桂糖11号Rubisco基因克隆、原核表达及纯化  

Cloning, prokaryotic expression and purification of Rubisco gene from sugarcane cultivar Guitang 11

作　　者：吴朝兴[1] 蒋媛[1] 王露蓉 顾彩彩[1] 牛俊奇[2] 孙波[1] 李杨瑞[1,3] 杨丽涛[1,3] 

机构地区：[1]广西大学农学院/亚热带农业生物资源保护与利用国家重点实验室,南宁530005 [2]玉林师范学院生命科学与技术学院,广西玉林537000 [3]广西农业科学院甘蔗研究所/中国农业科学院甘蔗研究中心/农业部广西甘蔗生物技术与遗传改良重点实验室/广西甘蔗遗传改良重点实验室,南宁530007

出　　处：《南方农业学报》2016年第4期524-529,共6页Journal of Southern Agriculture

基　　金：国家“863”计划项目(2013AA102604);广西八桂学者和特聘专家专项基金项目(厅发[2013]3号)

摘　　要：【目的】通过原核表达及纯化获得甘蔗1,5-二磷酸核酮糖羧化酶/加氧酶(Rubisco)的大、小亚基融合蛋白,为获得符合抗体制备条件的高纯度融合蛋白提供技术支持。【方法】以甘蔗品种桂糖11号(GT11)+1叶为材料,根据NCBI公布的甘蔗Rubisco大、小亚基基因(rbc L和rbc S)的编码域序列(CDS)设计特异性引物,PCR扩增rbc L和rbc S基因的CDS,然后连接至原核表达载体p ET-30a(+),构建重组质粒后转入原核细胞(大肠杆菌Rosetta)中诱导表达,用镍亲和层析柱对融合蛋白进行纯化,并比较分析桂糖11号与其他甘蔗品种在rbc L和rbc S基因的CDS及编码蛋白序列方面的差异。【结果】rbc L和rbc S基因的CDS长度分别为1431和510 bp,其中桂糖11号rbc L基因的CDS与Saccharum hybrid cultivar SP80-3280(Gen Bank登录号AE009947)、Saccharum hybrid cultivar Q155(Gen Bank登录号KU214867)的一致,桂糖11号rbc S基因的CDS与Saccharum hybrid cultivar GT28(Gen Bank登录号JN591757)的存在7个碱基差异,但仅有2个氨基酸发生错义突变。Rbc L和Rbc S融合蛋白以包涵体形式存在原核细胞中,用镍离子亲和层析纯化及超滤浓缩后其浓度均在1.0 mg/m L以上。【结论】从桂糖11号成功克隆Rubisco大亚基和小亚基基因的CDS,大亚基基因的CDS比小亚基的保守性更高,且均可在原核细胞中高度表达,经纯化浓缩获得的高纯度融合蛋白可用于制备Rubisco单克隆抗体。[ Objective ]In order to technical reference for getting high-purity fusion proteins that met preparation con- ditions of monoclonal antibody, the large and small subunits of ribulose-1,5-bisphosphosphate earboxylase/oxygenase （Rubisco） from sugarcane were expressed in prokaryote, and then purified and concentrated. [Method]Using leaf （＋1） of sugarcane cuhivar Guitang 11 （GT11 ） as material, the specific primers of large and small subunit genes（rbcL and rbcS） of nabisco were designed based on coding domain sequences（CDSs） of rbcL and rbcS genes published on the NCBI. Then the rbcL and rbcS genes were amplified by PCR, and inserted to prokaryotie expression vector pET-30a（＋） to construct recombinant plasmids, respectively. The recombinant plasmids were transferred into prokaryotie cell（Eseheffehia eoli Rosetta）, and the over-expressed fusion proteins were purified with Ni affinity chromatography. Meanwhile, the CDSs of nucleotide and amino acid of rbeS and rbcL genes between GT11 and other sugarcane eultivars were analyzed and eompared.[Result]The CDSs of rbcL and rbcS genes were 1431 and 510 bp in length, respectively. The CDS of GTll rbcL gene was identical to those of Saccharum hybrid cultivar SP-80-3280 （GenBank： AE009947.2） and Saccharum hybrid culti- var Q155 （GenBank： KU214867.1 ）, while CDS of GT11 rbcS gene was different in seven nucleotides from Saceharum hy- brid cuhivar GT28 （GenBank： JN591757.1 ）, only leading to two missense mutation. The RbcL and RbcS fusion proteins were expressed in the form of inclusion bodies in prokaryotie cell, and then purified with Ni affinity chromatography and eoneentrated to 1.0 mg/mL by ultrafihration. [ Conclusion ]The CDSs of rubisco rbcL and rbcS genes have been successfully cloned from sugarcane variety GT11. The rbcL gene CDS is highly conserved compared with rbcS gene CDS. Moreover, the rbcL and rbcS genes are over-expressed in the prokaryotic cell, and the high-purity fusion proteins are obtained success- fully by

关 键 词：甘蔗 1 5-二磷酸核酮糖羧化酶/加氧酶(Rubisco) 克隆 原核表达 融合蛋白 纯化 

分 类 号：S566.1[农业科学—作物学] Q785[生物学—分子生物学]