光敏启动子控制的酵母Hem1基因在烟草中表达  

Expression of Saccharomyces cerevisiae Hem1 Recombined with Arabidopsis thaliana hemA1 Promoter in Transgenic Tobacco

作　　者：张治平[1] 汪良驹[1] 姚泉洪[1] 

机构地区：[1]南京农业大学园艺学院,南京210095

出　　处：《西北植物学报》2007年第10期1929-1936,共8页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金资助项目(30471181)

摘　　要：拟南芥hemA1基因启动子是一种光响应型启动子,它控制着植物体内5-氨基乙酰丙酸(ALA)昼夜节律型合成.将该启动子与酿酒酵母Hem1基因构建的二价载体转入烟草中,获得转基因植株.以转二价基因的烟草T0代种子为材料,用不同浓度卡那霉素(Km)溶液浸种,结果显示,种子发芽率和子叶绿化率随着Km浓度升高而降低.将1 000 mg·L-1Km溶液中长出的162株抗性植株移植至盆钵中培养,GUS检测出的阳性比率为92%.用特异引物PCR法检测Km抗性-GUS阳性植株,发现含有拟南芥HemA1基因启动子的比率为92.5%,含有酿酒酵母Hem1基因的比率为88%,含有二价重组基因的比率为84.2%.RT-PCR检测表明,Hem1基因在黑暗中的表达量明显低于光照下,证明光敏启动子有效地控制了结构基因Hem1的表达.生理指标分析表明,与野生型相比,转基因植株ALA合成速率、叶绿素含量明显增加,叶绿素b/a比值提高.野生型植株基部叶片SPAD值显著降低,而转基因植株基部叶片SPAD值保持较高水平.以上结果说明,外源二价基因已经成功整合到烟草基因组中,并且能够在光照条件下过量表达,合成过量ALA.The hemAl promoter in Arabidopsis thaliana is a light responsive promoter that regulates the diurnal rhythmic biosynthesis of 5-aminolevulinic acid （ALA） in plants. A binary recombinant gene comprised of A. thaliana hemal promoter and Saccharomyces cerevisiae Hem1, encoding ALA synthase in yeast,was transformed into tobacco explants. The To generation seeds of transgenic tobacco were distinguished by a series concentrations of kanamycin （Km） solutions,and the Km-resistant plants from 1 000 mg/L Km solution were transplanted in pots and inspected by GUS. The results showed that 92% of the Km-resistant tobacco plants were GUS-positive. Further inspection by PCR with specific primers showed that 92.5 % of GUS-positive plants contained A. thaliana hemAl promoter and 88% contained S. cerevisiae Hem1,and therefore, 84.2% contained the complete binary recombinant gene. RT-PCR detection demonstrated that the expression of S, cerevisiae Hem 1 gene was conditioned because the relative abundance of Hem1 mRNA transcript was higher under light than that at dark,which was corresponded wity tne light- responsive property of the HemAl promoter. Furthermore,the endogenous ALA biosynthesis and the chlo- rophyll content in transgenic tobacco leaves,compared with the wild type, were obviously increased with higher Ch1 b/a ratios. The leaf SPAD values in the base of the wild type plants dropped distinctly whereas those in transgenic ones were kept in much higher levels. These results suggested that both exogenous genes could be inherited in transgenic tobacco plants,which grew normally under natural condition and over-expressed exogenous genes to overproduce endogenous ALA when exposed to light.

关 键 词：转基因烟草 拟南芥HemA1基因启动子 酿酒酵母Hem1 5-氨基乙酰丙酸(ALA) 叶绿素 

分 类 号：Q789[生物学—分子生物学]