巴西橡胶树HbPSKR2基因克隆及互作蛋白鉴定

Cloning and Interacted Protein Identification of HbPSKR2 Gene from Rubber Tree

磺肽素是一种高等植物特有的小肽类激素,广泛参与植物的生长发育、分裂分化、生物或非生物胁迫等生物学过程。磺肽素受体蛋白(phytosulfokine receptor,PSKR)是磺肽素的直接受体,对于磺肽素信号传导至关重要。本研究采用RT-PCR(reverse transcription-polymerase chain reaction)技术克隆了橡胶树的HbPSKR2基因,并对其进行生物信息学、基因表达模式、互作蛋白筛选及鉴定分析。结果显示HbPSKR2基因的开放阅读框全长3159 bp,编码1052个氨基酸,理论分子量为114.84 kDa,理论等电点为6.34。结构域分析显示HbPSKR2属于典型的跨膜蛋白,前640个氨基酸是由亮氨酸重复组成的天线结构,第692~714个氨基酸是跨膜结构域,第765~1052个氨基酸是膜内激酶结构域。对HbPSKR2膜内激酶结构域以及拟南芥和水稻的PSKR同源序列进行多重比对,结果显示均存在ATP binding site、CaM binding site、Activation segment、GC Centre等保守性位点。表达模式分析显示,HbPSKR2基因在橡胶树形成层区高丰度表达,其表达量在冠菌素处理前期显著上升。通过酵母双杂交技术筛选到12个与HbPSKR2互作的候选蛋白,并对其中的2个蛋白激酶(HbPBL8和HbPIX13)与HbPSKR2的互作关系进行荧光素酶互补成像验证。结果显示,在烟草中共转化HbPSKR2-nLUC/HbPBL8-cLUC和HbPSKR2-nLUC/HbPIX13-cLUC可以观察到强烈的荧光信号,进一步证明了HbPSKR2在体内可与HbPBL8激酶和HbPIX13激酶互作。橡胶树HbPSKR2基因克隆及互作蛋白鉴定将为深入揭示橡胶树乳管分化分子机制提供新的思路。

Phytosulfokine(PSK)is a small peptide hormone unique to higher plants,widely involved in biological processes such as plant growth and development,division and differentiation,biotic and abiotic stress.PSK receptor(PSKR)is a direct receptor for PSK and is crucial for PSK signal transduction.In the present study,HbPSKR2 was cloned from rubber tree by RT-PCR(reverse transcription-polymerase chain reaction),and its bioinformatics,gene expression pattern,interacted proteins screening and identification were also analyzed.Results showed that the open reading frame of reading frame of HbPSKR2 had a total length of 3159 bp and encoded 1052 amino acids with molecular weight 114.84 kDa and theoretical isoelectric point 6.34.Domain analysis showed that HbPSKR2 was a typical trans-membrane protein.The first 640 amino acids were the antenna structures composed of leucine rich repeat(LRR),the 692 to 714 amino acids were transmembrane domains,and the 765 to 1052 amino acids were kinase domains.Multiple comparisons were conducted on the membrane kinase domain of HbPSKR2 and the membrane kinase domain of PSKR homologous proteins in Arabidopsis and rice.The results showed the existence of conserved sites such as ATP binding site,CaM binding site,Activation segment,GC Centre.Expression pattern analysis showed that HbPSKR2 was highly expressed in the cambium region of rubber tree.The expression level of HbPSKR2 was significantly increased at the early stage of coronatine(COR)treatment.Twelve candidate proteins interacted with HbPSKR2 were screened by yeast two hybrid technology,and the interaction between two protein kinases(HbPBL8 and HbPIX13)and HbPSKR2 was further verified by luciferase complementary imaging.The strong fluorescence signal was observed by co-transformation HbPSKR2-nLUC/HbPBL8-cLUC and HbPSKR2-nLUC/HbPIX13-cLUC into tobacco,suggesting the interaction between HbPSKR2-HbPBL8 and HbPSKR2-HbPIX13 in vivo.The cloning of HbPSKR2 and identification of HbPSKR2 interacted protein would provide new insights into the molecular mechanism of laticifer differentiation in rubber tree.