杜氏盐藻两种碳酸酐酶基因启动子的克隆和功能研究  

Cloning and Functional Analyses of Promoters of Two Carbonic Anhydrase Genes from Dunaliella salina

作　　者：吕玉民[1] 姜国忠[1] 牛向丽[1] 侯桂琴[1] 张贵星[1] 薛乐勋[1] 

机构地区：[1]郑州大学,河南省分子医学重点学科开放实验室,郑州450052

出　　处：《Acta Genetica Sinica》2004年第10期1157-1166,共10页

基　　金：国家高技术研究发展计划项目 ("863计划") (编号 :2 0 0 2AA62 80 5 0 );河南省重大科技公关项目 (编号 :0 12 2 0 3 2 5 0 0 )资助~~

摘　　要：将克隆得到的杜氏盐藻DCA1和CA基因的启动子区与bar基因和NOSpolyA终止子片段融合 ,分别构建成 pMDDC B和pMDC B转基因杜氏盐藻表达载体。用基因枪法将两种表达载体转化入杜氏盐藻细胞 ,通过除草剂草丁膦筛选培养获得转化藻株 ,对转化藻株进行分析。对转化杜氏盐藻藻株的筛选培养结果表明 :pMDDC B和pMDC B载体中的外源bar基因能在杜氏盐藻细胞中稳定或瞬时表达。同时在氦气压力为 6 90kPa条件下 ,微弹轰击 2次比微弹轰击 1次或 3次的效果更好。对 pMDDC B转化杜氏盐藻得到的稳定表达的转化藻株进行的PCR和Southern印迹分析的结果表明 :外源的bar基因确已整合到杜氏盐藻基因组中。Northern印迹分析表明 :DCA1基因启动子驱动bar基因在杜氏盐藻细胞中的表达效率受氯化钠浓度梯度调控。推测首次克隆得到的DCA1基因启动子可能是一种活性高、安全性好的高渗诱导性启动子 ;杜氏盐藻DCA1和CA基因启动子区的GT高度重复序列 。The cloning vectors pMD-DCA1 and pMD-CA containing the promoters of duplicated carbonic anhydrase 1 (DCA1) and carbonic anhydrase (CA) genes,respectively,from Dunaliella salina,and expression vector pDM307 containing bar-NOS polyA fragment were digested with EcoRⅠ.The bar-NOS polyA fragment was fused,respectively,to the fragments of the vectors pMD-DCA1 and pMD-CA to form transgenic D.salina expression vectors pMDDC-B and pMDC-B.The micro-shots were prepared by coating two constructs (pMDDC-B and pMDC-B) with gold particle.Each sample was bombarded once,twice,and thrice,respectively,with micro-projectile gun at a rupture pressure of 690 kPa in helium gas.The screening culture of the bombarded alga cells was performed in PKS liquid and solid medium containing 3 mg/L phosphinothricin (PPT) to develop the transformed cells of D.salina.Analyses of the transformed cells were carried out through PCR,Southern blotting,and Northern blotting.The results of screening culture showed that the expression of the external bar gene of vectors pMDDC-B and pMDC-B was stable and transient,respectively,in the transformed D.salina cells.In the meantime,the transformed efficiency of particle bombardment twice was higher than that of once or thrice particle bombardment at a rupture pressure of 690 kPa in helium gas.PCR and Southern blotting analyses indicated that the external bar gene was integrated into the genome of the cells.Northern analysis indicated that expression efficiency of the bar gene driven by DCA1 promoter was regulated by the gradient concentration of sodium chloride,and the positive blotting signal intensity of the bar mRNA was highest in the medium containing 2 mol/L of sodium chloride.The findings of the present study suggest that promoter of the DCA1 gene may be an inducible promoter following a hyperosmotic shock with high activity and safety in the research of transgenic D.salina .The tandem GT sequences of the promoter region of DCA1 and CA genes may be related to the molecular mechanisms of the extreme halo-t

关 键 词：杜氏盐藻 双拷贝碳酸酐酶 碳酸酐酶 启动子 BAR基因 转化 

分 类 号：Q75[生物学—分子生物学]