Cloning And Function Analysis Of MYC1a/1b In Tobacco

MYC transcription factor is an important transcription factor family,which is involved in the response to stress,secondary metabolism,growth and development of plant.To investigate the regulatory mechanism of nicotine biosynthesis in tobacco,two differential proteins,MYC1a/1b,had been screened from roots with 2D electrophoresis.Like MYC2a/2b,they belong to MYC transcription factor family.It has been proved that MYC2a/2b are involved in the regulation of nicotine biosynthesis in tobacco.To explore the function of MYC1a/1b in regulating nicotine biosynthesis,the experiments are conducted and the main results are followings.1.MYC1a and MYC1b genes were successfully cloned from tobacco roots,and their sequences were analyzed with bioinformatics methods.2.Over-expression vectors and RNAi vectors of MYC1a and MYC1b were successfully constructed and transformed into tobacco leaves for transient expression.The expression of MYC1a?MYC1b and PMT were detected by qPCR.The qPCR results showed the expressions of MYC1a?MYC1b increased for 2.9 and 2.5 fold,and the expression of PMT increased for 4.6 and 4.73 fold in transgenic tobacco plant with over-expression of MYC1a or 1b.Therefore,over-expression of MYC1a or 1b can increase the expression of PMT.Compared to non-transgenic tobacco plant,the expression of MYC1a,MYC1b and PMT were decreased for 0.214,0.235 and 0.2 fold,indicating that the expression of PMT decreased after MYC1a or MYC1b was silenced.3.In order to elucidate the role of MYC1a/1b in regulating nicotine biosynthesis,the RNAi vectors and over-expression vectors of MYC1a and MYC1b were transformed into tobacco with Agrobacterium tumefaciens mediated leaf dish transformation method.Compared to non-transgenic tobacco plant,the expression of MYC1a,MYC1b and PMT were decreased for 0.26,0.41 and 0.09 fold in the RNAi transgenic tobacco plants,and the expression of MYC1a and PMT were increased for 2.28 and 11.15 fold in MYC1a over-expression transgenic tobacco plants,indicating that MYC1a and MYC1b have important role in regulating nicotine biosynthesis.4.MYC1b promoter sequences were successfully cloned from tobacco,the promoter sequences of MYC1a/1b were analyzed with bioinformatics methods.