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
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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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