Pine wood nematode RNAi regulatory gene and application thereof

A technology for regulating genes and pine wood nematodes, applied in the field of bioengineering, can solve the problems of fast onset, death of pine trees, and lack of effective and sustainable prevention and control measures, and achieve the effect of high larval mortality.

Active Publication Date: 2019-03-01
BEIJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] Pine wood nematode (Bursaphelenchus xylophilus) feeds on pine trees, causing pine wood nematode disease and causing massive death of pine trees. ...

Method used

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  • Pine wood nematode RNAi regulatory gene and application thereof
  • Pine wood nematode RNAi regulatory gene and application thereof
  • Pine wood nematode RNAi regulatory gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] On the NCBI website, through BLAST analysis, first find the protein with the highest homology to the Caenorhabditis elegans ETR-1 protein, the amino acid sequence of the protein is shown in SEQ ID NO: 3, the domain of the Caenorhabditis elegans ETR protein Such as figure 1 shown. Then by BLAST analysis, find the full-length sequence of the gene encoding the amino acid sequence shown in SEQ ID NO:3 (as shown in SEQ ID NO:1) and the full-length cDNA sequence (as shown in SEQ ID NO:2) in the pine wood nematode genome . The sequence was verified by PCR and RT-PCR, the full length of Bx-etr-1 gene (2748bp) and the full length of cDNA (1539bp) were obtained, and the size of its encoded protein was 512aa.

[0033] Total RNA of B. xylophilus was extracted using Trizol or RNA extraction kit. First-strand cDNA was synthesized according to PrimeScript™ II 1ststrand cDNA Synthesis Kit (TaKaRa, Japan). Design the forward and reverse primers etr-1-F / R of the etr-1 gene interferen...

Embodiment 2

[0037] The construction method of the recombinant expression vector containing cDNA fragment sequence comprises the following steps:

[0038] (1) Add HindⅢ and XbaI (HindⅢ-etr-1-F / XbaI-etr-1-R) to the 5' ends of the forward primer etr-1-F and the reverse primer etr-1-R, respectively, and amplify Upstream fragment; and the restriction site sequence (ApaI-etr-1-F / SpeI-etr- 1-R) (see Table 1), amplify the downstream fragment, and the reaction system for RT-PCR amplification is shown in Table 2.

[0039] Table 2

[0040]

[0041] The reaction conditions are

[0042]

[0043] (2) Use HindⅢ and XbaI enzymes to digest the plasmid and the upstream amplified fragment respectively, and perform ligation after digestion; then use ApaI and SpeI enzymes to digest the downstream amplified fragment and the ligated product, and perform ligation after enzyme digestion. The pDH-RH plasmid and the amplified fragment were both digested with HindⅢ and XbaI, and ApaI and SpeI, respectively,...

Embodiment 3

[0051] The construction method of recombinant expression bacterial strain comprises the following steps: the plasmid that correctly expresses Bx-etr-1 gene dsRNA that embodiment 2 obtains is transformed in Agrobacterium, in containing kanamycin (100mg / L) and rifampicin ( 50mg / L) positive clones were screened on the LB plate. The plasmid of the positive strain was extracted, and the upstream and downstream primers were used for PCR amplification to carry out molecular identification, and the positive strain of the forward and reverse insertion fragments was obtained.

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Abstract

The invention provides a pine wood nematode RNAi regulatory gene. The full-length sequence of the RNAi regulatory gene is shown in SEQ ID NO:1, and the cDNA full-length sequence of the RNAi regulatorygene is shown in SEQ ID NO:2. The invention also provides application of the pine wood nematode RNAi regulatory gene in preparing a pine wood nematode control bactericide. A high-efficiency RNAi regulatory gene segment is screened, a positive fungal transformant containing the gene segment is fed to pine wood nematodes to obtains the distinct RNAi phenotype, and the Bx-etr-1 gene expression of the pine wood nematodes is obviously down-regulated. The pine wood nematodes have abnormal embryonic development, the larval mortality is high, the motion trail is changed, the motion speed is low, andthe population quantity is decreased significantly.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a pine wood nematode RNAi regulation gene and application thereof. Background technique [0002] Pine wood nematode (Bursaphelenchus xylophilus) feeds on pine trees, and the pine wood nematode disease caused by it leads to the death of pine trees in large numbers. Moreover, its disease speed is fast, and its prevention and control is difficult. So far, there is no effective and sustainable prevention and control measure. [0003] RNA interference (RNAi) mainly refers to the phenomenon that exogenous or endogenous double-stranded RNA (dsRNA) can cause the corresponding target gene mRNA to decrease. The use of RNAi technology has been applied in the prevention and control of plant parasitic nematodes, that is, the control of the populations of agriculturally important root-knot nematodes and cyst nematodes through host plant-mediated RNAi has become a new control method wit...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/113C12N1/21C12N1/15A01K67/033C12R1/01C12R1/77
CPCA01K67/0336C12N15/113C07K14/4354C12N2310/141
Inventor 成新跃卢俊张茜王萍秦飞飞
Owner BEIJING NORMAL UNIVERSITY
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