摘要
选择标记基因的删除是植物转基因工程中重要的步骤之一,双T-DNA区双元载体转化法以其共整合频率高、删除标记基因效率高的优点在植物遗传转化中应用非常广泛。本文通过构建天麻抗真菌蛋白基因、脂质转移蛋白基因双T-DNA区双元载体,为培育不含选择标记基因的抗病新材料奠定基础。通过PCR的方法克隆GAFP基因,产物纯化测序确定序列准确后与pSB130-35S载体进行BamHⅠ、SacⅠ双酶切;pCAMBIA2300-LTP、pSB130-35S经EcoRⅠ、HindⅢ双酶切,酶切产物纯化后连接,连接产物转化大肠杆菌。采用基因特异性引物进行菌液PCR鉴定阳性克隆,重组质粒pSB130-GAFP、pSB130-LTP双酶切产物大小分别为540bp、1200bp,与预期产物大小一致,pSB130-GAFP、pSB130-LTP双T-DNA区双元载体构建成功。载体pSB130-GAFP、pSB130-LTP转化根癌农杆菌后可以直接用于植物的遗传转化。
Deletion of selectable marker genes is one of the important steps in plant genetic engineering.Two T-DNA binary vectortransformation is widely used in plant genetic transformation for the advantages of high efficiency of co-integration and high frequency of deletion of selectable markergenes.In this paper,marker-free transgenic plants were to be expectedby constructing twinT-DNA binary vectors of Gastrodia anti-fungal protein（GAFP） andlipidtransferproteingene（LTP）.GAFP gene withBamHⅠ,SacⅠ sites was clonedby PCRandpurifiedinorder to be confirmedby sequencing.Afterdouble digestionof GAFP gene andpSB130-35S vectorby BamHⅠ andSacⅠ,pCambia2300-LTP and pSB130-35S by EcoRⅠ and HindⅢ,digestion products were purified and ligated,respectively.Gene-specific primers were used foridentifying positive clone and double digestion of recombinant plasmid pSB130-GAFP,pSB130-LTP represented the band as the expected size of 540 bp,1 200 bp.Thus construction of twin T-DNA binary vectors of pSB130-GAFP,pSB130-LTP was completed.Vectors pSB130-GAFP and pSB130LTP couldbe useddirectly forplantgenetic transformationaftertransforming into Agrobacterium tumefaciens.
出处
《分子植物育种》
CAS
CSCD
2010年第5期976-980,共5页
Molecular Plant Breeding
基金
转基因重大专项(2009ZX08004-002B)
江苏省大学生实践创新训练计划共同资助
