长穗偃麦草逆境诱导cDNA文库的构建和初步鉴定  

Construction and Preliminary Screening of Full-length cDNA Library of Lophopyrum elongatum

作　　者：王佳佳[1] 万琪[1] 葛冬冬[1] 董辉[1] 刘康[1] 陈国清 

机构地区：[1]南京农业大学作物遗传与种质创新国家重点实验室/江苏省现代作物生产协同创新中心,江苏南京210095 [2]江苏省沿江地区农业科学研究所,江苏如皋226541

出　　处：《核农学报》2016年第5期878-886,共9页Journal of Nuclear Agricultural Sciences

基　　金：中央高校基本科研业务费专项资金(KYZ201202-2)

摘　　要：长穗偃麦草是小麦的野生近缘植物之一,具有抗多种生物胁迫和非生物胁迫的优良基因。为了从中发掘耐盐基因,分别用100、200、300、400 mmol·L^(-1)的Na Cl和20%、30%、50%(w/v)的PEG-6000处理长穗偃麦草幼苗,采用改良的SMART方法合成逆境诱导条件下的全长c DNA。采用Gateway技术中的BP反应构建长穗偃麦草全长入门c DNA混合文库,滴度为4.0×106cfu·m L^(-1),库容量为2.0×107cfu,插入片段平均长度为0.75 kb,重组率达98.9%。将该入门文库重组到植物转基因双元载体p MDC83中,获得目标文库,其原始滴度为6.0×106cfu·m L^(-1),库容量为3.0×107cfu,插入片段平均长度为0.45 kb,重组率达100%。通过农杆菌侵染萌发花粉再授粉的方法,获得转基因玉米T0种子,转化率达3%;用农杆菌侵染法将农杆菌与烟草BY-2细胞共培育,成功将目标文库转入到烟草BY-2细胞中,在培养基中加入潮霉素和不同浓度的Na Cl溶液,筛选获得耐盐细胞系69个。研究结果为大规模快速筛选鉴定植株水平和细胞水平上的抗盐基因奠定了基础。Lophopyrum elongatum is an important wild relatives of common wheat（ Triticum aestivum L.）,which bears a variety of genes responsible for biotic and abiotic stress resistance. For the purpose of inducing expression of stress response and tolerance genes,Lophopyrum elongatum seedlings were treated with 100 mmol L-（-1）,200 mmol L-（-1）1,300 mmol L-（-1）1,and 400 mmol L-（-1）1 of Na Cl and 20%（ w / v）,30%（ w / v）,50%（ w / v） of PEG- 16000,the full- 1length c DNAs were synthesized by using a modified SMART（ switching mechanism at 5＇end of RNA transcript） method.And a mixed full- 1length entry c DNA library of Lophopyrum elongatum was constructed with BP reaction in Gateway Technology,the titer is 4. 0 × 106 cfu m L-（-1）,the capacity is 2. 0 × 107 cfu,with an average of 0. 75 kb insert fragments and 98. 9% of recombination rate. This entry c DNA library was subsequently shuttled into the plant transgenic binary vector of p MDC83 to obtain the destination library by LR reaction. The original titer of the destination library was 6 × 106 cfu m L-（-1）1,the capacity is 3. 0 × 107 cfu,the average length of insert fragment was 0. 45 kb and the recombination rate was 100%. The transgenic T0 generation seeds of maize were obtained by pollinating the pollens which were germinated and infected by Agrobacterium carrying the destination c DNA library. The transgenic rate was 3%. Meanwhile,the destination library was transformed into BY- 12 cells through co- 1incubation of Agrobacterium and BY- 12 cell. 69 salt tolerant cell lines were obtained by screening with homomycin resistance coupled with 50 or 100 mmol L-（-1）1 Na Cl. This study established a rapid and high throughput method for screening and identification of the salt resistance genes at both plant level and cell level.

关 键 词：长穗偃麦草 耐盐 全长CDNA文库 烟草BY-2细胞 玉米 

分 类 号：S512.1[农业科学—作物学]