Tea geometrid vitellogenin gene vtg and uses thereof
A technology of vitellogenin and tea looper, which is applied in the fields of application, genetic engineering, plant gene improvement, etc., and can solve the problems that gene expression regulation technology has not yet been reported.
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Embodiment 1
[0023] Take 2-3 female adults (about 0.2 g) of the newly eclovened tea geometrid adults, put them in a mortar and grind them quickly with liquid nitrogen, add 1ml TRIZOL (Invitrogen Company) to mix well, and extract total RNA according to the steps in the TRIZOL reagent instructions . According to spectrophotometric analysis, the OD260 / OD280 ratio of the RNA sample was 1.8-2.0, and the concentration was about 850 μg / mL. Using 2 μL of the above RNA solution as a template, the first-strand cDNA was synthesized according to the instructions provided by the MMLV First-Strand cDNA Synthesis Kit (Shanghai Sangon Bioengineering Co., Ltd.).
[0024] Design and commission Shanghai Sangon Biological Products Co., Ltd. to synthesize primers with sequences of SEQ ID No: 3 (upstream primer) and SEQ ID No: 4 (downstream primer), using the first-strand cDNA synthesized above as a template, according to Table 1 The PCR is performed after adding various reactants shown in the first column, an...
Embodiment 2
[0028] Based on the nucleotide sequence of SEQ ID No: 1, TaKaRa siRNA Cocktail Kit (si-RNAase III TM ) (Bao Biological Engineering [Dalian] Co., Ltd.) to prepare vtg-siRNAs that can interact with the endogenous vtg of the tea geometrid. The prepared vtg-siRNAs were dissolved in water into an aqueous solution, and the above-mentioned aqueous solution was injected into the abdomen of the fifth-instar larvae, pupae, and female adults of the day of emergence of the tea geometrid with a microsyringe, and the injection dose was 10 ng of vtg-siRNAs per head of the tea geometrid, and The injection of pure water was used as the control. After 2 days of normal feeding, total RNA was extracted with TRIZOL (Invitrogen Company) reagent. Using SEQ ID No: 5 and SEQ ID No: 6 as primers, using the total RNA extracted from the injection-treated tea geometrid larvae, pupae and adults as templates, and performing RT-PCR with the MMLV one-step RT-PCR amplification kit, For specific operations, r...
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