杨树UGT198基因的生物信息学分析、基因克隆及功能初探

Bioinformatics analysis,gene cloning and function study of poplar UGT198 gene

【目的】研究UGT198基因是否参与调控烟草抗虫性,为未来探索其参与调控杨树抗虫性奠定基础。【方法】前期通过分析公共转录组数据发现PtrUGT198基因在杨树响应虫害的转录组中高调表达,对其进行生物信息学及进化分析。以107杨为材料,克隆UGT198基因,构建过量表达载体,利用农杆菌介导法转化烟草获得过量表达转基因株系。通过棉铃虫饲虫试验初步验证UGT198基因是否参与调控抗虫性,分析UGT198基因在调控抗虫性中发挥的功能。【结果】PtrUGT198基因完整的ORF区为1440 bp,编码479个氨基酸,理论分子量为53.14 kDa,等电点为5.92,编码不稳定疏水性蛋白。PtrUGT198蛋白的二级结构α⁃螺旋占41.75%,β⁃折叠占14.61%,β⁃转角占7.10%,无规卷曲占6.53%。PtrUGT198蛋白不存在跨膜区,预测其不属于膜蛋白,为非分泌性蛋白,该蛋白的磷酸化修饰以丝氨酸为主。同源序列对比结果显示PtrUGT198蛋白的C末端存在高度保守的植物次生产物糖基转移酶(Plant secondary product glycosyltransferase,PSPG)基序。系统进化树分析发现,PtrUGT198蛋白与同属植物毛白杨、银白杨和胡杨亲缘关系最近,其次和模式植物烟草有较近的亲缘关系。成功克隆杨树UGT198基因,构建pNC⁃UGT198基因过量表达载体,并转化烟草获得过量表达转基因株系,进行饲虫实验,得到高表达量烟草相对抗虫。【结论】首次成功克隆了UGT198基因,并初步解析其调控抗虫性的功能,为深入了解UGT198基因调控杨树抗虫机理提供参考价值。

【Objective】The present paper aimed to study whether UGT198 gene was involved in the regulation of insect resistance of(Nicoti⁃ana tabacum)tobacco,and lay a foundation for the future study of UGT198 gene in the regulation of poplar insect resistance.【Method】By analyzing the public transcriptome data,it was found that PtrUGT198 was highly expressed in the transcriptome of poplar responding to insect infestation,and the bioinformatics and evolutionary analysis were conducted.Using 107 poplar as material,UGT198 gene was cloned,over⁃expression vector was constructed,and overexpression transgenic strain of tobacco was obtained by Agrobacterium mediated transformation.The feeding experiments preliminarily verified whether PtrUGT198 gene participated in the regulation of insect resistance,and analyzed the function of UGT198 gene in the regulation of insect resistance.【Result】The complete ORF region of PtrUGT198 gene was 1440 bp,encoding 479 amino acids,theoretical molecular weight was 53.14 kDa,and isoelectric point was 5.92,encoding unstable hydrophobic protein.The secondary structure of PtrUGT198 protein consisted ofα⁃helix(41.75%),β⁃fold(14.61%),β⁃corner(7.10%)and random curl(36.53%).PtrUGT198 did not have a transmembrane region,which was predicted to be a non⁃secreted protein,and the phosphorylation of this protein was mainly serine.The results of homologous sequence comparison showed that highly conserved plant secondary product glycosyltransferase(PSPG)motif existed in the C⁃terminal of PtrUGT198 protein.Phylogenetic tree analysis showed that PtrUGT198 protein was the most closely related to Populus tomentosa,P.alba and P.euphratica in the same genus,followed by a close relationship with tobacco model plants.UGT198 gene of poplar was cloned,pNC⁃UGT198 gene overexpression vector was constructed,and overexpression transgenic strain of tobacco was obtained.【Conclusion】UGT198 gene was successfully cloned for the first time,and its function of regulating insect re⁃sistance was initially analyzed,which provided reference value for further understanding of the mechanism of regulating poplar insect resist⁃ance by UGT198.