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Application and method for squalene synthase gene overexpression of medicago sativa

A kind of alfalfa, squalene synthase technology, applied in application, genetic engineering, plant genetic improvement and other directions

Inactive Publication Date: 2019-03-19
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is no report on the related research of SQS gene in alfalfa

Method used

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  • Application and method for squalene synthase gene overexpression of medicago sativa
  • Application and method for squalene synthase gene overexpression of medicago sativa
  • Application and method for squalene synthase gene overexpression of medicago sativa

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Embodiment Construction

[0050] 1. Materials and methods

[0051] 1.1 Plant material cultivation

[0052]Medicago sativa L.'Zhongmu No.3' is a salt-tolerant alfalfa variety bred by Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences. Select plump alfalfa seeds to germinate in petri dishes, then cultivate them in Hongland nutrient solution, place them in an artificial intelligence climate box, temperature 25±1°C, light 16h (day) / 8h (night), humidity 65%, and cultivate for 4 weeks For the extraction of DNA and RNA.

[0053] 1.2 Primer design

[0054] According to the known cds sequence of squalene synthase gene of Medicago truncatula in NCBI, Primer 5.0 was used to design primers MsSQS-f and MsSQS-r to amplify the cDNA sequence of MsSQS gene. According to the alfalfa actin β-actin gene published by Genbank as the housekeeping gene and the cloned MsSQS gene as the target detection gene, a pair of primers Actin-f and Actin-r, SQS-qPCR-f and SQS-qPCR w...

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Abstract

The invention discloses an application and method for squalene synthase gene overexpression of medicago sativa. The method includes steps: (1) primer design; (2) vector pEASY-T3 plasmid cloning, wherein pGEX-4T-1V is used for prokaryotic expression, and escherichia coli E.coli Trans5alpha and E.coli Transetta are used for gene cloning and expression; (3) cloning of full-length cDNA of medicago sativa squalene synthase gene MsSQS; (4) biological information analysis of an MsSQS sequence; (5) expression analysis of the squalene synthase gene MsSQS; (6) determination of squalene synthase content;(7) methyl jasmonate induced saponin content determination; (8) subcellular localization analysis; (9) prokaryotic expression; (10) MsSQS gene over-expression vector construction and medicago sativagenetic transformation. By establishment of an efficient genetic transformation system, overexpression of the squalene synthase gene in medicago sativa is realized, the content of total saponins in transgenic medicago sativa is increased, and breeding materials are provided for cultivating high-saponin-content medicago sativa varieties.

Description

technical field [0001] The invention relates to the field of rate-limiting enzyme genes related to plant saponin synthesis, in particular to a method and application for overexpressing squalene synthase gene in alfalfa. Background technique [0002] Alfalfa saponin (Saponin), also known as saponin and saponin, is composed of saponin plus one or more sugar chains. It is a large class of glycoside compounds commonly found in nature, including triterpenoids, sterols, and steroids. It is distributed in species, ferns, plants, animals and marine life. Although terpenoids are ubiquitous in plants, humans have so far been unable to determine the complete pathway of terpenoid synthesis in any one species. [0003] Squalene synthase (Squalene Synthases, SQS) is a membrane-bound enzyme bound to the endoplasmic reticulum, which catalyzes the condensation of bimolecular farnesyl diphosphate (FPP) to generate squalene, which is a branch of triterpenes and sterols. The first key enzyme ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/82A01H5/00A01H6/54
CPCC12N9/1085C12N15/8243C12Y205/01021
Inventor 康俊梅张铁军张俏燕王珍龙瑞才杨青川
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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