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Plasmid for constructing interference vector and construction method and application thereof

A technology for interfering with vectors and plasmids, applied in the direction of introducing foreign genetic material, applications, botany equipment and methods using vectors, etc. It can solve problems such as negative effects on plant growth and development, and achieve the effect of improving quality and high interference efficiency

Inactive Publication Date: 2010-11-10
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have found that there are some defects in the expression of foreign genes under the control of constitutive promoters, which often have unexpected negative effects on plant growth and development

Method used

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  • Plasmid for constructing interference vector and construction method and application thereof
  • Plasmid for constructing interference vector and construction method and application thereof
  • Plasmid for constructing interference vector and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1, construction of plasmid pFGC-NAP

[0023] 1. Preparation of seed-specific promoter fragments

[0024] 1. Extraction of Rapeseed Genomic DNA

[0025] The young leaves of rapeseed (Brassica napus variety Gaoyou 605; purchased from Zhejiang Fengqi Seed Industry Co., Ltd.) were taken, and genomic DNA was extracted with a plant genomic DNA extraction kit (catalogue number: DP320-02).

[0026] 2. PCR amplification of NAPIN gene promoter fragment

[0027] Using genomic DNA as a template, a pair of primers were designed (the 5' ends of the two primers were introduced into Stu I and Asc I restriction recognition sequences and protective bases, respectively).

[0028] Upstream primer S1: 5'-GA AGGCCT TCTTCATCGGTGATTGAT-3' (the underline is the Stu I recognition sequence);

[0029] Downstream primer S2: 5'-A GGCGCGCC TGTATGTTTTTAATCTTG-3' (Asc I recognition sequence is underlined).

[0030] PCR amplification was performed using the above primers. A PCR produ...

Embodiment 2

[0051] Embodiment 2, construction and application of interference carrier

[0052] Δ12 fatty acid desaturase (FAD2) is a key enzyme for plants to produce polyunsaturated fatty acids. FAD2-1 is specifically expressed in seeds during embryonic development and is considered to be the key enzyme responsible for further desaturation of oil in seeds. Inhibiting the FAD2-1 gene specifically expressed in cotton seeds by means of RNA interference can greatly reduce the production of linoleic acid and α-linolenic acid in cottonseed fatty acids, thereby increasing the content of oleic acid that is beneficial to production. Therefore, in this example, the plasmid pFGC-NAP constructed in Example 1 was used to construct an RNA interference expression vector using cotton FAD2-1 as an interference target gene.

[0053] 1. Preparation of target gene interference fragment

[0054] 1. Extraction of cotton total RNA

[0055] Cotton immature embryos (Kezimian 201, purchased from the Cotton Resea...

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Abstract

The invention discloses a plasmid for constructing an interference vector, a construction method thereof and application thereof. The invention provides a plasmid pFGC-NAP obtained by inserting a DNA presented by sequence 1 in a sequence list between an Stu I enzyme cutting site and an Asc I enzyme cutting site of a plasmid pFGC1008. The interference vector can be obtained by respectively inserting a plus-sense DNA fragment and an anti-sense DNA fragment into a multiple cloning site of the plasmid pFGC-NAP, wherein the plus-sense DNA fragment and the anti-sense DNA fragment are inverted repeat sequences designed for a target gene to be interfered and expressed. The plasmid constructs the interference vector capable of specially expressing a promoter during a seed development period, has high interference efficiency, can control a spatial temporal specificity expression, can be used for a specific gene expressed in silent plant seeds, and improves quality of oil crop seeds or other plant seeds.

Description

technical field [0001] The invention relates to a plasmid used for constructing an interference vector, a construction method and application thereof. Background technique [0002] RNA interference is a cellular response process in which double-stranded RNA (double strained RNA, dsRNA) is introduced into cells to cause the degradation of specific target gene mRNA. It is a post-transcriptional gene silencing (PTGS) phenomenon. The discussion on the mechanism of RNAi is not perfect enough. The current research has a relatively clear conclusion: Dicer is a member of the specific ribonuclease family, and the exogenous dsRNA that is complementary to the endogenous mRNA binds to Dicer and is cut into 21~23nt Long, small fragments known as small interfering ribonucleic acid (siRNA). A part of siRNA and Dicer form an RNA-inducing silencing complex (RISC), and RISC then binds to the mRNA transcribed from the target gene and degrades the mRNA; some siRNA can be used as primers to syn...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/113A01H5/00
Inventor 郭仰东赵立群杨鹏刘莉莎温常龙王玉珏程琳李蔚李仁
Owner CHINA AGRI UNIV
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