烟草侧芽生长相关基因克隆及RNAi载体的构建转化  

Tobacco Plant Axillary Bud Related Gene Cloning and RNAi Expression Vector Constructing and Transforming

作　　者：陈兴江[1] 蔡刘体[1] 郑少清[1] 

机构地区：[1]贵州省烟草科学研究所,贵阳市金阳新区云潭北路550081

出　　处：《烟草科技》2013年第3期67-71,76,共6页Tobacco Science & Technology

摘　　要：为了培育打顶后不长侧芽的烟草植株,通过同源克隆,在烟草(品种K326)中得到了与侧芽生长相关基因的1个片断,将该片段序列通过BLAST检索Genbank同源比对,发现该基因同番茄的LS基因有非常高的同源序列,另外与水稻的MOC基因、拟南芥的LAS基因也有一定的同源性,命名为TLR基因。将TLR基因阅读框内640 bp的正向和反向片断构建到RNAi载体pHANNIBAL内含子两侧,再经NotI酶切回收约4300 bp目的片段,并插入到双元载体质粒pART27中,成功构建了含TLR基因片段正反向重复序列的植物表达载体pHANNIBAL-TLR-PART27。将pHANNIBAL-TLR-PART27质粒导入根癌农杆菌LBA4404中并转化烟草叶片细胞,没有得到转基因再生植株,推测这与TLR基因表达被抑制有关。In order to breed tobacco plant without axillary bud growing after topping, the fragment DNA sequence of tobacco axillary bud related gene （named as TLR gene） was cloned from flue-cured tobacco cv. K326 basing on homology gene cloning method, and its sequence was analyzed with BLAST search and Genebank homology comparison, it was found that that gene had high identity with LS gene of Lycopersicum esculentum, and had certain identity with MOC gene of Oryza sativa and LAS gene ofArabidopsis thaliana. The reverse and forward fragments （640 bp） in the reading flame of TLR gene were configured into right and left sides of intron in intermediated RNAi vector pHANNIBAL, respectively. After being cut by NotI＇ a fragment of about 4300 bp was recovered and inserted into a binary plasmid pART27, plant expression plasmid pHANNIBAL-TLR-PART27 with inverted repeated sequence containing TLR was successfully constructed. The pHANNIBAL-TLR-PART27 was introduced into Agrobacterium tumefaciens LBA4404, then transformed cells in tobacco leaf, but no transgenic plant was obtained, it was suspected that the expression of TLR gene was restrained.

关 键 词：侧芽 转化烟草 RNAI TLR基因 

分 类 号：S572.01[农业科学—烟草工业]