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Plant RNAi expression vector and construction method and application thereof

An expression vector and RNA structure technology, applied in the field of genetic engineering, can solve the problems of cumbersome, large molecular weight of plasmid vector, long experimental period, etc., and achieve the effect of simplifying construction steps, shortening experimental period and saving experimental cost.

Active Publication Date: 2018-07-31
ZHOUKOU NORMAL UNIV
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  • Claims
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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

Method used

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  • Plant RNAi expression vector and construction method and application thereof
  • Plant RNAi expression vector and construction method and application thereof
  • Plant RNAi expression vector and construction method and application thereof

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Embodiment 1

[0045] Example 1 Construction of plant RNAi expression vector pCambia2301-GW-RNAi

[0046] The pCambia2301 vector, the pHANNIBAL vector, and the pBS-Gateway-rfA vector are commercial vectors.

[0047] The construction of plant RNAi expression vector pCambia2301-GW-RNAi comprises the following steps:

[0048] Step 1: Single enzyme digestion of pHANNIBAL vector

[0049] The pHANNIBAL vector is an intermediate vector commonly used to construct RNAi expression vectors, such as figure 2 shown. The traditional method is to insert a 200-300bp fragment of the target gene into the multiple cloning sites on both sides of the intron in the pHANNIBAL vector in the opposite direction, and then use not Ⅰ Cut out the fragment from the CaMV35S promoter to the OCS terminator, connect it with the plant expression vector, and construct the final RNAi expression vector. The present invention uses the pHANNIBAL vector as an intermediate vector to construct a plant RNAi expression vector. Fi...

Embodiment 2

[0061] Embodiment 2 barley CEBiPConstruction and detection of gene RNAi silencing vector

[0062] 1. Plant material and cultivation conditions:

[0063] Plant material: Nicotiana benthamiana; culture conditions: greenhouse cultivation; 16h light (150μmol / (m 2 s)), temperature 21°C; 8h dark, temperature 19°C; relative humidity 55%.

[0064] 2. Experimental carrier:

[0065] The pCambia2301 vector, the pHANNIBAL vector, the pBS-Gateway-rfA vector, and the pDONR207 vector are commercial vectors. Using Gateway technology, the CEBiP The gene fragment was integrated into the donor vector pDONR207 to obtain the entry vector pENTY- CEBiP ; then the entry vector pENTY- CEBiP Carry out LR reaction with the carrier CTAPi-35ss-GW-3HA to obtain the carrier 35ss- CEBiP -3HA, carrier 35ss- CEBiP -3HA expresses CEBiP and 3HA tag fusion, which is convenient for later detection. HA is an antigen tag, which is an amino acid sequence from influenza virus.

[0066] 3. Barley CEBiP...

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Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a plant RNAi expression vector and a construction method and application thereof. The plant RNAi expression vector containing a CaMV35S promoter, an intron, an OCS terminator, and two opposite-direction Gateway boxes is constructed based on commercial plant expression vectors pCambia2301 and pHANNIBIAL. The plant RNAi expression vector can be used for construction of a plant gene silencing vector by using Gateway technology; the present invention also establishes the construction method of the plant RNAi expression vector and application of the plant RNAi expression vector in constructing of a target-gene-containing RNAi silencing vector by use of fusion PCR for a one-step Gateway reaction.

Description

technical field [0001] 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. Background technique [0002] Plant mutants are difficult to obtain. Some gene mutations may be lethal to plants. Gene silencing is relatively easy to obtain. RNA interference (RNA interference, RNAi) is a post-transcriptional gene silencing technique and is an effective method for studying functional genes. Reverse genetics tools. RNAi is a post-transcriptional gene silencing phenomenon mediated by double-stranded RNA (dsRNA) and involved in specific enzymes. This phenomenon was first discovered in nematodes by Andrew Fire and Craig Mello. It is a very conserved and The ubiquitous mechanism is to introduce a partially double-stranded RNA (dsRNA) with a homologous and complementary sequence to the transcription product mRNA of the target gene into cells, and then efficiently and s...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N5/10C12N15/66
CPCC12N15/8218C07K14/415
Inventor 李成伟廖立冰于德水张菊张怡徐克东刘坤谭光轩陈璨何勇付贝贝
Owner ZHOUKOU NORMAL UNIV
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