粘毛黄芩SvFNSⅡ-2基因克隆及生物信息学分析

Cloning and Bioinformatic Analysis of SvFNSⅡ-2 Gene in Scutellaria viscidula

目的:克隆获得粘毛黄芩中黄芩苷合成关键酶黄酮合成酶Ⅱ-2基因(FNSⅡ-2)全长,并进行生物信息学分析。方法:以粘毛黄芩总RNA为模板,采用RACE技术和序列克隆相结合的方法,获得粘毛黄芩FNSⅡ-2基因的cDNA完全编码序列并进行生物信息学分析;采用实时定量(qPCR)和高效液相色谱技术(HPLC)测定粘毛黄芩采收期不同部位FNSⅡ-2的表达量及黄芩苷的含量,并对二者进行回归及相关性分析。结果:获得SvFNSⅡ-2基因ORF区长度为1 518 bp(GenBank ID:MG737856),编码氨基酸505个,包含3个高度保守区域,分析预测编码稳定的亲水性蛋白为游离核糖体合成的胞内膜结合蛋白,二、三级结构均以α-螺旋为主体,且包含有多个磷酸化位点;qPCR和HPLC结果显示,SvFNSⅡ-2基因的表达和黄芩苷含量在根、茎、叶中均存在极显著差异(P<0.01),而且不同部位间SvFNSⅡ-2基因表达量和黄芩苷含量呈极显著正相关关系(r=0.999,P<0.01)。结论:首次克隆获得SvFNSⅡ-2基因cDNA全长,初步证明SvFNSⅡ-2是粘毛黄芩黄芩苷合成途径中重要的调控关键基因,为揭示黄芩苷分子合成机制及调控研究奠定了基础。

Objective:To clone and analyze the flavone synthetase Ⅱ-2 gene(FNSⅡ-2)in Scutellaria viscidula.Methods:The cDNA complete coding sequence of FNSⅡ-2 gene was obtained using rapid-amplification of cDNA ends(RACE)and sequence cloning.Further bioinformatic analysis of this gene was conducted.The expression of FNSⅡ-2 and the content of baicalin in different parts of Scutellaria viscidula was determined by real-time quantification PCR(qPCR)and HPLC,respectively.Regression and correlation analysis between them were carried out.Results:The length of ORF region of SvFNSⅡ-2 gene was determined as 1 518 bp(GenBank ID:MG737856),which encodes 505 amino acids consisting of three highly conserved regions.According to the prediction results,this gene encodes stable hydrophilic proteins that intracellularred membrane-binding proteins synthesized by free ribosomes.The protein products mainly contain α-helix in their secondary and tertiary structures and had several phosphorylation sites.The results of qPCR and HPLC showed that highly significant differences(P<0.01)exist between the expression of SvFNSⅡ-2 as well as the content of baicalin in leaves and roots.For each part,the expression of SvFNSⅡ-2 and the content of baicalin were highly positively correlated(r=0.999,P<0.01).Conclusion:The complete sequence of SvFNSⅡ-2 cDNA is first cloned,which is considered to play an important role in baicalin synthesis pathways of Scutellaria viscidula.These results will lay solid foundation for revealing the molecular mechanism and regulation pathways of baicalin biosynthesis in Scutellaria viscidula plants.