橡胶树HbRAN1基因的克隆与表达分析

Isolation and Characterization of a Gene Coding a Small RAN GTPase in Hevea brasiliensis

利用本课题组获得的二代测序(FLX454)转录组数据库,筛选并克隆获得到1条969 bp的核苷酸序列,该序列编码1个由221个氨基酸组成的25.11 ku蛋白,氨基酸同源性分析发现,此氨基酸序列具有GTPase活性区域,属于小G蛋白RAN亚家族,且与蓖麻、烟草、水稻、小麦等的RAN1基因序列高度同源,命名为HbRAN1(GenBank登录号:KC577150)。实时荧光定量PCR分析结果表明,该基因受割胶、伤害、高温、低温、干旱及乙烯利等因素调控,同时其表达模式与橡胶树死皮程度相关,但对SA、GA、JA、ABA、CTK、2,4-D等激素处理应答不明显。结果表明,HbRAN1基因编码1个典型的RAN蛋白,可能在橡胶树抗逆过程中起作用,可作为橡胶树抗逆分子育种的靶标基因。

After searching the secondary generation FLX454 latex transcriptome database,the full length cDNA of a Hevea RAN GTPase（named as HbRAN1,GenBank accession： KC577150） was cloned.The full-length cDNA of HbRAN1 was first obtained from latex of H.brasiliensis,which is 969 bp long,encodes a protein of 25.11 ku（221aa） and contains the typical domains of Ran GTPases.At the amino-acid sequence level,HbRAN1 exhibits high homology to RcRAN1,NtRAN1,OsRAN1,TaRAN1 and AtRAN1.By Real-time RT-PCR analysis,the transcription level of HbRAN1 was regulated by tapping treatment,wounding,high temperature,chilling,drought stresses and ethylene.In addition,HbRAN1 showed obviously higher expression level in TPD（Tapping Panel Dryness） trees than that in healthy rubber trees.However,its expression did not affect by other hormones（SA、 GA、 JA、 ABA、 CTK and 2,4-D）.The results suggested the HbRAN1 was one typical RAN member of small GTPase superfamily,and was implicated in stress-responses in Hevea tree,which may serve as a target gene for Hevea molecular breeding.