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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。
In order to study the function of sugarcane A20 / AN1 zinc finger protein gene ShSAP1 and its resistance to sugarcane breeding, we constructed the RNAi vector of ShSAP1 gene And the genetic transformation of sugarcane, with a view to reverse genetics ShSAP1 functional analysis. The fragment of ShSAP1 zinc finger coding region was inserted into both sides of the intron of the intermediate vector pTCK303 in forward and reverse directions respectively to construct the intermediate vector pTCK-iShSAP1, and then the interference fragment was ligated into pCAMBIA-GUS to obtain the RNAi expression vector pCAMBIA- iShSAP1. The vector was transfected into Agrobacterium tumefaciens EHA105. The embryogenic callus was infected with Agrobacterium tumefaciens by Agrobacterium tumefaciens, and some of the transformed seedlings were identified by PCR. After restriction endonuclease analysis and sequencing, the RNAi vector pCAMBIA-iShSAP1 was successfully constructed. After screening the transformed seedlings with PPT resistance, 58 resistant plants were obtained and the resistant seedlings were subjected to PCR detection of Bar gene. Six strains PCR positive plants. This study completed the construction of RNAi vector of ShSAP1 and genetic transformation of sugarcane, which laid a foundation for the function of ShSAP1.