毛白杨(Populus tomentosa)中2个谷甾醇糖基转移酶基因的克隆与表达分析

Cloning and Expression Analysis of Two Sitosterol Glycosyltransferase Genes in Populus tomentosa

谷甾醇糖基转移酶(sitosterol glycosyltransferase,SGT)是能与膜结合催化植物谷甾醇糖基化的一种糖基转移酶。谷甾醇–葡萄糖苷(sitosterol-glucoside,SG)能在纤维素合酶(cellulose synthase,Ces A)的作用下,作为纤维素合成的引物,以UDP-葡萄糖(UDP-Glucose)为底物合成葡聚糖链。为了研究毛白杨(Populus tomentosa)PtSGT基因的功能,根据毛果杨(Populus trichocarpa)的同源基因Ptr SGT1和Ptr SGT3设计了PCR引物,以毛白杨组培苗叶片c DNA为模板克隆了毛白杨PtSGT1和PtSGT3 CDS序列,长度分别为1 851 bp、1 911 bp,分别编码616个氨基酸、636个氨基酸。结构分析表明,毛果杨(P.trichocarpa)同源基因Ptr SGT1和Ptr SGT3均含有14个外显子和13个内含子,结构与长度差异明显,分别定位于14号和2号染色体上。氨基酸序列同源性分析和进化分析表明,毛白杨(P.tomentosa)的PtSGT1和PtSGT3均与毛果杨和胡杨(Populus euphratica)相应蛋白具有较高的同源性。Real-time PCR分析显示,PtSGT基因家族的所有基因在根、茎、叶中均有表达,总体上呈组成型表达模式;PtSGT3在各组织中表达量显著高于其他成员,在茎中表达量最高,推测PtSGT3基因在谷甾醇–葡萄糖苷合成中有着重要作用。该实验研究结果为进一步解析毛白杨纤维素合成中这2个基因的功能奠定了基础。

Sitosterol glycosyltransferase（SGT） is a glycosyltransferase that catalyzes the glycosylation of plant sitosterol with membranes. The sitosterol-glucoside（SG） can act as a primer for cellulose synthesis under the action of cellulose synthase（Ces A） and forms glucan chains using UDP-glucose as a substrate. In order to study the function of Pt SGT gene in Populus tomentosa, PCR primers were designed according to the homologous genes Ptr SGT1 and Ptr SGT3 of Populus trichocarpa. The c DNA sequences of Pt SGT1 and Pt SGT3 CDS were cloned from c DNAs of P. tomentosa seedlings, and their lengths were 1 851 bp and 1 911 bp, encoding 616 and 636 amino acids, respectively. Structural analysis showed that the homologous genes Ptr SGT1 and Ptr SGT3 of P. trichocarpacontained 14 exons and 13 introns, with significant differences in structure and length, and were located on chromosomes 14 and 2, respectively. Amino acid sequence homology analysis and phylogenetic analysis showed that Pt SGT1 and Pt SGT3 have high homology with P. trichocarpa and Populus euphratica. The results of Real-time PCR showed that all genes in the Pt SGT gene family were expressed in roots, stems and leaves, with a constitutive expression pattern overall. The relative expression of Pt SGT3 was higher than other 3 members in poplar tissues, the highest expression in stems. It was speculated that Pt SGT3 played an important role in the synthesis of sitosterolglucoside. The results of this experiment laid a foundation for further analysis of the functions of these two genes in cellulose synthesis of P. tomentosa.