文献类型：期刊文献

中文题名：油楠倍半萜类合成酶基因SgSTPS3的克隆与表达分析

英文题名：Cloning and Expression Analysis of Sesquiterpene Synthase Gene SgSTPS3 in Sindora glabra

作者：陈朝黎[1,2] 余纽[1] 李荣生[1] 邹文涛[1] 董明亮[1] 朱茂成[1] 杨锦昌[1]

第一作者：陈朝黎

机构：[1]中国林业科学研究院热带林业研究所,广州510520;[2]南京林业大学,南京210037

年份：2021

卷号：57

期号：11

起止页码：68-78

中文期刊名：林业科学

外文期刊名：Scientia Silvae Sinicae

收录：CSTPCD;;Scopus;北大核心:【北大核心2020】；CSCD:【CSCD2021_2022】；

基金：中央级公益性科研院所基本科研业务费专项基金(CAFYBB2018QB003,CAFYBB2017SY023)。

语种：中文

中文关键词：萜类合成酶基因;表达模式;激素调控;油楠

外文关键词：terpene synthetase gene;expression pattern;hormone regulation;Sindora glabra

分类号：S718.46

摘要：【目的】油楠茎部树脂油富含大量倍半萜类化合物,萜类合成酶是萜类化合物生物合成途径中的关键酶之一。对油楠倍半萜合成酶基因SgSTPS3的克隆与表达分析,有利于探明油楠树脂油的形成机制,为解析林木萜类生物合成与代谢调控机制提供新的理论依据。【方法】基于前期油楠转录组数据库,从成熟油楠植株茎部克隆获得油楠倍半萜类合成酶基因SgSTPS3,并对其进行生物信息学分析。利用原核细胞进行诱导表达,GC-MS鉴定其表达产物。通过实时荧光定量PCR分析该基因在油楠不同年龄(0.5、1.5、15、30年)和不同器官(根、韧皮部、木质部、嫩叶、老叶)中的表达特性,以及该基因对不同激素水杨酸(SA)、茉莉酸甲酯(MeJA)和激素组合SA+MeJA的响应模式。【结果】获得SgSTPS3基因,CDS序列全长1701 bp,编码566个氨基酸(GenBank登录号:MW345633)。蛋白多序列比对显示SgSTPS3蛋白与苏木亚科古巴香胶树CoTPS3和兰氏香脂树ClTPS3同源性最高,达87.68%。生物化学分析结果表明SgSTPS3蛋白为多功能酶,以法尼基焦磷酸(FPP)为底物时,主要催化产生倍半萜环苜蓿烯、可巴烯及顺式-β-可巴烯,以香叶基焦磷酸(GPP)为底物时,产生单萜芳樟醇。SgSTPS3基因的表达量在不同年龄、不同器官中存在显著差异:同一年龄的各器官,0.5和1.5年生植株根部表达量最高,15和30年生植株韧皮部表达量最高;不同年龄的同一器官,根部表达量随年龄的增大而减小,木质部表达量随年龄增大而增大,30年生植株韧皮部显著高于其他年龄的植株,嫩叶和老叶无明显规律。对2年生油楠幼株叶片进行激素诱导处理,喷施激素SA(50μmol·L^(-1))时,该基因在12 h表达量最高,为初始表达量的35.1倍,72 h最低;喷施MeJA(100μmol·L^(-1))时,该基因在6 h表达量最高,24 h最低;混合激素SA(50μmol·L^(-1))+MeJA(100μmol·L^(-1))作用下,SgSTPS3基因表达量变化与对照组趋势接近,诱导效果较差。【结论】SgSTPS3为倍半萜类合成酶基因,具有多底物酶催化功能,在幼苗的根部及成年植株的韧皮部表达量较高;不同激素对SgSTPS3基因的调控作用差异显著,SA(50μmol·L^(-1))诱导效果最佳,MeJA(100μmol·L^(-1))诱导效果次之。
【Objective】The oleoresin in the stem of Sindora glabra contains rich sesquiterpenoids,while terpene synthase is one of the key enzymes in the terpene biosynthesis pathway.Cloning and expression analysis of S.glabra sesquiterpene synthase gene SgSTPS3 is helpful to explore the formation mechanism of S.glabra oleoresin,which will provide a new theoretical basis for analyzing the mechanisms of terpenoid biosynthesis and metabolism in forest trees.【Method】Based on the previous transcriptome data,the sesquiterpene synthase gene SgSTPS3 was cloned from the stem of mature S.glabra,and analyzed by bioinformatics tools.Prokaryotic expression in Escherichia coli cells,and identification by-products by GC-MS.The expression levels of this gene at different ages(0.5,1.5,15,30 years old)and in differet organs(root,phloem,xylem,tender leaves,old leaves)of S.glabra were also analyzed by quantitative real-time PCR.To analyze the response pattern of this gene to hormones,salicylic acid(SA),methyl jasmonate(MeJA),and the combination of SA+MeJA were applied.【Result】The full-length cDNA sequence of SgSTPS3 gene including 1701 bp was cloned and encoded 566 amino acids(GenBank accession number:MW345633).The multiple sequence alignments indicated that SgSTPS3 had the highest homology with Copaifera officenalis CoTPS3 and Copaifera langsdorffii ClTPS3,reaching 87.68%.Biochemical analysis showed that its protein was a multifunctional enzyme.When farnesyl pyrophosphate(FPP)was used as a substrate,it was mainly catalyzed and produced sesquiterpene cyclosativene,copaene and cis-β-copaene;when geranyl pyrophosphate(GPP)was used as the substrate,it was mainly produced monoterpene linalool.The expression level of SgSTPS3 gene was significantly different in different organs and at different ages:in different organs of same plant age,the highest expression level appeared in the root of 0.5 and 1.5 years old plants,and the hightest in the phloem of 15 and 30 years old plants;for the same organ at different ages,the expression of roots decreased with age,while the expression in the xylem increased with age;the expression in phloem of 30 years old plants was significantly higher than that of the other three ages,but there was no significant rule between young and old leaves.Hormone treatment was performed on the leaves of 2 years old S.glabra,the highest expression level of this gene appeared in 12 h after salicylic acid(SA,50μmol·L^(-1))spraying,35.07 folds compared to the control,and the lowest was in 72 h.In addition,its expression was highest at 6 h and lowest at 24 h after methyl jasmonate(MeJA,100μmol·L^(-1))treatment.Moreover,the expression of SgSTPS3 gene was similar to that of the control group,under the treatment of SA(50μmol·L^(-1))+MeJA(100μmol·L^(-1)),with a poor induction effect.【Conclusion】SgSTPS3 was a sesquiterpene synthetase gene with multi-substrate enzyme catalytic function,and its expression level was higher in the root of seedlings and the phloem of adult plants.The regulation of different hormones on SgSTPS3 gene was significantly different,among which SA(50μmol·L^(-1))had the best induction effect,and MeJA(100μmol·L^(-1))came the second.