RNAi vector for expressing dsRNA of gene HaAK in transgenic plant and application of RNAi vector

A transgenic and genetic technology, applied in the field of agricultural biology, can solve the problems of increasing drug resistance of cotton bollworm, endangering life safety and health, and reducing the quality of crop products.

Active Publication Date: 2015-06-10
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The indiscriminate application of highly toxic and high-residue pesticides has not only led to a decline in the quality of crop products and an increase in the resistance of cotton bollworms, but has also endangered people's lives and health

Method used

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  • RNAi vector for expressing dsRNA of gene HaAK in transgenic plant and application of RNAi vector
  • RNAi vector for expressing dsRNA of gene HaAK in transgenic plant and application of RNAi vector
  • RNAi vector for expressing dsRNA of gene HaAK in transgenic plant and application of RNAi vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1, the cloning of HaAK gene:

[0021] (1) Get 1-2 heads of cotton bollworm, and extract total RNA with TRIzol method;

[0022] (2) Synthesizing the first strand of cDNA;

[0023] (3) Obtain the gene fragment sequence from the transcriptome of cotton bollworm, and after performing homology comparison in the NCBI database, design F1 / R1 according to the sequence using Primer premier 5.0 software;

[0024] (4) F1: 5'-ATGGTGGACGCCGCAACAAT-3';

[0025] (5) R1: 5'-TTACAGCGACTTCTCAATT-3'.

[0026] (6) Using the first strand of the cotton bollworm cDNA synthesized in step (2) as a template, and using the primers in (4) and (5) to perform RT-PCR amplification. The RT-PCR reaction program was: denaturation at 94°C for 5 min; 35 cycles (1 min at 94°C, 1 min at 56°C, 1 min at 72°C); 10 min at 72°C. Reaction system (25μL): 10×Ex PCR Buffer 2.5μL, 2.5mM dNTP Mixture 2.0μL, 10μM upstream primer F11μL, 10μM downstream primer R11μL, cDNA template, 2μL, 5U / μL Ex Taq 0.5μL, ...

Embodiment 2

[0029] Embodiment 2, the construction of RNAi plant expression vector

[0030](1) Based on the cloned HaAK gene sequence of the cotton bollworm, design the specific forward primer F2: 5′-CACCATGGTGGACGCCGCAACAAT-3′ (add four bases to the specific primer F1), and use R1: 5′- Using TTACAGCGACTTCTCAATT-3' as the reverse primer, the plasmid pMD-HaAK was amplified by PCR with Prime STAR™ HS DNA polymerase. The amplification reaction system is (total system 25 μL): 0.25 μL of 5×Prime STAR enzyme, 2.5 μL of 5×Prime STAR Buffer, 2 μL of dNTPs, 1 μL of each primer F2 / R1, 0.5 μL of template pMD18-HaAK, supplemented with ddH 2 0 to 25 μL. The amplification conditions were: 94°C for 5 min, 30 cycles (94°C for 1 min, 56°C for 1 min, 72°C for 1 min), and 72°C for 10 min.

[0031] (2) Recover the amplified blunt-end PCR product, and carry out TOPO directional cloning with the pENTR / D-TOPO vector to obtain pENTR / D-HaAK.

[0032] (3) Transform Escherichia coli strain E.coli TOP 10 competent...

Embodiment 3

[0035] Example 3, Agrobacterium-mediated transformation of Arabidopsis

[0036] (1) Preparation of Agrobacterium Competent Cells

[0037] Agrobacterium LBA4404 stored at -80°C was streak-inoculated on YEB solid medium containing 50 μg / mL rifampicin (Rif), cultured at 28°C for 48 hours, single clones of Agrobacterium LBA 4404 were picked, and inoculated in 5ml YEB (50 μg / mL Rif) liquid medium, cultured at 28°C and 200rpm for 48h, inoculated the overnight culture solution into the YEB liquid medium (erlenmeyer flask) containing 50μg / mL Rif with an inoculum size of 1:100, continued at 28°C and 200rpm Grow to OD 600 The value is about 0.5, about 6-10h. Transfer the bacterial solution to a 50mL sterilized centrifuge tube, put it in an ice bath for 30min, centrifuge the bacterial solution at 5000rpm at 4°C for 5min, and discard the supernatant. Add 10 mL of pre-cooled 0.1M (sterilized) NaCl to the centrifuge tube to thoroughly suspend the Agrobacterium cell pellet (the whole ope...

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Abstract

The invention relates to an RNAi vector for expressing the dsRNA of a gene HaAK in a transgenic plant and application of the RNAi vector, and belongs to the field of agricultural biotechnologies. The invention provides a method for controlling helicoverpa armigera by selecting a helicoverpa armigera energy metabolism key enzyme gene arginine kinase gene HaAK as a target gene of RNAi. The invention particularly relates to an RNAi vector for expressing the dsRNA of helicoverpa armigera HaAK in the transgenic plant and the application of the RNAi vector to a silent target gene. The vector is constructed by inserting two HaAK segments into a plant expression vector in two opposite directions; and the transgenic plant expressing the dsRNA of the gene HaAK is cultivated under agrobacterium mediation. The comparison between the transgenic plant and a wide plants shows that the growth and development of helicoverpa armigera eating the transgenic plant can be affected.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology; more specifically, the invention relates to an RNAi vector expressing double-stranded RNA (dsRNA) of cotton bollworm Ha4K in transgenic plants and its application in silencing the target gene of cotton bollworm. Background technique [0002] In our country, spraying insecticides on cotton bollworm is still the main control method at present. The indiscriminate application of highly toxic and high-residue pesticides has not only led to a decline in the quality of crop products and an increase in the resistance of cotton bollworms, but has also endangered people's lives and health. Therefore, there is an urgent need to develop safe, effective and new strategies to control the harm of pest populations in production. [0003] RNA interference (RNA interference, RNAi) refers to the phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA (double-stra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/70C12N15/74A01H5/00
Inventor 刘峰赵伊英汪小东孙杰
Owner SHIHEZI UNIVERSITY
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