苹果sMdCAX1基因超表达载体的构建及遗传转化

Construction of s Md CAX1 Gene Overexpression Vector and Its Genetic Transformation in Apple

为使sMdCAX1基因能在苹果中高效表达,改善苹果对Ca^(2+)吸收转运的能力,本研究构建了sMdCAX1植物超表达载体并将其通过农杆菌介导法转入长富6号苹果中。根据MdCAX1基因序列设计引物,从长富6号中克隆MdCAX1基因,通过PCR方法截除编码MdCAX1基因的N末端序列,获得sMdCAX1片段。利用重叠PCR方法将sMdCAX1序列中的SacI位点进行定点突变,并在序列两端分别添加BamHI和SacI酶切位点,获得长度为1272bp的突变序列sMd1+2Mu。利用BamHI和SacI双酶切sMd1+2Mu和植物表达载体pYH4215,目的片段经回收、连接、转化和筛选,获得植物双元表达载体pYH4215-sMd1,并将该载体转化农杆菌菌株EHA105。以长富6号无菌试管苗叶片为受体,进行遗传转化获得了转基因植株,经鉴定,有6个株系呈GUS染色阳性,其中2个株系呈PCR阳性。进一步对PCR鉴定阳性的株系进行RT-PCR分析,表明sMdCAX1基因在这2个株系中得到表达。本试验结果为进一步研究苹果MdCAX1基因的功能和通过转基因方法提高苹果果实钙含量奠定了基础。

To enhance the expression of the sMdCAX1 gene in apple and improve the ability of Ca^(2+),absorption and transportion,an overexpression vector containing sMdCAX1 gene were constructed and transfered to Nagafu No. 6 by Agrobacterium-mediated method. In this research, Md CAX1 gene was cloned from apple Nagafu No. 6 and the nucleotide sequence encoding the N-terminal was removed from Md CAX1 by PCR method. The N-terminal truncated Md CAX1 was named as sMdCAX1. For constructing of the binary vector harboring sMdCAX1 fragment,the Sac I sites in sMdCAX1 fragment were mutated by overlapping PCR method under the condition of its encoding amino acid sequence unchanged. Bam H I and Sac I restriction sites were added at the 3' and 5' ends of the mutated fragment,respectively,and named s Md1 + 2Mu. The s Md1 + 2Mu fragment and p YH4215 vector were digested by Bam H I + Sac I respectively. The objective bands were recovered,ligated,transformed and screened. The plant over-expression vector p YH4215- s Md1 was obtained and introduced to apple cultivar Nagafu No. 6 with Agrobacterium tumefaciens EHA105 mediated transformation. The putative transgenic plantlets were identified by GUS histochemical staining and PCR testing. Two transformants were testified that sMdCAX1 was integrated to the apple genome. Moreover,sMdCAX1 gene was proved expressed in the 2 transgenic plants in transcriptional level by RT-PCR analysis. The study would contribute to further analysis of the Md CAX1 functions and laid a foundation for improving calcium content in apple fruits by transgenic approach..