龙眼胚性愈伤组织DlGRAS4与DlGRAS54基因的克隆及表达分析  

Cloning and Expression Analysis of DlGRAS4 and DlGRAS54 from Embryogenic Callus of Dimocarpus longan Lour.

作　　者：陈裕坤[1] 林玉玲[1] 田奇琳[1] 林丽霞[1] 赖瑞联[1] 赖钟雄[1] 

机构地区：[1]福建农林大学园艺植物生物工程研究所,福州350002

出　　处：《西北植物学报》2014年第2期215-224,共10页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(31272149);国家自然科学基金(31071787);高等学校博士学科点专项科研基金(20093515110005);福建省科技平台建设项目(2008N2001)

摘　　要：以龙眼松散型胚性愈伤组织为实验材料,采用RT-PCR结合RACE法,克隆了植物特异转录调控因子DlGRAS4和DlGRAS54的cDNA全长序列和DNA序列,并进行生物信息学以及表达分析。结果表明:DlGRAS4全长1 668bp,开放阅读框(ORF)1 296bp,编码431个氨基酸(GenBank登录号:KC127683);DlGRAS54全长2 113bp,ORF 1 659bp,编码552个氨基酸(GenBank登录号:KC127684);DlGRAS54的DNA序列在5′-UTR含有1个1 533bp的内含子。生物信息学分析表明:DlGRAS4和GRAS54是不稳定亲水蛋白,不含信号肽,具有跨膜结构和GRAS家族的保守结构域,亚细胞定位于细胞膜中;与毛果杨、葡萄、蓖麻和可可等具有较高的同源性。系统进化树分析显示,DlGRAS54与拟南芥AtPAT1、葡萄VvPAT1、毛果杨PtPAT1、大豆GmPAT1处于同一分枝;DlGRAS4与拟南芥AtSCL23、玉米ZmSCL23处于同一分枝。推测DlGRAS4和DlGRAS54是GRAS基因家族的成员。qPCR结果表明,DlGRAS4在整个发育阶段转录水平呈"W"型变化趋势,在球形胚和子叶形胚阶段的表达量达最大值;DlGRAS54在整个发育阶段的转录水平呈"M"型变化趋势,在球形胚和鱼雷形胚阶段表达量达最大值,表明DlGRAS4和DlGRAS54主要在发育的中晚期起作用。外源赤霉素处理后DlGRAS4和DlGRAS54的表达量明显上调,说明DlGRAS4和DlGRAS54对赤霉素处理能做出积极的反应。The RT-PCR combined with RACE method was used to clone the complete cDNA sequences and DNA sequences of plant-specific transcription factor DlGRAS4 and DlGRAS54 from embryogenic callus of Dimocarpus longan. The complete cDNA sequence of DlGRAS4 was 1 668 bp, encoding 431 amino acids, the complete cDNA sequence of DlGRAS54 was 2 113 bp,encoding 552 amino acids. DNA sequence analy- sis indicated that 5＇-UTR of DlGRAS54 has a 1 533 bp intron. The sequences of both nucleotides and ami- no acids of the two members were high homologous with those of the known GRAS genes in other species. D1GRAS4 and D1GRAS54 located in plasma membrane; they had no signal peptide, had transmembrane structure and the GRAS protein domain. Anglicizing phylogenetic tree of GRAS in plants indicated that D1-GRAS54 and AtPAT1, VvPAT1, PtPAT1, GmPAT1 belonged tO the same branch; D1GRAS4 and At- SCL23, ZmSCL23 belonged to the same branch. Therefore, they putatively belonged to GRAS family, qPCR results indicated that DIGRAS4 showed approximately a ＂W＂ curve,it expressed at the highest levels in the globular embryos and cotyledonary embryos cultures. DlGRAS54 showed approximately an ＂M＂ curve, it expressed at the highest levels in the globular embryos and topedo embryos cultures, suggesting that DI- GRAS4 and DlGRAS54 play a major role at the middle and late developmental stages of longan somatic em- bryogenesis. DIGRAS4 and DlGRAS54 showed up-regulated expression patterns under exogenous GA3 treatment showed that they made the positive response with GAg treatment.

关 键 词：龙眼 体细胞胚胎发生 GRAS 克隆 生物信息学 实时荧光定量PCR 

分 类 号：Q785[生物学—分子生物学] Q786