Nucleotide sequence and application thereof in enhancing harmful organism-resistant capability of plants
A nucleotide sequence and nucleotide technology, applied in the field of bioengineering, can solve the problems of easy pollution of the environment by pesticides, and achieve the effect of facilitating promotion and low cost
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Embodiment 1
[0060] Example 1V-ATPase subunit E and COO2 gene sequence homology comparison
[0061] Using the NCBI database, the selected V-ATPase subunit E gene fragment (SEQ ID NO: 4) was compared to the nucleic acid sequence, and the results showed that the sequence was similar to that of the bean aphid (Acyrthosiphon pisum) V-type protonATPase subunit E-like 100% homology to sequence (NCBI accession number: NM_001162178.2); 95% homology to Myzuspersicae V-type proton ATPase subunit E sequence (NCBI accession number: XM_022312248.1); (Diuraphis noxia) V-type proton ATPase subunit E sequence (NCBI accession number: XM_015522279.1) homology 92%; and sorghum aphid (Melanaphis sacchari) V-type protonATPase subunit E sequence (NCBI accession number: XM_025342771.1) 94% homology; 86% homology with Siphaflava V-type proton ATPase subunit E sequence (NCBI accession number: XM_025565024.1).
[0062] The nucleic acid sequence alignment of the selected COO2 gene fragment (SEQ ID NO:5) showed that...
Embodiment 2
[0064] Embodiment 2 contains the synthesis of the RNAi carrier of V-ATPase subunit E and COO2 double gene
[0065] Synthesize the V-ATPase subunit E gene fragment, the COO2 gene fragment and the V-ATPase subunit E and COO2 double gene fragment respectively, respectively construct the forward gene of the hairpin structure, the intron sequence and the reverse gene, and construct it into the pDE1005 vector ( Purchased from the multiple cloning sites of Beijing Baierdi Biotechnology Co., Ltd., pDE1005:proUBI:V-ATPase subunit E, pDE1005:proUBI:COO2 and pDE1005:proUBI:V-ATPasesubunit E+COO2 gene silencing vectors were obtained respectively, The gene was synthesized by Shanghai Sangon Bioengineering Company. The schematic diagram of constructing the RNAi vector of V-ATPasesubunit E and COO2 double gene fragment is shown in Figure 1.
[0066]In an optional embodiment, the pDE1005 vector has its own rice intron, the maize ubiquitin-1 promoter is located upstream of the rice intron, an...
Embodiment 3
[0067] Example 3 Agrobacterium tumefaciens mediates V-ATPase subunit E, COO2 and V-ATPase subunit E+COO2 double-gene RNAi vector transformation Fielder spring wheat
[0068] 1.1. Preculture of immature embryos
[0069] Immature seeds (immature embryo size 1.0-1.2mm) 13-14 days after flowering and pollination are surface-sterilized with 70% alcohol for 1-2 minutes, sterilized with 15% sodium hypochlorite for 15 minutes, and rinsed with sterile water 4-5 times.
[0070] 1.2. Co-cultivation of Agrobacterium and immature embryonic callus
[0071] At room temperature, centrifuge at 3500rpm for 10min to collect Agrobacterium cells, discard the supernatant, and resuspend with 1 / 10 WCC resuspension solution (i.e. MS basic medium) at a ratio of 1:2. The immature wheat embryos were transferred to the Agrobacterium bacterial solution for 30min infection, the calli were transferred to sterile filter paper in a sterilized petri dish, and co-cultivated under dark conditions at 25°C for 2 d...
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