Method for improving insect resistance of plants by using RNA interference technique and special DNA fragment of method

A technology of insect resistance and fragmentation, applied in the biological field, can solve the problems of insect resistance evolution, single excellent insect resistance gene, and unsatisfactory insect resistance of transgenic plants, so as to achieve the effect of reducing damage and improving insect resistance

Active Publication Date: 2019-06-04
HEBEI UNIVERSITY
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Problems solved by technology

[0010] The purpose of the present invention is to provide a method for improving plant insect resistance by using RNA interference technology and its special DNA fragment, s...

Method used

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  • Method for improving insect resistance of plants by using RNA interference technique and special DNA fragment of method
  • Method for improving insect resistance of plants by using RNA interference technique and special DNA fragment of method
  • Method for improving insect resistance of plants by using RNA interference technique and special DNA fragment of method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The gene sequence was obtained by RT-PCR (reverse transcription-polymerase chain reaction) technology.

[0047] 1. PCR primer design

[0048] The PCR primer sequence of chitin deacetylase 5a gene (HaCDA5a) of cotton bollworm is CDA5aF: 5'-ATGAAGTTGTTCGGGCTTCTTG-3', CDA5aR: 5'-CAGTTGTAGATTTATTGTCCAAG-3'.

[0049] 2. Total RNA extraction and cDNA synthesis and amplification of cotton bollworm

[0050] The total RNA of the 5th instar cotton bollworm was extracted with TRIzol reagent, and the first strand of cDNA was synthesized with reverse transcriptase. PCR reaction system: 2 μL 10×Buffer, 1 μmol / L upstream / downstream primers, 0.1 mmol / L dNTPs, 1 unit Taq polymerase, 0.5 μL cDNA, ddH 2 O to make up to 20 μL. PCR reaction program: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 2 min, 35 cycles; final extension at 72°C for 10 min.

[0051] 3. PCR product recovery, TA cloning and identification

[00...

Embodiment 2

[0054] 1. Preparation of target sequence

[0055] PCR technology was used to amplify the above positive clones to prepare target sequences, and the primer sequences were:

[0056] CDAI5aF: 5'-accaggtctcaggagATGAAGTTGTTCGGGCTTCTTG-3',

[0057] CDAI5aR: 5'-accaggtctcatcgtTACTCCAAGCCATATTCCTG-3',

[0058] The lowercase part of the 15 bases at the 5' end of the primer is a partial sequence required by the Golden gate cloning technique. The size of the amplified target gene sequence is 578bp (HaCDA5a, shown as sequence 2 in the sequence listing).

[0059] 2. PCR amplification to obtain target gene fragments

[0060] The PCR amplification system is: 0.6 μL of plasmid template, 2.4 μL of corresponding upstream and downstream primers, 15 μL of 2×Taq MasterMix, ddH 2 Make up 30 μL with O; the PCR amplification program is: 95°C, 3min; 95°C, 30s; 52°C, 30s; 72°C, 40s, 35 cycles; 72°C, 10min. The amplified PCR product was subjected to agarose gel electrophoresis, and the DNA electrop...

Embodiment 3

[0062] Construction of plant hpRNA expression vector

[0063] The backbone of the plant expression vector used in this example is pRNAi-GG, and there is an hpRNA expression cassette on the vector plasmid: driven by the CaMV35S promoter and terminated by the Nos terminator; the transcribed region has two bacterial ccdB separated by an intron sequence Gene; each ccdB gene sequence has two Bsa I endonuclease (IIS type) recognition sites at both ends.

[0064] Plant expression vectors were constructed using Goldgate technology. Put the target sequence PCR amplification product and pRNAi-GG plasmid in one tube, and add Bsa I and T4 DNA ligase (using T4 ligase reaction buffer) at the same time for GoldenGate reaction. The reaction system is: pRNAi-GG plasmid 200 ng , target sequence PCR product 50 ng, T4 ligase 350 U (TAKARA company), T4 ligase buffer 1 μL (TAKARA company), Bsa I 5 U (NEB company), ddH 2 O to make up 10 μL. After transformation, sequencing identification and othe...

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Abstract

The invention provides a method for improving insect resistance of plants by using an RNA interference technique and a special DNA fragment of the method. The DNA fragment is shown as formula (I), andSEQ forward -X-SEQ backward (I), wherein the SEQ forward is any one fragment at least comprising 21bp in the overall length cDNA fragment of bollworm chitin deacetylase 5a genes; SEQ backward is in backward complementary with the SEQ forward; the X is an intervening sequence between the SEQ forward and SEQ backward, and is not complementary with the SEQ forward and SEQ backward, and the overall length cDNA of the bollworm chitin deacetylase 5a gene is shown as the sequence 1 in a sequence table. For the first time, the bollworm chitin deacetylase 5a gene is used as the RNA interference targetgene, and is applied to breeding of genetically-modified plants. Insect test results prove that after switching for 2-5 days, the body weight of young bollworms is 1/10 smaller than that of the control group, and the mortality rate of the young insects of 5 days on genetically modified tobacco leaves achieves 68%, and the mortality rate of wild type tobacco is only 21%.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for improving plant insect resistance by using RNA interference technology and a special DNA fragment thereof. Background technique [0002] The ingestion of dsRNA (double-stranded RNA) by eukaryotic cells will cause the degradation of the homologous mRNA in the cell, thereby blocking the expression of the corresponding gene. This phenomenon is called RNA interference (RNA interference, RNAi). Plant-mediated RNAi technology is to introduce the gene expression cassette expressing insect gene dsRNA (or hpRNA, that is, hairpin structure RNA) into plants through transgenic technology. After the pests eat the plant material containing the pest gene dsRNA / hpRNA , the ingested dsRNA / hpRNA will cause RNAi in pests, which may inhibit the growth and development of insects and even kill pests, so as to achieve the purpose of protecting plants. [0003] In 2007, Mao YB et al. expressed...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N15/82A01H5/00A01H6/60
Inventor 李继刚史月芝刘娟娟张石凯李洪宇
Owner HEBEI UNIVERSITY
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