A kind of plant RNAi expression vector and its construction method and application

Abstract

The invention belongs to the technical field of genetic engineering, and in particular relates to a plant RNAi expression vector and its construction method and application. Based on the commercialized plant expression vectors pCambia2301 and pHANNIBIAL, the present invention constructs a plant RNAi expression vector containing a CaMV35S promoter, an intron, an OCS terminator, and two Gateway boxes in opposite directions. Construction of the plant target gene silencing vector; the present invention also establishes the construction method of the plant RNAi expression vector, and the application of the one-step Gateway reaction using fusion PCR to construct the RNAi silencing vector containing the target gene.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a plant RNAi expression vector and a construction method and application thereof. Background technique [0002] Plant mutants are difficult to obtain, some gene mutations may be lethal to plants, and gene silencing is relatively easy to obtain. RNA interference (RNAi) is a post-transcriptional gene silencing technology and an effective method for studying functional genes. Reverse genetics tool. RNAi is a post-transcriptional gene silencing phenomenon mediated by double-stranded RNA (dsRNA) involving specific enzymes, which was first discovered in C. elegans by Andrew Fire and Craig Mello et al. The ubiquitous mechanism is to introduce a partially double-stranded RNA (dsRNA) with a homologous complementary sequence to the target gene's transcription product mRNA into cells, and then efficiently and specifically degrade the homologous mRNA into 21 A small fragment of...

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Problems solved by technology

[0003] The traditional method of constructing plant RNAi expression vectors is enzyme digestion and ligation, cutting and recycling the target gene fragments, which are limited by the cutting point of restriction endonucleases, which is time-consuming and cumbersome, and small fragments are difficult to recover , multiple fragments are not easy to connect, the molecular weight of the plasmid vector is relatively large, the success rate of constructing the vector by enzyme cutting and ligation is not high

[0004] Gateway technology is a commercialized technology of Invitrogen. It is based on the site-specific recombination reaction of phage. The recombination site is added to the target fragment, and the PCR product is mixed with the donor vector containing the recombination site for BP reaction to obtain the entry vector ( Entry clone), the entry vector can perform LR reaction with the target vector of different purposes to obtain the corresponding expression vector, that is to say, the use of this technology to obtain the expression vector usually requires two steps of BP reaction and LR reaction, the experimental cycle is longer and the cost is relatively high. high

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