樟树MVA途径基因克隆与表达模式初探

Cloning genes of MVA pathway and preliminary survey of expression pattern in Cinnamomum camphora

【目的】克隆樟树MVA途径系列基因,初步研究其表达模式及分析其表达水平与倍半萜含量的关系,为解析樟树精油倍半萜类成分生物合成机制提供理论依据。【方法】基于转录组与基因组数据,PCR克隆MVA途径系列基因。进一步借助荧光定量PCR,聚类分析系列基因在橙花叔醇型樟树不同组织与不同化学型樟属植物叶组织中的表达模式。挑选代表性单株,关联分析在不同时令下系列基因表达量与倍半萜含量。【结果】橙花叔醇型樟树叶、叶柄、花与花梗等4个组织精油主要成分为倍半萜类化合物,小枝与树皮精油主要成分为单萜类化合物,而根组织精油主要成分则为苯丙素类化合物。樟树MVA途径11个序列基因在不同组织中可分为3种不同表达模式。其中,HMGS2、HMGR2与IDI1等3个基因具有类似的表达模式,即在富含倍半萜类化合物组织中优势表达。进一步分析HMGS2、HMGR2与IDI1等3个基因在不同化学型樟属叶组织表达模式,结果显示三者在倍半萜型单株叶组织中表达量是其它化学型的3.26~6.07倍。在小群体分析中,不同时令下HMGS2、HMGR2与IDI1等3个基因表达丰度与倍半萜累积速度相关,但与单株间倍半萜含量无明显相关性。【结论】樟树部分MVA途径系列基因即HMGS2、HMGR2与IDI1等存在共表达模式,在转录水平上三者对樟树倍半萜类化学型形成及不同组织倍半萜类化合物差异性分布具有重要调控作用,但不同单株间叶组织中倍半萜类化合物含量多寡可能还受其它因素调控。

【Objective】The leaf essential oils from C.camphora(trans-Nerolidol type)was rich in sesquiterpenoids.Biosynthesis of plant sesquiterpenoids precursors were mainly controlled by the Mevalonic acid(MVA)pathway.At present,genes of MVA pathway of C.camphora had not been isolated completely and especially there were few studies on expression patterns and regulatory mechanism on sesquiterpene synthesis of these genes.Isolation these genes,preliminary characterization their expression pattern and the relationship between expression levels and sesquiterpenoid contents in C.camphora leaves were our research goals.This research would provide a basis for the biosynthesis mechanism of sesquiterpenoids in camphor tree.【Method】Cloning genes of the MVA pathway based on transcriptome and genomic data of C.camphora by PCR.Furthermore,clustering analysis of the expression patterns in different tissues of C.camphora and in leaves from different chemical types of cinnamomum were performed by real time PCR.Finally,correlation analysis between the expression levels and sesquiterpenoid contents in leaves under different months were proposed among representative individuals.【Result】The data showed that the main components of essential oils from leaf,petiole,flower and pedicel were sesquiterpenoids,and that from twig and bark were monoterpenoids,and that from roots were phenylpropanoids,respectively.Eleven genes of the MVA pathway of C.camphora were isolated and could be divided into three different expression patterns in different tissues.Of them,HMGS2、HMGR2 and IDI1 had similar expression patterns,which were significantly up-regulated in leaves,petioles,flowers and pedicels relative to twig,bark and root.Monoterpenoids-and phenylpropanoids-chemotype leaf tissues of Cinnamomum were also sampled to subject to assay.The relative expression levels of HMGS2、HMGR2 and IDI1 in leaves of the sesquiterpenoids chemotype were 3.26-6.07 times than that of leaves of other chemotypes. Furthermore, six plants were screened to perform correlation analysis. Theexpression abundance of HMGS2, HMGR2 and IDI1 were were positively related to accumulation rate of sesquiterpenoids. However,the expression levels were not associated with sesquiterpenoid contents in leaves among different individuals.【Conclusion】Therewere coexpression patterns of some genes including HMGS2, HMGR2 and IDI1 in C. camphora, and they played important roles inregulating the formation of sesquiterpenoid chemotype and the distribution of sesquiterpenoid in different tissues at the transcriptionallevel. However, other factors may be also involved in the regulation of sesquiterpenoid contents in leaves of different individuals.