Genes regulating flavonoid synthesis and their encoded proteins and their applications
A technology of flavonoids and genes, applied in the fields of application, genetic engineering, plant genetic improvement, etc.
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Embodiment 1
[0034] The cloning of tobacco NtMYB59 gene sequence comprises the following steps:
[0035] (1) Tobacco seedling leaf tissues were immediately frozen with liquid nitrogen after harvesting, and then stored in a -80°C refrigerator. After being fully ground with liquid nitrogen, the tobacco RNA rapid extraction kit (Gene Answer, RT0110) was used for extraction, and the reagents were strictly followed. The box instructions were used to extract total RNA from tobacco leaves. During the extraction process, DNase I (Omega, #E1091-01) was used to digest and remove genomic DNA. After the extraction was completed, the quality of RNA was detected by agarose gel, and the concentration of RNA samples was determined by Nanodrop. Select samples with good quality and concentration to synthesize the first strand of cDNA. The synthesis process uses PrimeScript TM II 1stStrand cDNA Synthesis Kit (TAKARA, 6210A), in strict accordance with the instructions of the kit, to obtain cDNA samples, whic...
Embodiment 2
[0047] The construction of tobacco NtMYB59 gene expression vector comprises the following steps:
[0048] (1) Sequence the correct plasmid and pCAMBIA1301 plasmid using XbaI and KpnI double enzyme digestion, digest at 37°C for 2 hours, and then undergo agarose gel electrophoresis to recover the target gene fragment and the cut pCAMBIA1301 plasmid, and configure the connection in a microcentrifuge tube System: digested pCAMBIA1301 plasmid 1μl, target gene fragment 5μl, T4buffer 1μl, T4 ligase 1μl, dd-H 2 O2 μl. Ligation reaction was carried out overnight at 16°C, transformed into Escherichia coli competent cells, and positive clones were selected to extract plasmids.
[0049] (2) After the plasmid is identified by PCR, it is transformed into competent Agrobacterium and used for transgenic tobacco. The construction of the antisense expression vector is to use the following primers to amplify a fragment of about 380bp in the coding region of the NtMYB59 gene, and then reversely ...
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