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RNAi expression vector of Plutella xylostella tor gene and its construction method and application

A technology of expression vectors and plant expression vectors, applied in application, genetic engineering, plant genetic improvement, etc., to achieve the effect of delaying pest resistance and reducing the use of pesticides

Inactive Publication Date: 2018-12-11
CHONGQING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Construction of TOR gene RNAi vector to interfere with the TOR gene of diamondback moth has not been studied in diamondback moth

Method used

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  • RNAi expression vector of Plutella xylostella tor gene and its construction method and application
  • RNAi expression vector of Plutella xylostella tor gene and its construction method and application
  • RNAi expression vector of Plutella xylostella tor gene and its construction method and application

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Experimental program
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Embodiment 1

[0031] The construction of embodiment 1 Plutella xylostella TOR gene RNAi vector

[0032] 1. Acquisition and synthesis of TOR gene RNAi fragment of diamondback moth

[0033] Through the Plutella xylostella genome database ( http: / / iae.fafu.edu.cn / DBM / ), find out the full-length CDS sequence of the TOR gene of Plutella xylostella: TOR1: 7140bp; TOR2: 6882bp. Using the CDS of Plutella xylostella TOR to perform homologous alignment in NCBI, find out the three most conserved sequences, and fuse the three sequences. Add an Asc I-Not I restriction site at the 5' end of the fused sequence, add a SbfI-BbvC I restriction site at the 3' end, and name the sequence TOR-RNAi (658bp), and the sequence is as follows: As shown in the list Seq ID No: 1, the sequence was synthesized.

[0034] 2. Construction of Plutella xylostella TOR gene RNAi vector

[0035] Follow the steps below:

[0036] (1) Synthesize the sequence shown in the sequence table Seq ID No: 2, the sequence is named 35S-IN...

Embodiment 2

[0055] The acquisition of embodiment 2 transgenic Arabidopsis plants

[0056] Follow the steps below:

[0057] (1) The pE35S-TORRI vector obtained in Example 1 is transformed into the Agrobacterium LBA4404 bacterial strain, and the transformation steps are: 1) adding the pE35S-TORRI vector plasmid DNA (using plasmid Extracted by DNA extraction kit, refer to the kit manual for operation method) 5 μL, mix gently and then ice-bath for 30 minutes; 2) Put it in liquid nitrogen for 1 minute, then immediately put it in a 37°C water bath for 5 minutes; 3) Take it out and centrifuge put the tube on ice for 3 minutes; 4) add the liquid in the centrifuge tube to 500 μL LB liquid medium in the ultra-clean workbench, shake and culture at 28 ° C and 200 rpm for 4 h; 5) take out the bacterial solution in the Ping and Kanamycin 50 mg / L LB solid medium were plated, and after drying, they were cultured upside down in a 28°C incubator. Colonies were visible in about 2 days.

[0058] A single co...

Embodiment 3

[0061] Example 3 Research on insect resistance of transgenic Arabidopsis

[0062] 1. Group rearing of diamondback moth

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Abstract

The invention discloses an RNAi (ribonucleic acid interference) expression vector for TOR (target of rapamycin) genes of plutella xylostella, a method for constructing the RNAi expression vector and application thereof. The RNAi expression vector comprises separate polynucleotide, a vector and a recombinant vector. Sequences of the separate polynucleotide are shown as SEQ ID NO:1 and SEQ ID NO:2; the vector comprises the polynucleotide with the sequences shown as SEQ ID NO:1 or SEQ ID NO:2; the recombinant vector comprises sequences shown as SEQ ID NO:7, and pEarleyGate 303 is used as a skeleton of the RNAi expression vector for the TOR genes of the plutella xylostella. The method for preparing the RNAi expression vector includes displacing and inserting EcoR I, AsiS I, P35S promoters, Not I, the sequences SEQ ID NO:1, Sbf I, fourth introns of ACTIN, BbvC I, reverse complement sequences of the sequences SEQ ID NO:1, Asc I, T35S terminators and EcoR I, which are sequentially connected with one another, in recombination sites.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to an RNAi expression vector and its construction method and application, in particular to an RNAi expression vector of Plutella xylostella TOR gene and its construction method and application. Background technique [0002] The diamondback moth (Plutella xylostella), also known as the small caterpillar with two pointed ends, is an important pest of cruciferous vegetables such as cabbage, rape, cauliflower, cabbage, and radish, and is also a widely distributed pest in the world. The first instar larvae only eat the mesophyll, leaving the epidermis, forming transparent spots on the vegetable leaves; the 3rd to 4th instar larvae can eat the vegetable leaves into holes and notches, and in severe cases, the whole leaf is eaten into a net shape. Due to its multiple generations, strong reproductive ability, wide range of hosts, and high level of drug resistance, diamondback moth gradually r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N15/63C12N15/66C12N15/82A01H5/00A01H6/20
Inventor 任茂智李林宣冯丽
Owner CHONGQING UNIV
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