Application of soybean VQ motif coding gene GmVQ58
A technology for encoding genes, soybeans, applied in the field of genetic engineering
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Embodiment 1
[0023] 1) Cloning of soybean VQ gene GmVQ58
[0024] Take the soybean variety Williams 82 as the sample object, take its leaves, grind them with a mortar, add them to a 1.5mL EP tube filled with lysate, shake fully, transfer to a 1.5mL EP tube, and extract total RNA (Total RNA Kit (Tiangen, Beijing, China). Identify the total RNA quality with formaldehyde denaturing gel electrophoresis, and measure the RNA content with a spectrophotometer. The total RNA obtained is a template, according to the reverse transcription kit (TaKaRa Primer Script provided by Japan TaKaRa Company) TMRT reagent kit, Japan) instructions for reverse transcription, after obtaining the first strand of cDNA, PCR amplification, the PCR program is as follows: 95 ° C pre-denaturation for 3 minutes, 95 ° C denaturation for 15 seconds, 60 ° C annealing for 15 seconds , 72°C for 90 seconds, a total of 35 cycles, and finally 72°C for 5 minutes, followed by a constant temperature of 12°C to obtain the cDNA of Will...
Embodiment 2
[0033] 1) Cloning of soybean VQ gene GmVQ58
[0034] Using the total RNA of leaves of soybean variety Williams 82 as a template, the first strand of cDNA was synthesized by reverse transcription, and then PCR was amplified. The primer sequences are shown in SEQ ID NO.9 and SEQ ID NO.10. The PCR program was as follows: pre-denaturation at 95°C 3 minutes, denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 90 seconds, a total of 35 cycles, and finally incubation at 72°C for 5 minutes, followed by constant temperature at 4°C. After sequencing, the soybean GmVQ58 gene without stop codon was obtained CDS sequence.
[0035] 2) Construction of subcellular localization vector
[0036] When constructing the subcellular localization vector, the CDS sequence of soybean GmVQ58 gene without stop codon was inserted into the pFGC5941 expression vector containing GFP tag. The vector has a 35S promoter, which can strongly induce the expression of the ...
Embodiment 3
[0039] Genetic engineering application of embodiment 3 gene GmVQ58
[0040] 1) Cloning of soybean VQ gene GmVQ58
[0041] Using the total RNA of leaves of soybean variety Williams 82 as a template, the first strand of cDNA was synthesized by reverse transcription, and PCR amplification was performed. The primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4. The PCR program was as follows: pre-denaturation at 95°C 3 minutes, denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 90 seconds, a total of 35 cycles, and a final incubation at 72°C for 5 minutes, followed by constant temperature at 4°C. The CDS sequence of soybean GmVQ58 gene, its sequence is shown in SEQ ID NO.1.
[0042] 2) Construction of plant expression vectors
[0043] When constructing the overexpression vector, the GmVQ58 gene sequence and Invitrogen's Technology with Clonase TM The pDONR221 vector in the II kit was subjected to BP reaction, and PCR sequencing o...
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