穿龙薯蓣DnSQS基因的克隆及表达分析

Cloning and Expression Analysis of DnSQS Gene in Dioscorea nipponica

角鲨烯合成酶(SQS)是植物甾醇和萜类化合物合成的关键酶之一。为了揭示穿龙薯蓣SQS基因的功能,该研究以穿龙薯蓣cDNA为模板,采用PCR方法克隆DnSQS基因,并对得到的序列进行生物信息学分析。采用HPLC方法检测不同组织的薯蓣皂苷元含量,采用实时荧光定量PCR技术分析DnSQS基因在不同组织、激素诱导及非生物胁迫下的表达特征。结果表明:(1)成功克隆得到穿龙薯蓣2个基因DnSQS1与DnSQS2,二者分别编码409和433个氨基酸,但编码的蛋白二级结构均以α螺旋和无规则卷曲为主,都存在由α螺旋折叠形成的保守催化中心以及2个富含天冬氨酸的功能结构,都具有2个跨膜螺旋结构,并且都定位于内质网。(2)DnSQS1和DnSQS2与盾叶薯蓣DzSQS的亲缘关系较近,2个DnSQS蛋白在进化上相对保守。(3)不同组织的薯蓣皂苷元含量存在差异,其含量大小依次为叶>根状茎>地上茎>根;DnSQS1与DnSQS2基因在不同组织中的表达也具有显著差异,其中DnSQS1在地上茎中表达水平最高,DnSQS2在叶中表达水平最高。(4)以激素MeJA、ABA、SA、Eth及非生物胁迫H_(2)O_(2)、NaCl、PEG-6000处理叶片,2个DnSQS基因均可诱导表达;而GA_(3)处理下抑制了2个DnSQS基因的表达。研究表明,穿龙薯蓣的2个DnSQS基因可能参与薯蓣皂苷元的合成,并在逆境胁迫机制中发挥重要作用。

Squalene synthase(SQS)is one of the key enzymes in the synthesis of phytosterols and terpenoids.In order to reveal the function of SQS gene in Dioscorea nipponica,we used the cDNA of D.nipponica as template to clone the DnSQS gene by PCR,and analyzed the obtained sequence by bioinformatics.Simultaneously,the content of diosgenin in different tissues was detected by HPLC,and the expression characteristics of DnSQS gene in different tissues,hormone induction and abiotic stress were analyzed by real-time fluorescence quantitative PCR.The results showed that:(1)two genes(DnSQS1 and DnSQS2)of D.nipponica were successfully cloned.They encode 409 and 433 amino acids,respectively.However,the secondary structures of the encoded proteins consist mostly ofα-helix and random coil,both of which have a conserved catalytic core generated byα-helix folding,two functional structures rich in aspartic acid,two transmembrane helix structures and are localized in the endoplasmic reticulum.(2)DnSQS1 and DnSQS2 were closely related to DzSQS,and the evolution of the two DnSQS proteins was reasonably conserved.(3)The contents of diosgenin in different tissues were different,and the levels of the content were in the order of leaf>rhizome>aboveground stem>root;the expression levels of DnSQS1 and DnSQS2 genes in different tissues were also significantly different,with DnSQS1 being expressed at the highest level in aboveground stems and DnSQS2 being expressed at the highest level in leaves.(4)The expression of two DnSQS genes could be induced by MeJA,ABA,SA,Eth,H_(2)O_(2),NaCl and PEG-6000,while GA_(3) treatment inhibited the expression of two DnSQS genes.Studies have shown that the two DnSQS genes may be involved in the synthesis of diosgenin and play an important role in the mechanism of adversity stress.