红缨子高粱籽粒酵母文库构建及TAN2基因互作蛋白的筛选鉴定OA

[Objective]The TAN2-interacting proteins were screened by constructing Hongyangzi sorghum grain yeast library to provide a scientific basis for analyzing the regulatory network of proanthocyanidin(PA)biosynthesis and seed development.[Method]The primary cDNA library and pGADT7-based yeast secondary library were constructed by extracting total RNA and isolating mRNA on 10 and 20 days after flowering.The pGBKT7-TAN2 bait vector was constructed via homologous recombination.The interacting-proteins were screened by the yeast two-hybrid system after toxicity and self-activation validation,and the key candidate genes were screened by the subcellular localization prediction and expression patterns.[Result]A high-quality yeast secondary library with library titer of 9.14×106 cfu/mL and inserting more than 750 bp target bands was successfully constructed.308 positive clones were obtained by the yeast two-hybrid system,and 189 transcripts corresponding to 175 genes were obtained after sequence alignment.Subcellular localization prediction indicates that there were 18 genes nuclear localization proteins.The transcriptome data showed that 11 genes including YABBY transcription factor(SbiHYZ.08G177700),MYB transcription factor(SbiHYZ.09G146900),and bHLH transcription factor(SbiHYZ.03G139500)and so on were highly expressed at the early stage of grain development.[Conclusion]The yeast cDNA nuclear library is successfully constructed by extracting total RNA and isolating mRNA from Hongyingzi sorghum grains.The identified nuclear localization candidate proteins interacted with TAN2 may involved in biosynthesis of proanthocyanidins in sorghum grains and development of seed coat.