甘蔗锌指蛋白基因ShSAP1的RNAi载体构建及对甘蔗的转化

Construction of RNAi Vector of Sugarcane Zinc Finger Protein Gene ShSAP1 and Its Transformation in Sugarcane

植物中具有A20/AN1锌指结构域的蛋白参与了逆境应答,为研究甘蔗A20/AN1型锌指蛋白基因ShSAP1的功能及在甘蔗抗逆育种方面的价值,本研究通过构建ShSAP1基因的RNAi载体并进行甘蔗的遗传转化,以期通过反向遗传学进行ShSAP1的功能分析。将ShSAP1锌指编码区片段分别以正向和反向插入到中间载体pTCK303内含子的两侧,构建中间载体pTCK-iShSAP1,而后把干扰片段连入pCAMBIA-GUS中,获得RNAi表达载体pCAMBIA-iShSAP1,将该载体转导根癌农杆菌EHA105,通过农杆菌介导法侵染甘蔗胚性愈伤组织,并对部分转化幼苗进行了初步的PCR鉴定。经过限制性内切酶分析和测序验证,RNAi载体pCAMBIA-iShSAP1构建成功;转化幼苗经PPT抗性筛选后,获得了58株抗性植株,对抗性幼苗进行了Bar基因的PCR检测,得到6株PCR阳性植株。本研究完成了ShSAP1的RNAi载体构建及对甘蔗的遗传转化,为ShSAP1的功能研究打下了一定的基础。

The A20/AN1 type zinc finger proteins are reported to involve in stress response in plants. RNAi expression vector of sugarcane A20/AN1 zinc finger protien gene ShSAP1 was constructed and transformed into sugarcane for the functional characterization and evaluation of its application value on sugarcane breeding for stress tolerance. A 550 bp fragment amplified from the zinc finer coding region of ShSAP1 was inserted into both sides of intron fragment of pTCK303 in reverse and forward direction, respectively. After identification by enzyme digestion and sequencing, the RNA interference fragment from intermediate vector pTCK-iShSAP1 was linked into expression vector pCAMBIA-GUS to form RNA interference expression vector pCAMBIA-iShSAP1 driven by Ubi promoter. The recombinant was transformed into sugarcane mediated by Agrobacteria tumefaciens EHA105. Fiftyeight seedlings were obtained after PPT resistance selection and 6 seedlings showed positive after PCR detection.