摘要
环烯醚萜合酶(IS)是环烯醚萜类成分合成途径的关键酶,催化10-oxogeranial生成烯醇化物中间体epi-iridodial。该文通过同源比对,在地黄转录组中筛选出4条与长春花CrIS同源的cDNA序列,长度分别为1 527,1 743,1 425,1 718 bp,依次命名为RgIS1,RgIS2,RgIS3,RgIS4。它们编码389~392个氨基酸残基,蛋白质相对分子质量44. 30~44. 74 k Da,等电点pI在5. 30~5. 87,亚细胞定位预测均分布在细胞质中。序列分析表明地黄4个环烯醚萜合酶的氨基酸序列均包含6个保守的短链脱氢酶/还原酶(SDR)基序,其三维结构包含N端Rossmann结构域和C端伸展结构域。进化分析表明,RgIS3与CrIS、橄榄OeIS聚在同一类群,另外3个环烯醚萜合酶与其他植物的15个蛋白亲缘关系较近。实时荧光定量PCR分析表明,RgIS1和RgIS3在展开叶中表达量均最高,RgIS2在茎中表达量最高,而RgIS4在块根中表达量最高。RgIS1和RgIS2在QH1的非菊花心部位表达量较高,RgIS3在2个品种的非菊花心部位表达量均较高,而RgIS4在QH1菊花心有较高的表达量。RgIS1,RgIS2和RgIS3在MeJA处理后的毛状根均显著上调表达。因此,该研究为进一步研究地黄环烯醚萜合酶的分子功能奠定基础,同时有助于揭示地黄环烯醚萜类成分的合成机制。
Iridoid synthase(IS),the key enzyme in the natural biosynthesis of vegetal iridoids,catalyzes the irreversible cyclization of 10-oxogeranial to epi-iridodial.In this study,we screened the Rehmannia glutinosa transcriptome data by BLASTn with Catharanthus roseus CrIS cDNA,and found four c DNA fragments with length of 1 527,1 743,1 425,1 718 bp,named RgIS1,RgIS2,RgIS3 and RgIS4,respectively.Bioinformatics analysis revealed that the four iridoid synthase genes encoding proteins with 389-392 amino acid residues,protein molecular weights were between 44.30-44.74 k Da,and theoretical isoelectric points were between 5.30 and 5.87.Subcellular localization predictions showed that the four iridoid synthase were distributed in the cytoplasm.Structure analysis revealed that R.glutinosa iridoid synthases contain six conserved short-chain dehydrogenase/reductase(SDR)motifs,and their 3 D models were composed typical dinucleotide-binding'Rossmann'folds covered by helical C-terminal extensions.Using the amino acid sequences of four R.glutinosa iridoid synthases,phylogenetic analysis was performed,the result indicated that RgIS3,CrIS and Olea europaea OeIS were grouped together,the other R.glutinosa iridoid synthases and fifteen proteins in other plants had close relationship.Real-time fluorescent quantitative PCR revealed that RgIS1 and RgIS3 highly expressed in unfold leaves,however,RgIS2 and RgIS4 highly expressed in stems and tuberous roots,respectively.RgIS3 showed higher expression levels in non-radial striations(nRS)of the two cultivars,and RgIS1 and RgIS2 had higher expression levels in nRS of QH,while RgIS4 had less expression levels in nRS of QH1.RgIS1,RgIS2 and RgIS3 were up-regulated by Me JA treatment,although the time and degree of response differed.Our findings are helpful to reveal molecular function of R.glutinosa iridoid synthases and provide a clue for studing the molecular mechanism of iridoid biosynthesis.
作者
杨超飞
李欣容
智惊宇
耿晓桐
洪利亚
王丰青
谢彩侠
YANG Chao-fei;LI Xin-rong;ZHI Jing-yu;GENG Xiao-tong;HONG Li-ya;WANG Feng-qing;XIE Cai-xia(College of Agronomy,Henan Agricultural University,Zhengzhou 450002,China;School of medicine,Henan University of Chinese Medicine,Zhengzhou 450046,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2019年第12期2472-2479,共8页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(81872950,81473299)
河南省自然科学基金项目(162300410157)
关键词
地黄
环烯醚萜合酶基因
克隆
序列分析
表达特性
Rehmannia glutinosa
iridoid synthase gene
cloning
sequence analysis
expression characterization
