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Rubber tree phytoene dehydrogenase gene VIGS silencing system, and construction method and application thereof

A phytoene and rubber tree technology, applied in application, genetic engineering, plant gene improvement, etc., can solve the problem of reducing the expression level of HbPDS gene, achieve an effective virus-induced silencing system, simple operation, avoid labor and low efficiency Effect

Active Publication Date: 2020-08-28
INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This system can effectively reduce the expression level of the HbPDS gene in rubber trees, and can obtain virus-induced gene silencing traits, thereby solving the problem of effectively verifying the functions of rubber tree genes, and laying the foundation for mining rubber tree genes with important agronomic traits and cultivating excellent rubber tree varieties

Method used

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  • Rubber tree phytoene dehydrogenase gene VIGS silencing system, and construction method and application thereof
  • Rubber tree phytoene dehydrogenase gene VIGS silencing system, and construction method and application thereof
  • Rubber tree phytoene dehydrogenase gene VIGS silencing system, and construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Cloning of target gene fragment of HbPDS and construction of expression vector

[0029] S1. The rubber tree leaves are from the Reyan 7-33-97 rubber tree normally tapped in the experimental farm of the Chinese Academy of Tropical Agricultural Sciences. The total RNA was extracted according to the method recorded in [Li Huiliang. The molecular basis of the difference between the juvenile clone of rubber tree and its donor (senile clone) [D]. Haikou: Hainan University, 2010]. The synthesis of the first strand of cDNA was carried out according to the instructions of the PrimeScript RTreagent Kit of Bao Bio Company.

[0030] S2. According to the sequence information of the coding gene of Hevea phytoene dehydrogenase (HbPDS), we use the bioinformatics tool software pss-RNAit (https: / / plant grn.noble.org / pssRN Ait / ) to predict the hevea phytoene dehydrogenase The siRNAs in the coding gene of lycopene dehydrogenase (HbPDS) selected a sequence with a size of 409bp. Combined w...

Embodiment 2

[0046] Agrobacterium Transformation of Recombinant Vector and Its Genetic Transformation

[0047] S1. Take the pTRV2-HbPDS recombinant plasmid obtained in Example 1 and add it to GV3101 competent cells, let it stand on ice for 10 minutes, freeze it in liquid nitrogen for 5 minutes, warm it in a water bath at 37°C for 5 minutes, and put it on ice for 2 minutes. The LB liquid medium without antibiotics was placed in a shaker at 28°C and 150rpm for 1.5h recovery culture, and then 200μl of bacterial liquid was taken and spread on antibiotic-containing (50mg / l Kan+50mg / l Rif ) on LB solid medium. After 48 hours, single clones were picked from the plate and cultured overnight in 300 μl LB liquid medium containing antibiotics (50mg / l Kan+50mg / l Rif), and then PCR identification of positive clones was carried out to obtain GV3101 with pTRV2-HbPDS recombinant plasmid Agrobacterium.

[0048] S2. The positive clones screened and the LB liquid medium containing antibiotics (50mg / l Kan+5...

Embodiment 3

[0053] Determination of VIGS Silencing System Efficiency in Hevea

[0054] S1, such as image 3 As shown, after 2 months of infection, the new leaves gradually appeared bleaching phenomenon, and then the degree of bleaching deepened.

[0055] S2. Extraction of total RNA and cDNA synthesis of control plants and transgenic plants: the extraction of total RNA from rubber tree leaves was carried out according to the method in the literature. The synthesis of the first strand of cDNA was carried out according to the instructions of the PrimeScript RTreagent Kit of Bao Bio Company.

[0056] S3. Fluorescence quantitative PCR analysis of HbPDS gene in different materials: SYBR Premix ExTaq II was used for fluorescence quantification, and the actin gene HbACT gene was used as an internal reference. A sequence design outside the targeted silencing region was selected for the determination of the HbPDS gene in Hevea. The primers used for quantitative PCR are:

[0057] HbPDS1F: ATGACT...

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Abstract

The embodiment of the invention discloses a rubber tree phytoene dehydrogenase gene VIGS silencing system, and a construction method and application thereof, and belongs to the technical field of biology. The rubber tree phytoene dehydrogenase gene VIGS silencing system has a nucleotide sequence as shown in SEQ ID NO: 1. The method in the invention is simple to operate; a set of rapid and effective virus induced silencing system is established; and the defects that the traditional genetic transformation needs heavy labour and is low in efficiency are effectively avoided. The system can effectively reduce the expression level of a HbPDS gene in a rubber tree, can obtain virus-induced gene silencing traits, further solves the problem of effectively verifying functions of rubber tree genes, and lays a foundation for excavating the rubber tree genes with important agronomic traits and cultivating excellent rubber tree varieties.

Description

technical field [0001] The embodiment of the present invention relates to the field of biotechnology, in particular to a VIGS silencing system for the rubber tree phytoene dehydrogenase gene and its construction method and application. Background technique [0002] Hevea brasiliensis is a plant of the genus Hevea in the Euphorbiaceae family, native to the Amazon forest. China's rubber planting areas are mainly distributed in Hainan, Guangdong, Guangxi, Yunnan and other regions, among which Hainan is the main rubber planting area. The rubber tree is a deciduous tree. The milk ducts in the bark produce the latex of the milky sap. The latex is the main secondary metabolite of the Brasilian rubber tree and the main source of natural rubber. The economic life of rubber trees is about 30 years, and the growth life is about 60 years. It takes 20 to 30 years to breed a new rubber tree variety through conventional hybrid breeding. The establishment of a genetic transformation syst...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/53A01H5/00A01H6/38
CPCC12N15/8218C12N9/001C12Y103/99
Inventor 李辉亮彭世清郭冬王颖朱家红
Owner INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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