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Nilaparvata lugens gene NlABCB as well as encoding product and applications thereof

A brown planthopper and genetic technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problem of no brown planthopper being cloned, and achieve the effect of promoting biological control

Active Publication Date: 2016-08-10
INST OF FOOD CROPS HUBEI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, so far, no relevant target gene of brown planthopper has been cloned at home and abroad, and the purpose of controlling brown planthopper can be achieved by silencing the gene through artificial feeding of dsRNA

Method used

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  • Nilaparvata lugens gene NlABCB as well as encoding product and applications thereof
  • Nilaparvata lugens gene NlABCB as well as encoding product and applications thereof
  • Nilaparvata lugens gene NlABCB as well as encoding product and applications thereof

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Experimental program
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Embodiment 1

[0026] Embodiment 1: Obtaining and sequence analysis of NlABCB gene

[0027] 1) The total RNA of N. lugens was extracted using the total RNA extraction kit RNAiso Plus. Using the extracted total RNA as a template, 5' and 3' RACE cDNAs were synthesized using the RACE kit, respectively.

[0028] 2) Outer PCR reaction program: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, and extension at 72°C for 10 min after 20 cycles. The Inner PCR reaction program is the same as the above program, but changed to 30 cycles.

[0029]3) The PCR product was cloned into the pMD19-T vector of Takara Company to obtain the pMD19-N1ABCB vector, transformed into Top10 Escherichia coli, and the Top10 bacterial liquid containing the pMD19-N1ABCB vector was sent to Beijing Qingke Company for sequencing. NCBI Blast (http: / / www.ncbi.nlm.nih.gov / BLAST / ) was used for sequence comparison analysis to obtain the sequence SEQ ID No.1 ...

Embodiment 2

[0030] Example 2: Expression of brown planthopper L4440-NlABCB-dsRNA and its RNAi effect

[0031] Step 1: Prokaryotic expression vector of BPH L4440-NlABCB

[0032] 1) Amplification of fragments

[0033] The highly expressed NlABCB sequence was selected to design primers, the upstream primer was added to the Nco I site, and the upstream primer was added to the Sma I site. The target gene was amplified using the brown planthopper cDNA as a template, and the high-fidelity enzyme KOD PLUS was used to amplify in a 25 μL reaction system. The reaction conditions were as follows: pre-denaturation at 94°C for 2 min, followed by 28 cycles of PCR amplification (94°C for 15 s, 55°C for 30 s, and 68°C for 30 s). Ordinary Taq was used for adding A reaction, ligated transformation and identification of positive clones. After the sequenced correct fragment plasmid was double digested with Nco I and Sma I, it was gel-cut, purified and recovered, and placed at 4°C for later use.

[0034] 2)...

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Abstract

The invention discloses a nilaparvata lugens gene NlABCB as well as an encoding product and applications of the nilaparvata lugens gene NlABCB. The gene NlABCB has the DNA sequence shown as the SEQ ID No.1. The sequence is composed of 1468 nucleotide basic groups, and comprises a complete encoding frame composed of 459 nucleotides, and protein with small molecular weight of 153 amino acid residues is encoded. The research finds that the gene is relevant to the digestion feeding and survival of nilaparvata lugens. A dsRNA carrier of L4440-NlABCB is constructed, after conversion of E. coli HT115 and through the IPTG induction, the NlABCB-dsRNA is formed, and after the nilaparvata lugens is fed with the artificial feed containing the NlABCB-dsRNA, the survival rate of nilaparvata lugens is obviously reduced. The nilaparvata lugens gene NlABCB and an encoding product of the nilaparvata lugens gene NlABCB can be widely applied in the crop breeding, biopesticides and transgenic animal culture.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a brown planthopper gene N1ABCB and its encoded product and application. Background technique [0002] Rice (Oryza sativa) is one of the main food crops in the world, and more than half of the world's population uses rice as a staple food. Experts estimate that by 2025, the world's rice production will have to increase by 60% to meet the demand. my country is a big rice producing country, with an annual rice planting area of ​​about 30 million hectares, accounting for 1 / 4 of the world's total rice planting area. Therefore, the safety of rice planting is inseparable from my country's economic development and people's lives. [0003] Brown planthopper (Nilaparvata lugens BPH) is an insect belonging to the family Homoptera (Homoptera: Delphacidae), also known as brown rice lice. The brown planthopper mainly sucks the phloem juice of rice, and the first damage is the leaf sheath tis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/435
CPCC07K14/43563
Inventor 游艾青闸雯俊周雷李三和杨国才陈志军刘凯徐华山李培德
Owner INST OF FOOD CROPS HUBEI ACAD OF AGRI SCI
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