龙眼UGD6基因克隆及其表达特性分析

该试验采用RT-PCR和RACE技术,对龙眼多糖合成的关键基因尿苷二磷酸-葡萄糖6-脱氢酶基因(DlUGD6)进行分离克隆、生物信息学分析和亚细胞定位研究,并采用qRT-PCR技术,对其在龙眼体细胞胚胎发生、合子胚发育及不同组织器官中的表达模式进行分析。结果表明:(1)DlUGD6基因的cDNA序列全长1 860bp,包含开放阅读框1 443bp,编码480个氨基酸(GenBank登录号KU198438);生物信息学分析显示,DlUGD6属于稳定的酸性亲水蛋白,不含信号肽,具有跨膜结构和3个典型的保守结构域,属于UDP-葡萄糖/GDP-甘露糖脱氢酶家族;进化树分析表明,DlUGD6与柑橘亲缘关系较近。(2)洋葱内表皮GFP荧光定位观察发现,DlUGD6定位于细胞质;qRT-PCR结果显示,DlUGD6在龙眼非胚性愈伤组织中表达量相对较高,且在其他体胚发育阶段也均有稳定表达;在合子胚发育中子叶胚形成后第8天(S3)和第24天(S7)时表达量最高,整体呈"W"型;在不同组织器官中,DlUGD6在花药和茎中的表达量最高,且整体上生殖器官中的表达水平高于营养器官。研究认为,DlUGD6基因可能参与龙眼生长发育各个阶段中细胞壁多糖合成。

Cloning and Expression Characteristics of UGD6 in Dimocarpus longan Lour.

To investigate the gene structure, characteristics and potential biological function of uridine diphosphate glucose dehydrogenase 6 gene (UGD6) in Dimocarpus longan Lour. (longan), we cloned the key gene DlUGD6 of polysaccharide synthesis in longan by RT PCR and RACE PCR. Its bioinformatics, subcellular localization and expression patterns in somatic embryogenesis, embryonic deveploment and different tissues were also analyzed. The results showed that: (1)the cDNA length of DlUGD6 was 1 860 bp, which contained an open reading frame of 1 443 bp and encoded 480 amino acids (GenBank accession number KU198438). Bioinformatics analysis showed that DlUGD6 belonged to the family of UDP glucose/GDP mannose dehydrogenase, which had three typical conserved domains. According to phylogenetic tree analysis, DlUGD6 kept close genetic relationship with Citrus sinensis. (2)Observation of fluorescence localization by GFP in onion epidermis indicated that DlUGD6 was localized in cytoplasm. The results of qRT PCR indicated that DlUGD6 was up expressed in non embryogenic callus and it kept stable in other stages of somatic embryogenesis in longan. The expression level of DlUGD6 in S3 and S7 stages of embryo development was the highest. In addition, the expression of DlUGD6 kept the highest in anther and stem of longan; As a whole, it was higher in genital organs than that in vegetative organs. These results suggested that DlUGD6 might involve in synthesis of cell wall polysaccharides in growth and development of longan.