转lpaat和gpd1基因工程藻的快速筛选  

Rapid screening of the transgenic algae haboring lpaat and gpd1

作　　者：王潮岗[1] 李逸[1] 胡章立[1] 

机构地区：[1]深圳大学生命与海洋科学学院,深圳市海洋生物资源与生态环境重点实验室,广东深圳518060

出　　处：《深圳大学学报（理工版）》2016年第1期33-40,共8页Journal of Shenzhen University(Science and Engineering)

基　　金：国家自然科学基金资助项目(31470389;31470431)~~

摘　　要：根据莱茵衣藻密码子偏好性对源于油菜的溶血磷脂酸酰基转移酶基因(lpaat)和酵母的甘油3-磷酸酰基转移酶基因(gpd1)进行优化,获得适合莱茵衣藻表达的c-lpaat和c-gpd1基因,将其连接到p H124质粒中,构建相应的衣藻表达载体.通过珠磨法进行遗传转化,经Zeomycin抗性筛选和聚合酶链式反应(polymerase chain reaction,PCR)验证,获得导入c-lpaat和c-gpd1的转基因藻.利用半定量反转录PCR(reverse transcription-PCR,RT-PCR)技术筛选高效表达c-lpaat和c-gpd1的转基因藻,目的基因的转录水平分别提高了56.38%和75.95%,其藻细胞的脂肪酸含量也得到相应增加,与基因表达情况一致.证明莱茵衣藻可以有效表达外源高等植物的基因,利用半定量RT-PCR可以快速筛选出潜在的高油脂含量转基因工程藻株.Lpaat from Brassica napus and gpd1 from Saccharomyces cerevisiae were optimized according to the codon of Chlamydomonas reinhardtii to obtain c-lpaat and c-gpd1. The modified DNA fragments were amplified and inserted into plasmid p H124 to construct Chlamydomonas expression vectors. The expression vectors were then introduced into Chlamydomoas with the glass-bead-method and the transgenic algae containing c-lpaat and c-gpd1 was selected after screening with zeomycin resistance and polymerase chain reaction（ PCR） analysis. Finally,the transgenic algae in which c-lpaat and c-gpd1 were expressed effectively was screened by semi-quantitative reverse transcription-PCR（ RT-PCR）. The transcription level of c-lpaat and c-gpd1 in transgenic algae was increased by56. 38% and 75. 95%,respectively,resulting in correspondingly increased lipid content. In conclusion,the exogenous genes from the plants could be effectively expressed in Chlamydomonas reinhardtii and the transgenic algae with high lipid could be screened quickly by semi-quantitative RT-PCR. These results lay the foundation of quickly screening for transgenic algae with high lipid content.

关 键 词：基因工程 转基因藻 莱茵衣藻 lpaat基因 gpd1基因 三酰甘油合成 

分 类 号：Q786[生物学—分子生物学]