蓖麻三磷酸甘油脱氢酶基因(RcGPDH)的克隆及功能分析

Cloning and characterization of glycerol-3-phosphate dehydrogenase gene (RcGPDH) from castor bean

为了阐明蓖麻三磷酸甘油脱氢酶基因(glycerol-3-phosphate dehydrogenase,RcGPDH)在蓖麻油累积过程中的作用,本研究根据已报道的蓖麻种子表达序列标签(Expressed Sequence Tags,EST)设计引物,通过RACE(rapid-amplification of cDNA ends)方法克隆蓖麻RcGPDH基因的全长并对其序列进行分析,构建了该基因的酵母表达载体pYES2.1/V5-His-TOPO-RcGPDH,以载体pYES2.1/V5-His/lacZ质粒DNA为对照,转化酵母野生型菌株BY4742,运用分光光度法测定转基因酵母的生长曲线,通过香草醛法测定稳定生长期的转基因酵母的油脂含量。结果表明,转基因菌株比转空载体对照菌株生长慢,两者均在培养18h后进入平台期;两个菌株的油脂含量没有明显的差别,表明RcGPDH基因对酵母油脂的累积没有起到积极的作用,在蓖麻种子中可能还存在另一个GPDH同源基因参与三脂酰甘油(TAG)的合成。

In order to elucidate the role of glycerol-3-phosphate dehydrogenase gene（RcGPDH） from castor bean in castor oil accumulation,we designed primers according to express sequence tags（EST） and cloned the full cDNA of RcGPDH gene by RACE（rapid-amplification of cDNA ends）.The yeast expression vector pYES2.1 /V5-His-TOPO-RcGPDH was constructed and transformed to wild yeast strain BY4742,using plasmid pYES2.1/V5-His/lacZ as control.We drew the growth curves of transformed yeast strains with spectrophotometer at 600nm,and determined their oil content using vanillin method.Results showed that they grew stably after cultured 18h,but the strain with RcGPDH gene grew slower than control.The oil content didn＇t change between the two yeast strains,which suggested that RcGPDH gene was not involved in yeast oil accumulation.These results suggested that other GPDH homologous genes may be involved in triacylglycerls（TAG） synthesis in castor seeds.