Chilo venosatus walker ecdysis regulation transcription factor cDNA and cloning method and recombinant application thereof
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A technique for sugarcane stalk borer and regulating transcription, which is applied in the field of biogenetic engineering technology, and can solve the problems of insect death, difficulty, and high technical requirements
Inactive Publication Date: 2012-07-18
广西康田农业科技股份有限公司
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Problems solved by technology
Although the injection method of RNAi has made great achievements in insect functional genes, there are still many problems as follows: First, the injection method of RNAi requires high technical requirements for experimental operators, is difficult and intensive, and it is very easy to cause data loss due to improper operation. Do not allow and kill insects
In the GenBank database, there is no record or research report on the molt regulator gene of the sugarcane borer
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Embodiment 1
[0065] Sugarcane borer ( Chilo sacchariphagus ) Key genes for growth and development CsHR3 clone.
[0066] 1. Extraction of total RNA: The total RNA of the prepupal stage larvae of the sugarcane stem borer was extracted in one step by the Trizol method of Invitrogen Company.
[0067] 2. Synthesize cDNA: refer to the cDNA kit instructions of Dalian Bao Bioengineering Co., Ltd., specifically: take 5 μl of total RNA, 1 μl of Random primer, 2 μl of substrate dNTPs, and use RNase-free H 2 Add O to 10 μl, mix well, incubate at 65°C for 5 minutes, quickly place on ice to cool for 3 minutes; then add 4 μl of 5×Primer Script Buffer, 0.5 μl of RNase inhibitor, and 0.5 μl of AMV reverse transcriptase to the above mixture , and use RNase-free H 2 O was added to 20 μl, reacted at 42°C for 60 minutes, extinguished at 70°C for 15 minutes, cooled at 4°C, and stored at -20°C for later use.
[0068] 3. PCR amplification of sugarcane barworm CsHR3 The middle segment of the gene: design deg...
Embodiment 2
[0090] Construct bacteria-mediated RNAi recombinant vectors L4440-CsHR3-I1, L4440-CsHR3-I2, and select the green fluorescent protein gene at the same time EGFP As a negative control, clear water was used as a negative control. Bacteria expressing dsRNA feeding experiment, divided into 4 treatment groups, respectively fed 3rd and 5th instar larvae of sugarcane borer with the same growth, and repeated the experiment 3 times. The specific operation is as follows:
[0091] 1, CsHR3 Selection of target interfering gene fragments
[0092] Considering the molting-regulating transcription factor HR3 It is a gene expression regulatory factor that plays an important role in the process of insect molt, so this embodiment selects the sugarcane molt molt regulator transcription factor CsHR3 genes as targets, the selected CsHR3 The gene contains two dsRNA interference fragments of non-LBD functional domain and LBD functional domain (ie interference fragment 1 and 2, the nucleotide se...
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Abstract
The invention belongs to the field of biological gene engineering. A chilo venosatus walker ecdysis regulation transcription factor CsHR3 contains sequences shown by SEQ ID NO.1 and SEQ ID NO.2. The cloning method provided by the invention comprises the following steps of: extracting total RNA (ribonucleic acid) from chilo venosatus walker larvas in prepupal period, and carrying out reverse transcription to synthesize cDNA; designing a degenerate primer according to conserved sequences of different lepidopterous insect ecdysis regulation transcription factors, carrying out PCR (polymerase chain reaction) amplification on the intermediate segment of the chilo venosatus walker CsHR3 gene, designing a specific primer by adopting 3'-RACE (rapid-amplification of cDNA ends) technology, and then obtaining the 1266bp segment of the CsHR3 gene successfully. According to the invention, dsRNA is expressed in RNaseIII defective escherichia coli HT 115 by adopting a bacterium mediate RNAi technology to obtain dsRNA with biological activity, and the chilo venosatus walker ecdysis regulation transcription factor CsHR3 gene is silenced by feeding the chilo venosatus walker larvas, thus chilo venosatus walker can not complete ecdysis or normal growth and development process of the chilo venosatus walker is destroyed and biological prevention and control on the chilo venosatus walker is realized; or the chilo venosatus walker ecdysis regulation transcription factor is converted into a crop, and the crop with insect resistance is obtained.
Description
technical field [0001] The invention belongs to the cloning and expression technology in the technical field of biogenetic engineering, and specifically relates to the key gene for the growth and development of the sugarcane moth moth - molt regulation transcription factor CsHR3 Gene cloning and bacteria-mediated RNAi expression CsHR3 dsRNA to silence molting-regulating transcription factors in sugarcane barworm CsHR3 The gene prevents the sugarcane borer from molting or disrupts its normal growth and development process. Background technique [0002] With the rapid growth of world population, how to increase crop yield is an urgent problem. According to statistics, grain losses caused by insect pests account for 20% of the entire grain harvest. Therefore, how to effectively control insect pests is an important factor in improving crop yield. Although the extensive use of chemical pesticides can suppress insect pests, it is easy to cause insect resistance, and it will c...
Claims
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