Green shrimp vitellogenin vg gene, encoded protein and its application
A technology of vitellogenin and green shrimp, applied in the fields of biotechnology and developmental regulation, can solve the problems of Vg gene silencing and expression that have not yet been reported, and achieve the effect of delaying time
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Embodiment 1
[0026] Embodiment 1: Obtaining the full-length cDNA of Vg gene of freshwater shrimp
[0027] Extraction of total RNA: Select the livers of 3 to 4 mature female shrimps, quickly put them into a pre-cooled mortar filled with liquid nitrogen, and grind them rapidly. Total RNA was extracted using RNAiso Plus extraction reagent from Takara Company combined with the traditional phenolic extraction method, and the extraction method was referred to the instruction manual. The quality of the RNA was detected by agarose gel electrophoresis, and the OD260 / 280 ratio of the sample was analyzed by a spectrophotometer to be between 1.8 and 2.0, and the concentration of the RNA was determined.
[0028] cDNA first-strand synthesis: refer to the Takara M-MLV reverse transcription kit to synthesize the first-strand cDNA.
[0029] Cloning of the full-length cDNA sequence of the freshwater shrimp Vg gene:
[0030] According to the obtained full-length cDNA sequence of Vg gene (Macrobrachium rose...
Embodiment 2
[0047] Embodiment 2: the synthesis of green shrimp Vg gene dsRNA
[0048] Based on the nucleotide sequence of SEQ ID NO.1, use NCBI online dsRNA primer design software (http: / / www.flyrnai.org / cgi-bin / RNAi_find_primers.pl) to design and prepare double-stranded RNA within the open reading frame Primers (SEQ ID NO.3-SEQ ID NO.4), according to the instructions of the Transcript AidTM T7 High YieldTranscription kit (Fermentas, Inc., USA) for in vitro transcription and synthesis of dsRNA. The prepared dsRNA was dissolved in DEPC water, its concentration was measured and its purity was detected on 1.5% agarose gel. The above dsRNA solution was injected into the pericardial cavity of freshwater shrimp at a dose of 4 μg / g. The control group was injected with the same volume of DEPC water, and the female shrimp used in the experiment were all in the early stage of ovarian development (just after ovulation). Samples (N ≥ 3) were collected on days 1, 3, 5, 7, 9, 11, 13, 15, and 17 post-...
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