决明1-脱氧-D-木酮糖-5-磷酸合酶和1-脱氧-D-木酮糖-5-磷酸还原异构酶的功能鉴定与表达分析

Functional identification and expression analysis of 1-deoxy-D-xylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductorisomerase in Cassia obtusifia

本研究根据决明三代转录组测序获得的1-脱氧-D-木酮糖-5-磷酸合酶(CoDXS)和1-脱氧-D-木酮糖-5-磷酸还原异构酶(CoDXR)基因的全长序列设计特异性引物,PCR扩增获得CoDXS、CoDXR基因的OFR,构建原核表达载体pTrc-CoDXS、pTrc-CoDXR在含有质粒PAC-BETA的TOP10重组菌中进行表达,通过TOP10的菌斑颜色变化来验证CoDXS和CoDXR基因的功能。利用实时荧光定量PCR来检测CoDXS、CoDXR基因在决明不同组织中以及六种胁迫条件下的表达情况。研究结果表明CoDXS、CoDXR基因的OFR分别为2127、1416 bp,编码的氨基酸数目分别为708、471,功能鉴定结果显示含有CoDXS、CoDXR基因编码区的TOP10菌斑呈现出深橘黄色;CoDXS基因的表达趋势为:花>茎>种子>叶>根,CoDXR基因的表达趋势为:叶>花>茎>根>种子。对CoDXR和CoDXS基因在不同的胁迫条件下的相对表达量检测分析发现,在盐胁迫条件下:CoDXS基因的表达量整体呈现出下降趋势,CoDXR基因的表达量波动范围较小;在ABA胁迫条件下:CoDXS基因的表达量呈现出先下降上升趋势,CoDXR基因的表达量呈现出上升趋势;在MeJA胁迫条件下:CoDXR和CoDXS基因的表达量均呈现出先下降后上升的趋势;在PEG6000胁迫条件下:对CoDXS基因表达量的影响较小,CoDXR基因的表达量呈现出上升趋势;在高温胁迫条件:CoDXS基因呈现出下降趋势,对CoDXR基因的表达影响较小;低温胁迫条件下:CoDXR和CoDXS基因的表达均呈现出下降趋势。

The specific primers were designed based on the full-length sequence of 1-deoxy-D-xylulose-5-phosphate synthase(CoDXS)and 1-deoxy-D-xylulose-5-phosphate reductoisomerase(CoDXR)genes from Cassia obtusifia.The OFR sequences of CoDXS and CoDXR were cloned by PCR.Prokaryotic expression vectors of pTrc-CoDXS and pTrc-CoDXR were constructed and expressed in TOP10 containing plasmid PAC-BETA.The function of CoDXS and CoDXR genes were verified by the color change of TOP10.The expression levels of CoDXS and CoDXR genes in different tissues and six stress conditions were analyzed by fluorescent quantitative PCR.The results showed that the CoDXS gene contains a 2127 bp OFR and encodes a predicted protein of 708 amino acid.The CoDXR gene contains a 1416 bp OFR and encodes a predicted protein of 471 amino acid.The results of functional identification showed that TOP10 containing CoDXS and CoDXR genes appeared dark orange.Relative real-time PCR analysis indicated that CoDXS showed the highest transcript abundance in the flowers,moderate level in the stems,lower level in the seeds,and the lowest level in the roots.The CoDXR showed the highest transcript abundance in the leafs,moderate level in the flowers,lower level in the stems,and the lowest level in the seeds.Under salt stress:the expression level of CoDXS gene showed a downward trend,and the expression level of CoDXR gene fluctuated within a small range.Under ABA stress conditions:the expression level of CoDXS gene showed a downward and upward trend,and the expression level of CoDXR gene showed an upward trend.Under MeJA stress conditions:the expression levels of CoDXR and CoDXS genes showed a downward and upward trend.Under PEG6000 stress conditions:it had little effect on the expression level of CoDXS gene,and the expression level of CoDXR gene showed an upward trend.Under the heat stress conditions:the expression level of CoDXS gene showed a downward trend,and had little effect on the expression level CoDXR gene.Under cold stress conditions:the expression levels of CoDXS and CoDXR genes showed a downward trend.