摘要
肿瘤血管生成抑制因子Arresten是人Ⅳ型胶原蛋白α1链羧基末端NC1结构域多肽片段,可抑制新血管生成。研究拟实现该基因对烟草细胞的转化,为进一步利用植物细胞表达该蛋白奠定基础。①采用RT-PCR法从人胎盘组织克隆Arresten基因cDNA,克隆产物经酶切后定向插入到植物双元表达载体pCAMBIA1301的NcoⅠ和BstEⅡ位点之间,构建成含有目的基因的植物表达载体pCA,经PCR、限制性内切酶检测及序列测定正确后,转化根癌农杆菌LBA4404,获得携带目的基因的重组农杆菌。②采用叶盘转化法,以重组农杆菌转化烟草。在hygromy cin选择压力下获得烟草转化不定芽和完整植株,经PCR检测初步证实,Arresten基因已导入烟草基因组中。该研究首次将Arresten基因导入高等植物基因组中,为避免利用原核细胞或动物细胞表达Arresten的潜在问题,以及进一步利用植物进行该基因的大规模廉价表达奠定了基础。
The aim of this study was to transfer the arresten-cDNA into Nicotiana tabacum to prepare for the expression of the tumor angiogenesis inhibitor Arresten in plant. The target gene coding for tumor angiogenesis inhibitor Arresten was cloned from placenta via RT-PCR. After purified with chromatography column, the products of RT-PCR was cloned into T-vector and sub-cloned into Nco Ⅰ/BstE Ⅱ sites of the recipient plasmid pCAMBIA1301 which was digested with the restriction endonucleases Nco Ⅰ/BstE Ⅱ , and therefore a plant expression vector named pCA, was constructed. These recombinant plasmids pCA were determined by PCR, digestion with the restriction endonucleases and sequencing,then pCA was transferred into Agrobacterium tumefacien LBA4404 by freeze-thaw method. Moreover,the transference with the new engineering bacterium to Nicotiana tabacum was studied. The adventitious shoots and complete plants obtained under the selection pressure of hygromycin were confirmed with PCR of genome of Nicotiana tabacum, which showed that the target gene was transferred into the plants.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2007年第4期86-90,共5页
Journal of Northwest A&F University(Natural Science Edition)
