基于叶绿素代谢途径的不同类型厚朴转录组与蛋白质组学研究

Comparative transcriptome and proteome profiling of chlorophyll metabolism pathway in four types of Magnolia officinalis

厚朴是一种传统中药材,在其多年的种植过程中,由于不同种源种子的交换,导致厚朴叶片呈现较多的分化类型。笔者经走访调查发现四川省大部分地区分布有厚朴(CH)、凹叶厚朴(DA)、小凹叶厚朴(XA)及柳叶厚朴(LY)4种类型,且不同类型厚朴在生长速率、化学成分、叶形及叶色等方面存在明显差异。该研究以川产不同类型的厚朴叶(DA,XA,LY,CH)为研究对象,测定其叶绿素含量,采用高通量技术对不同类型的厚朴叶片组织进行转录组学测序,使用一种包含TMT标记和LC-MS/MS的整合方法来量化不同类型厚朴叶整个蛋白质组的动态变化。结果表明CH的叶绿素含量最低,DA的叶绿素含量最高;转录组和蛋白质组学检测发现DA中参与叶绿素合成途径的谷氨酰-tRNA还原酶、尿卟啉原脱羧酶(UROD)、氧依赖性原卟啉原-Ⅲ氧化酶(ODCO)、原卟啉原氧化酶(PPO)、镁螯合酶亚基ChlD、原卟啉镁Ⅸ单甲酯[氧化]环化酶(MPPMC)等较CH,XA,LY中均显著上调,与DA在4个类型中叶绿素质量分数最高(37.56 mg·g^-1 FW)检测结果一致。将参与叶绿素合成途径相关的部分限速酶如ODCO,PPO,MPPMC等进行平行反应检测(parallel reaction monitoring,PRM)验证,结果表明DA中限速酶的含量均高于其他3个类型。因此,该研究基于不同类型厚朴叶色差异,通过对不同类型厚朴叶片中叶绿素代谢途径进行初步研究,从分子水平上探究引起叶色差异的相关基因和蛋白,以期为研究不同类型厚朴在生长发育上的差异奠定基础。

Magnolia officinalis is a traditional Chinese medicine,with many years of cultivating process,M.officinalis leaves show more differentiation types due to the exchange of seeds from different provenances."Da Ao"(DA),"Xiao Ao"(XA),"Chuan Hou"(CH),and"Liu Ye"(LY)are the main types of M.officinalis in Sichuan province of China,and there were obvious differences in growth rate,chemical composition,leaf shape and leaf colour.This study selected different types of M.officinalis leaves(DA,XA,LY and CH)from Sichuan to determine their chlorophyll content.Transcriptomic level sequencing of different types of M.officinalis leaf tissues was by high-throughput sequencing analysis and proteomics used an integrated approach involving TMT labelling and LC-MS/MS to quantify the dynamic changes of the whole proteome of M.officinalis.The results showed that CH had the lowest chlorophyll content while DA had the highest chlorophyll content.Furthermore,transcriptome and proteomics results showed that chlorophyll synthesis pathway in DA glutamine-tRNA reductase,urinary porphyrins decarboxylase(UROD),oxygen-dependent protoporphyrin(ODCO),the original-Ⅲ oxidase protoporphyrin oxidase(PPO),magnesium chelating enzyme subunit ChlD,protoporphyrin magnesiumⅨmonomethyl ester[oxidative]cyclase(MPPMC)were significantly higher than CH,XA and LY,consistent in the results of determination of chlorophyll content(chlorophyll content was highest of 37.56 mg·g^-1 FW).Some rate-limiting enzymes related to the chlorophyll synthesis,such as ODCO,PPO and MPPMC were tested by Parallel Reaction Monitoring(PRM),and the results showed that the rate-limiting enzyme content in DA was higher than that in other three types.Therefore,based on the differences in leaf color of four types of M.officinalis,the research conducted a preliminary study on the chlorophyll metabolism pathway in leaves of different types of M.officinalis,and explored relevant genes and proteins causing leaf color differences from the molecular level,so as to lay a foundation for studying the differences in growth and development of different types of M.officinalis.