A kind of pine xylophilus RNAi regulation gene and its application

A technology that regulates genes and pine xylophilus, applied in the field of bioengineering, can solve the problems of rapid onset, death of pine trees, and no effective and sustainable prevention and control measures, and achieve the effect of high larval mortality

Active Publication Date: 2022-04-01
BEIJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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. 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

Method used

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  • A kind of pine xylophilus RNAi regulation gene and its application
  • A kind of pine xylophilus RNAi regulation gene and its application
  • A kind of pine xylophilus RNAi regulation gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] On the NCBI website, through the BLAST analysis, first find the highest homologous protein with the beautiful hinges ETR-1 protein, the amino acid sequence of the protein, such as SEQ ID NO: 3, the domain of the beautiful hiped nematode ETR protein Such as figure 1 Indicated. Then BLAST analysis, the gene full length sequence (e.g., SEQ ID NO: 1 shown) and the cDNA full length sequence (as shown in SEQ ID NO: 2) in the pose nematode genome found. . The sequence was verified by PCR and RT-PCR to obtain a full length of BX-ETR-1 gene (2748 bp) and cDNA (1539 bp), which coded protein size was 512AA.

[0033] The total RNA of the pine material nemmode was extracted using Trizol or RNA extraction kit. The synthesis of the first chain of CDNA was explained in accordance with Primescript TM II 1ststrand CDNA Synthesis Kit (Takara, JAPAN). The positive reverse primer ETR-1-F / R of ETR-1 gene interference fragment is designed, as shown in SEQ ID NO: 5, SEQ ID NO: 6 (see Table 1), w...

Embodiment 2

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

[0038] (1) Adding HindIII and XBAI (HindII-ETR-1-F / XBAI-ETR-1-R) in the 5 'end of the forward primer ETR-1-F and the reverse primer ETR-1-R, respectively, amplification Upstream fragment; and the 5 'end of the forward primer ETR-1-F and the reverse primer ETR-1-R (APAI-TR-1-F / SpeI-ETR-) 1-r) (see Table 1), amplified downstream fragment, and the RT-PCR amplification reaction system is shown in Table 2.

[0039] Table 2

[0040]

[0041] Reaction conditions

[0042]

[0043] (2) The plasmid and upstream amplification fragment were performed using HindII, XBAI enzymes, and the enzyme digestion was performed after the enzyme digestion; then the downstream amplification fragment and the ligation product were used, and the enzyme was sized. The PDH-RH plasmid and amplification fragment were bisased using HindIII and XBAI, and APAI and SPEI, us...

Embodiment 3

[0051] The construction method of recombinant expression strain includes the steps of: transforming the plasmid of the BX-ETR-1 gene DSRNA obtained in Example 2 to Agrobacterium, which contains kanamycin (100 mg / L) and rifampicin ( Fifty-positive clones were screened on the LB plate of 50 mg / L). The plasmid of the positive strain was extracted, and the molecular identification was used to obtain a positive strain in which the fragment was inserted into the fragment was obtained by the upper and lower printed PCR amplification.

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Abstract

The invention provides a pine xylophilus RNAi regulation gene, the full-length sequence of the RNAi regulation gene is shown in SEQ ID NO:1, and the full-length cDNA sequence of the RNAi regulation gene is shown in SEQ ID NO:2. The present invention also provides the application of the pine wood nematode RNAi regulation gene in the preparation of pine wood nematode control bacteria agent. The present invention screens out a highly efficient RNAi regulation gene segment, and feeds the positive fungal transformants containing the gene segment to pine wood nematodes to obtain an obvious RNAi phenotype, the gene expression of pine wood nematode Bx-etr-1 is significantly down-regulated, and the pine wood nematode The nematodes have abnormal embryonic development, high larval mortality, changed trajectory, slowed movement speed, and significantly decreased population.

Description

Technical field [0001] The present invention is a technical field of bioengineering, and therefore specifically, a rock nematode RNAi regulatory basis and its application. Background technique [0002] Bursaphelenchus Xylophilus feeding hazards in pine trees, caused by pine nematode disease, resulting in a large amount of dying in pine trees, and its pathogenesis is fast, the prevention and control is difficult, and there is no effective and sustainable control. measure. [0003] RNA Interference (RNAI) mainly refers to an exogenous or endogenous double-stranded RNA (DSRNA) can cause a phenomenon that is lowered to reduce the target gene mRNA. The use of RNAi technology in the prevention and control of plant parasitic nematodes, that is, the number of populations of RNAi-controlled agricultural roots and cytoplatoeetica, have become new prevention and control with extensive application prospects through host plant-media way. In the study of pose nematode, the fungi-mediated RNAi ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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