转1174AALdico-2+CTP耐草甘膦优异棉花种质系的创制及其特性  

The Creation and Characteristics of Cotton Germplasm Lines Transgenic 1174AALdico-2+CTP Gene with Excellent Glyphosate Tolerance

作　　者：王宛如 曹跃芬 盛况 陈进红[1,2] 赵天伦 祝水金[1,2] WANG WanRu;CAO YueFen;SHENG Kuang;CHEN JinHong;ZHAO TianLun;ZHU ShuiJin(College of Agriculture and Biotechnology,Zhejiang University,Hangzhou 310029;Hainan Institute,Zhejiang University,Sanya 572000,Hainan)

机构地区：[1]浙江大学农业与生物技术学院,杭州310029 [2]浙江大学海南研究院,海南三亚572000

出　　处：《中国农业科学》2023年第17期3261-3276,共16页Scientia Agricultura Sinica

基　　金：国家转基因重大专项(2016ZX08005-005);国家棉花产业体系岗位科学家(CARS-15-27);江苏省作物协同创新中心、海南省重点研发计划(SQ2021XDNY0696)。

摘　　要：【目的】棉田杂草是限制棉花生长的因素之一,与棉花争夺营养、水分和光照,不仅影响棉花的生长发育,还影响棉花的产量和品质。通过基因工程途径培育高耐草甘膦优异棉花种质,实现棉田间化学除草,提高植棉的经济效益。【方法】将来源于耐辐射奇球菌(Deinococcus radiodurans)的EPSPS基因1174AALdico-2连接叶绿体转运肽,以35S为启动子,将2个目的基因串联后构建棉花转化载体。运用农杆菌活体转化技术,将目的基因导入棉花品种中棉所49,获得阳性转化体植株。以受体材料中棉所49和转化体自交分离得到的非转基因NON为对照,对转化体植株进行分子特征检测、草甘膦耐受性鉴定、农艺性状和经济性状等综合评价,以筛选出优良的转基因耐草甘膦棉花材料。【结果】通过农杆菌活体转化方法获得138个阳性转化体,对转化体植株进行目的基因PCR检测、Southern blot、Western blot分析和ELISA检测,从中筛选出17个分子特征明确、外源基因表达量高的阳性转化体。Southern blot和核苷酸测序结果表明,转化体插入位点和拷贝数各不相同,其中,ZD131、ZD185和ZD207等3个转化体为单拷贝插入位点,外源基因插入位点分别位于棉花D亚组第7染色体、D亚组第13染色体和A亚组第12染色体。草甘膦耐受性鉴定结果表明,17个阳性转化体均高耐草甘膦,其中ZD131、ZD185和ZD207等3个转化体的耐受性在3个世代之间稳定遗传,可耐4倍的田间草甘膦推荐剂量。农艺性状和经济性状评估结果表明,ZD131、ZD185和ZD207等3个转化体植株生长发育正常,表现出铃大、衣分高、结铃性强等特点,皮棉产量超过对照,纤维品质达到优质棉水平。【结论】将来源于耐辐射奇球菌的EPSPS基因1174AALdico-2与叶绿体转运肽连接,多基因串联后构建转化载体,农杆菌活体转化技术转入中棉所49,通过一系列筛选后获得3份优良的转基因耐草甘�【Objective】Weeds are one of the factors limiting cotton growth in cotton production,which not only competes with cotton for nutrition,water,and light,affecting the growth and development of cotton but also the yield and quality of cotton.The aim of this study is to develop excellent cotton germplasms with high glyphosate tolerance by genetic engineering and provide technical support for the commercialization of glyphosate-tolerant cotton to realize chemical weed control and enhance the economic benefits of cotton production.【Method】To construct the cotton transformation vector,the EPSPS gene 1174AALdico-2 from Deinococcus radiodurans was connected to the chloroplast transit peptide(CTP),35S was used as the promoter,and the two target genes were linked in series.The target gene was transformed into the CCRI 49 by Agrobacterium tumefaciens in vivo transformation,and the positive transformants were obtained.The positive transgenic plants obtained were evaluated for glyphosate tolerance,molecular characterization,agronomic and economic traits,in order to obtain outstanding transgenic cotton germplasms with excellent glyphosate tolerance,using the recipient cultivar,CCRI 49,and the non-transgenic NON isolated from the transformants during selfing as the controls.【Result】138 positive transformants were obtained by Agrobacterium tumefaciens in vivo transformation.All the transformants were tested for target gene PCR,Southern blot,Western blot,and ELISA detection etc.,and 17 positive transformants with clear molecular characteristics and high expression of foreign genes were identified.Southern blot and nucleotide sequencing results revealed that there was great variation in insertion site and copy numbers among the 17 transformants.Among them,ZD131,ZD185,and ZD207 had single-copy insertion sites,and the foreign genes were located on D7,D13,and A12,respectively.The results of glyphosate tolerance identification revealed that the glyphosate tolerance of the three transformants,ZD131,ZD185,and ZD207,was stably

关 键 词：棉花 EPSPS 叶绿体转运肽 草甘膦 皮棉产量 纤维品质 

分 类 号：S562[农业科学—作物学]