灰毡毛忍冬MADS-box家族基因SVP克隆及表达分析

Cloning and expression analysis of MADS-box family gene SVPfrom Lonicera Macranthoides

目的克隆获得灰毡毛忍冬Lonicera macranthoides MADS-box家族基因SHORT VEGETATIVE PHASE(SVP),并对其进行生物信息学、时空表达特异性和蛋白原核表达等特性分析。方法根据灰毡毛忍冬转录组数据设计特异性引物,通过PCR技术克隆Lm SVP基因全长;运用生物信息学方法分析其编码蛋白的序列特征;利用实时荧光定量技术(reverse-transcription polymerase chain reaction,q RT-PCR)分别检测该基因在灰毡毛忍冬2个品种“龙花”和“金翠蕾”叶片、花早期和晚期中的表达水平;最后构建p TOPO-D1-Lm SVP原核表达载体,转化至大肠杆菌BL21(DE3)中进行蛋白表达。结果Lm SVP基因全长1472bp,开放阅读框(open reading frame,ORF)长720 bp,编码239个氨基酸,蛋白质相对分子质量为26712.63。进化树分析表明,Lm SVP蛋白与中华猕猴桃、小粒咖啡的SVP蛋白亲缘关系最近。q RT-PCR结果显示,Lm SVP基因在“龙花”和“金翠蕾”品种中的表达均存在组织特异性表达,其在叶中的表达量显著高于花中;而在花发育不同时期,Lm SVP基因表达量没有明显差异。Lm SVP在大肠杆菌中成功表达出蛋白,蛋白表达结果显示其相对分子质量约为26710,与预测相符合。结论通过对Lm SVP基因的全长克隆、序列分析和表达特性鉴定,为进一步研究该基因在调控花蕾型灰毡毛忍冬蕾期长及花冠不开放的功能提供实验基础。

ObjectiveTo clone the MADS-boxfamily gene SHORT VEGETATIVE PHASE(SVP)of Lonicera macranthoides,analyze the characteristics of bioinformatics,spatiotemporal expression and protein prokaryotic expression.MethodsBased on the transcriptome sequencing data of L.macranthoidesin the previous study,the full-length cDNA of LmSVPwas cloned by PCR.The bioinformatics methods were used to analyze the sequence characteristics of their encoded proteins.Real-time quantitative reverse-transcription polymerase chainreaction(qRT-PCR)was used to detect the expression level of LmSVPin leaves,early and late flowers of the“Longhua”and“Jincuilei”varieties of L.macranthoides.And then,the pTOPO-D1-LmSVP prokaryotic expression vector was constructed and transformed into E.coilBL21(DE3)to express.Results The full-length of LmSVPgene was 1472 bp,containing an open reading frame(ORF)of 720 bp and encoding 239 amino acids.The relative molecular weight of LmSVP protein was 26712.63.Phylogenetic analysis indicated that the LmSVP protein was most closely related to Actinidia chinensisand Coffea arabica.The qRT-PCR results showed that the expression level of LmSVPgenes were specific in both“Longhua”and“Jincuilei”variety,and expression in leaves were significantly higher than in flowers.At different stages of flower development,the expression level of LmSVPgenes were not significantly different.LmSVPsuccessfully expressed a recombination protein with molecular weight of about 26710 in the E.coliBL21(DE3),which was consistent with the prediction.Conclusion Through the full-length cloning,sequence analysis and expression characterization of LmSVPgene,it provided experimental basis for further study on the function of LmSVPgene in regulating the bud length and corolla non-opening of L.macranthoides.