绞股蓝皂苷生物合成后修饰酶基因的cDNA克隆和组织表达特征

cDNA Cloning and Tissue Expression Characteristics of Post-modifying Enzyme Genes in Gypenosides Biosynthesis

从绞股蓝转录组挖掘可能与绞股蓝皂苷生物合成相关修饰酶基因细胞色素P450(cytochrome P450,CYP)及UDP-糖基转移酶(UDP-dependent glycosyltransferase,UGT)基因的cDNA进行克隆并开展了以下研究。根据课题组前期绞股蓝转录组数据,筛选CYP及UGT Unigenes,利用RT-PCR克隆ORF全长,并结合生物信息学对其编码蛋白进行功能分析,最后通过荧光定量PCR验证其在根、茎、叶的差异性表达。结果克隆得到两个ORF序列,分别命名为CYP94A1和UGT91A1;CYP94A1序列包含1509 bp的开放阅读框架,编码一条含503个氨基酸残基的肽链,具有CYP保守结构域;UGT91A1序列包含1383 bp的开放阅读框架,编码一条含461个氨基酸残基的肽链,具有UGT保守结构域。这两个基因的转录表达均具有组织特异性,在叶或茎中的表达均高于根。CYP94A1和UGT91A1的克隆、转录表达及生物信息学分析为进一步挖掘绞股蓝中CYPs、UGTs及功能验证提供了帮助,增加对CYP和UGT两个超基因家族的认识。

The cDNA of cytochrome P450(CYP)and UDP-dependent glycosyltransferase(UGT)genes,which may be related to the modification gene of gypenosides biosynthesis,were cloned from the transcriptome of Gynostemma pentaphyllum and the following studies were carried out.According to the previous transcriptome data of Gynostemma pentaphyllum in our research group,we screened CYP and UGT Unigenes,and cloned their full-length ORFs by RT-PCR,and analyzed the encoded proteins function combined with bioinformatics.At last,the differential expression of the two genes in root,stem and leaf was verified by real-time PCR.In result,two ORF sequences were cloned and named as CYP94 A1 and UGT91 A1,respectively.The length of CYP94 A1 sequence is 1509 bp,encoding a peptide chain containing 503 amino acid residues with a CYP conserved domain.The UGT91 A1 sequence contains 1383 bp,encoding a peptide chain containing 461 amino acid residues with a UGT conserved domain.The transcriptional expression of both genes is tissue specific,which is higher in leave or stem than in root.The cloning,transcriptional expression and bioinformatics analysis of CYP94 A1 and UGT91 A1 provide help for further excavation of CYPs,UGTs and functional verification in Gynostemma pentaphyllum,and contributing to the understanding of CYP and UGT superfamily.