant expression vector, transforms the fragile embryogenic callus of rubber tree, and obtains the transgene plant method. The present invention clones PHEV2.1 and HbHMGR1 genes from excellent varieties of rubber trees, constructs the laticifer-specific plant expression vector pCAMBIA2301-PHEV2.1-HbHMGR1, transforms rubber tree fragile embryogenic callus, and obtains rubber tree transgenic embryoid bodies and Transgenic plants, using GUS staining, PCR and reverse PCR to prove that the transgenic plants have been obtained, and the detection of fluorescent quantitative PCR technology proves that the expression level of HbHMGR1 gene in the transgenic embryoid body and transgenic plants of rubber tree is significantly increased, which provides a basis for improving rubber production by genetic engineering methods Foundation.
Description
technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a method for obtaining transgenic plants of rubber trees by using a lactiferous duct-specific promoter. Background technique [0002] Rubber tree is an important tropical economic crop, and the natural rubber it produces is an important strategic material. In recent years, with the rapid development of the national economy and national defense, the self-sufficiency rate of natural rubber in my country has declined year by year, and the dependence on imports has continued to rise (Wang Shaoming et al., 2011). However, the area suitable for rubber tree planting is limited, and the potential for expanding the planting area to increase rubber production is extremely small. Therefore, it is more urgent to carry out research on improving rubber tree varieties and increasing rubber yield per unit area. Natural rubber biosynthesis is an important factor aff...
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IPC IPC(8): C12N15/84C12N15/66C12Q1/6895A01H5/00A01H6/38
CPCC12N9/0006C12N15/66C12N15/8205C12N15/8223C12N15/8261C12N2800/101C12N2800/60C12N2820/007C12Q1/6895C12Q2600/13C12Q2600/158C12Y101/01088
Inventor 李哲贺永国黄绵佳马晓晓曾宪海林位夫周建南刘洁琼
Owner RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI