摘要
目的:研究欧洲花楸植保素糖基化修饰的相关基因,从欧洲花楸悬浮细胞中克隆糖基转移酶(glycosyltransferase,GT)基因,进行序列分析和原核表达。方法:基于欧洲花楸转录组数据设计特异性引物,克隆得到2条Sa UGTs基因的c DNA序列,构建HIS-MBP-pET28a-SaUGTs原核表达载体,诱导表达Sa UGTs重组蛋白。结果:克隆得到2条糖基转移酶基因Sa UGT1和Sa UGT2序列,完整开放阅读框为1 458 bp和1 431 bp,分别编码485和476个氨基酸,相对分子质量为54. 27 k Da和53. 49 k Da,理论等电点为5. 50和5. 63。生物信息学分析显示,无信号肽,含有糖基转移酶家族保守结构域(PSPG)。系统进化结果显示Sa UGT1和Sa UGT2蛋白与拟南芥UGT85家族亲缘关系较近。实时荧光定量PCR检测显示,欧洲花楸悬浮细胞经酵母提取物(YE)诱导后,Sa UGT1和Sa UGT2的相对表达量明显上调,分别在24 h和12 h达到最大值。通过IPTG诱导,在大肠埃希菌中成功表达Sa UGT1和Sa UGT2重组蛋白,并得到了纯化的重组蛋白。结论:该研究首次在欧洲花楸中克隆得到糖基转移酶基因,并成功构建了原核表达载体,为进一步研究该类基因的功能奠定了基础。
Objective: To obtain the glycosyltransferase gene involved in modification reaction of phytoalexin from Sorbus pohuashanensis suspension cell,and conduct sequence analysis and prokaryotic expression analysis. Method: Based on the transcriptome data,specific primers were designed to obtain 2 cDNA sequences of SaUGTs genes,construct prokaryotic expression vector HIS-MBP-pET28 a-SaUGTs and induce the expression of recombinant SaUGTs protein. Result: Sa UGT1 and Sa UGT2 sequences were cloned and obtained from glycosyltransferases,then bioinformatic analysis of the sequence and prokaryotic expression analysis were conducted. SaUGT1 gene contained 1 458 bp open reading frame(ORF),encoding a polypeptide of 485 amino acids,with a relative molecular weight of 54. 27 kDa and theoretical isoelectric point(pI) of 5. 50. SaUGT2 gene contained 1 431 bp ORF, encoding a polypeptide of 476 amino acids, with a relative molecular weight of53. 49 kDa and theoretical pI of 5. 63. Bioinformatics analysis indicated that SaUGT1 and SaUGT2 protein had no signal peptide,and the conserved domains of glycosyltransferase family were detected. Phylogenetic results showed that SaUGT1 and SaUGT2 proteins had the closest relationship with the UGT85 family of A. thaliana. Differential expression analysis revealed that the relative expression levels of SaUGT1 and SaUGT2 were increased significantly after being induced by yeast extract(YE),with the highest expression level found at 24 h and 12 h. The recombinant SaUGT1 and SaUGT2 proteins were successfully expressed in Escherichia coli DE3 cells and finally,the recombinant SaUGT1 and SaUGT2 proteins were purified through Ni2 +affinity chromatography. Conclusion:The glycosyltransferase gene was cloned from the S. aucuparia for the first time,and the prokaryotic expression vector was successfully constructed,laying foundation for further study of the function of this gene.
引文
[1]中国科学院《中国植物志》编辑委员会.中国植物志.第36卷[M].北京:科学出版社,1974:284.
[2]贾宏丽,周良云,王升,等.茉莉酸通路参与Hpa1诱导的欧洲花楸细胞植保素生物合成[J].中国中药杂志,2018,43(14):2928-2934.
[3]魏杰,石佳,侯潇,等.欧洲花楸的化学成分及药理作用研究进展[J].辽宁大学学报:自然科学版,2014,41(4):362-368.
[4]陈永钧,龙晓英,潘素静,等.黄酮类化合物的药效机制及构效关系研究进展[J].中国实验方剂学杂志,2013,19(11):337-344.
[5]黄蕾,肖文娟,刘超,等. 3种重金属对欧洲花楸悬浮细胞生物量的影响[J].中国实验方剂学杂志,2013,19(24):226-229.
[6] Hrazdina G,Borejsza-Wysocki W,Lester C. Phytoalexin production in an apple cultivar resistant to Venturia inaequalis[J]. Phytopathology,1997,87(8):868-876.
[7] Chizzali C,Khalil M N,Beuerle T,et al. Formation of biphenyl and dibenzofuran phytoalexins in the transition zones of fire blight-infected stems of Malus domestica cv.‘Holsteiner Cox’ and Pyrus communis cv.‘Conference’[J]. Phytochemistry,2012,77:179-185.
[8] Chizzali C,Beerhues L. Phytoalexins of the pyrinae:biphenyls and dibenzofurans[J]. Beilstein J Org Chem,2012,8(1):613-620.
[9] ZHOU L,YANG J,YANG G,et al. Biphenyl phytoalexin in Sorbus pohuashanensis suspension cell induced by Yeast extract[J]. Molecules,2016,21(9):1180.
[10] Campbell J A,Davies G J,Bulone V,et al. A classification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities[J]. Biochem J,1997,326(3):929.
[11] Vogt T,Jones P. Glycosyltransferases in plant natural product synthesis:characterization of a supergene family[J]. Trends Plant Sci,2000,5(9):380-386.
[12] Tiwari P,Sangwan R S,Sangwan N S. Plant secondary metabolism linked glycosyltransferases:an update on expanding knowledge and scopes[J]. Biotechnol Adv,2016,34(5):714-739.
[13]李攀.拟南芥和玉米糖基转移酶基因参与非生物胁迫耐性的功能研究[D].济南:山东大学,2017.
[14] Augustin J M,Drok S,Shinoda T,et al. UDPglycosyltransferases from the UGT73C subfamily in Barbarea vulgaris catalyze sapogenin 3-O-glucosylation in saponin-mediated insect resistance[J]. Plant Physiol,2012,160(4):1881-1895.
[15] LI X,Shin S,Heinen S,et al. Transgenic wheat expressing a barley UDP-glucosyltransferase detoxifies deoxynivalenol and provides high levels of resistance to Fusarium graminearum[J]. Mol Plant Microbe Interact,2015,28(11):1237-1246.
[16]秦晶晶,孙春玉,张美萍,等.植物UDP-糖基转移酶分类、功能以及进化[J].基因组学与应用生物学,2018,37(1):440-450.
[17] Schmittgen T D,Livak K J. Analyzing real-time PCR data by the comparative C(T)method.[J]. Nat Protoc,2008,3(6):1101-1108.
[18] Kubo A,Arai Y,Nagashima S,et al. Alteration of sugar donor specificities of plant glycosyltransferases by a single point mutation[J]. Arch Biochem Biophys,2004,429(2):198-203.
