几丁质酶基因克隆及其野生蕉转化  

Cloning of Chitinase Gene and its Genetic Transformation of Wild Banana(Musa itinerans Cheesm.)

作　　者：胡春华[1] 魏岳荣[1] 刘凯[1] 易干军[1] 黄秉智[1] 黄永红[1] 

机构地区：[1]广东省农业科学院果树研究所香蕉研究中心,广州510640

出　　处：《分子植物育种》2010年第4期719-724,共6页Molecular Plant Breeding

基　　金：国家科技支撑项目(2008BAD92B06);科技部国际合作项目(2009DFA31470);广东省科技攻关项目(2007A020200003-3);广东省农科院院长基金(20090104)共同资助

摘　　要：本研究根据GenBank公布的木霉几丁质酶基因(chitinase)序列设计一对引物,采用RT-PCR方法从木霉(Trichoderma spp.)中克隆获得了几丁质酶基因全序列,编码区共1275bp,推测其编码424个氨基酸,该基因与哈茨木霉(Trichoderma harzianum)的内切几丁质酶基因chit42(GenBank accession No.L14614)具有99%的同源性。在此基础上,将获得的几丁质酶基因从pGEM-T载体中用XbaⅠ和BamHⅠ切下,克隆到pBI121植物表达载体的XbaⅠ和BamHⅠ位点,构建了植物表达载体pBI-chit并将其转入根癌农杆菌菌株EHA105。该菌株转化野生蕉(Musa itinerans Cheesm.)胚性细胞悬浮系,经过抗性筛选、胚的诱导和萌发,获得成熟体细胞胚和再生苗。通过GUS组织化学法检测和PCR方法鉴定,结果表明外源基因已经成功转入到野生蕉中。本研究为下一阶段转化香蕉栽培品种和筛选抗香蕉枯萎病新种质奠定一定的基础。Using RT-PCR,a chitinase gene was cloned from Trichoderma spp.by a pair of primers designed according to the sequence of chitinase gene of Trichoderma harzianum released on the GenBank.The open reading frame（ORF） sequence of the chitinase gene was found to be 1 275 bp long which encoding 424 amino acids.The result of sequence analysis showed that the cloned chitinase gene had 99% similarity with that of the chitinase gene from Trichoderma harzianum deposited in GenBank accession No.L14614.Then,the fragments containing the chitinase were excised from pGEM-T cloning vector with restriction enzyme XbaⅠ and BamHⅠ and inserted into the site between the same two restriction enzymes on pBI121 vectors.The constructed plasmids were then transformed into EHA105 strain of Agrobacterium tumefaciens by using the freeze-thaw method,and then we did transform of the embryogenic cell suspensions of the wild banana via Agrobacterium-mediated transformation.After selection,embryogenic development and germination transgenic buds were obtained and confirmed by histochemical GUS assay and PCR.The results suggested that we successfully construct the plant expression vector of chitinase and transferred into wild banana,which would be an important base for further genetic transformation of banana with resistance to fusarium wilt.

关 键 词：野生蕉 几丁质酶基因 克隆 转基因 

分 类 号：S668.1[农业科学—果树学]