棉花nodulin-like基因及其启动子的结构特征和遗传表达

用同尾酶反向PCR技术(Ⅱ-PCR)和快速分离目的基因cDNA 5'未知序列方法(RICUP)首次从陆地棉品种Y18(Gossypium arboreum L.Y18)中分离到nodulin-like基因的全长cDNA、DNA和启动子序列.结果表明,该基因全长为2 353 bp,具有5个内含子和6个外显子.cDNA全长1480 bp,含有一个1125 bp的ORF结构,编码375个氨基酸,在GenBank nr数据库中没有与之同源的基因序列,在EST数据库中仅有一条733 bp的亚洲棉纤维EST序列(GenBank登录号:BF271235)与之有92%的同源性.Nodulin-like基因的启动子全长1 969 bp,启动子区域具有Initiator、TATA box、CAAT-like box和富含AT序列等启动子特征序列.Southern blot结果表明:该基因在棉花基因组中有一个拷贝.Northern blot结果表明:该基因在棉花的蕾、花、纤维和铃壳等生殖器官中呈优势表达.本研究有望为改良棉花生殖器官的农艺性状提供靶基因,并为解决目前转基因抗虫棉"前期抗虫性强、后期抗虫性弱"这一生产实际问题提供有效的表达调控元件.

Structural Characteristic and Genetic Expression of nodulin-like Gene and Its Promoter in Cotton

Full-length DNA, cDNA and promoter sequences of nodulin-like gene have been isolated and characterized by isocaudarner inverse PCR (Ⅱ-PCR) and rapid isolating cDNA 5' unknown sequence and promoter (RICUP) methods from cotton. Sequence analysis indicated that nodulin-like gene was 2 353 bp in size, including five introns and six exons. Its full-length cDNA was 1 480 bp, containing an 1 125-bp ORF structure, which encoded a 40.23-kD polypeptide with predicted isoelectric point of 8.433. A check against nr database in GenBank indicated that no DNA sequence was found to share homology with it. Searched in EST database, a 733-bp EST sequence of Gossypium arboreum L. (GenBank asscession number BF271235) was found to share 92% homology with it. Promoter of nodulin-like gene was 1 969 bp in length with several typical sequences, such as initiator, TATA box, CAAT-like box and AT-rich sequence. Southern blotting analysis suggested that one copy of nodulin-like gene was integrated into cotton genome. Northern blotting analysis showed that nodulin-like gene expressed preferentialy in fiber and reproductive organs of cotton, such as bud, flower and boll. Our work provides researchers with candidate genes to improve agronmic traits of cotton and also provide efficient expression elements for problem solving in transgenic Bt cotton, namely resistance to bollworm declining dramatically at late developmental stage of cotton.