Flavone synthetase gene and polypeptide encoded thereby
A technology of gene encoding and synthase, which is applied in the field of flavonoid synthase gene and the polypeptide encoded by it, and can solve the problems that have not yet been discovered.
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Embodiment 1
[0017] Cloning, expression and identification of Gfns II-1 daidzein synthase gene
[0018] Step 1, cloning of Gfns II-1 daidzein synthase gene
[0019] 1. Processing of soybean tissue
[0020] The soybean variety Hefeng 47 was used, which was obtained from the Hejiang Institute of Agricultural Sciences, Heilongjiang Academy of Agricultural Sciences, with the approval number: Heixindou 2004003. The seeds were sterilized and inoculated on MSB5 solid medium to germinate for 5 days, wherein MSB5 medium specifically consisted of: inorganic components of MS medium (Murashige and Skoog, 1962) + organic components of B5 medium (Gamborg et al, 1968); The plants were placed in MSB5 liquid medium added with 0.4M glucose for 24 hours, and the roots were taken as materials for extracting DNA or RNA.
[0021] 2. TRIzol method to extract total RNA in roots
[0022] (1) Take by weighing 0.5g soybean treatment root, after quick-freezing with liquid nitrogen, grind into fine powder rapidly, ...
Embodiment 2
[0142] Cloning, expression and identification of Gfns II-2 daidzein synthase gene
[0143] The treatment of soybean tissue and the extraction of total RNA from roots by TRIzol method are the same as those in Example 1.
[0144] Step 1, the cloning of Gfns II-2 gene cDNA
[0145] Using the total RNA in the root as a template, use PrimeScript TM The 1st Strand cDNA Synthesis Kit was reverse transcribed into cDNA. Use the primers listed in the forward primer 5'-ATGATATCTGAGTCCCTTGTTAG-3' and the reverse primer 5'-CTACACTTGAGGAAAAGAGGTGGG-3' and high-fidelity Taq enzyme PCR to obtain a fragment of about 1600bp, connect it into the pMD18-T vector, and introduce it into E. coli DH5α . Sequencing verification obtained SEQ ID NO.2, and the sequencing results used BLAST and FASTA software in the GCG software package (Wisconsin group, USA) to search the existing database (Genebank+EMBL) to know its nucleic acid sequence and encoded protein. The homology of the FNSII gene in Medicag...
Embodiment 3
[0244] Construction and functional identification of Gfns II-1 and Gfns II-2 gene RNAi plant expression vectors
[0245] Step 1, construction of Gfns II-1 and Gfns II-2 gene RNAi plant expression vector
[0246] In view of the high homology of the Gfns II-1 and Gfns II-2 genes, about 300 bp of the nearly identical 3' ends of the two gene sequences were taken as the target fragment for constructing the RNAi plant expression vector. Design primers to amplify the target fragment, and introduce restriction endonuclease sites on the upstream and downstream primers, depending on the vector selected, in order to construct an expression vector. Using the Gfns II-1 obtained in Example 1 as a template, constructing an RNAi vector only requires one gene as a template. After PCR amplification and digestion, it was ligated in different directions to the pHannibal plasmid containing the CaMV35S promoter and nos terminator, separated by an intron fragment in the middle. This is an RNAi expr...
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