毛白杨蔗糖合酶基因PtSS3的克隆及其表达模式分析

Cloning and expression pattern analysis of PtSS3 gene in Populus tomentosa

为研究毛白杨蔗糖合酶PtSS3基因的表达模式以及PtSS3蛋白的结构和功能,采用RT-PCR技术,以毛白杨叶片总RNA逆转录的cDNA为模板进行PCR扩增,获得毛白杨PtSS3的基因序列,测序结果显示该基因序列全长为2 474 bp,包括一个完整的阅读框,编码823个氨基酸。通过NCBI的Blast检索和Bioediter软件分析,Pt SS3与毛果杨、胡杨、橡胶树、麻风树的核酸和氨基酸序列的同源性分别达到85%－98%和87%－98%,氨基酸序列中包含有蔗糖合酶和糖基转移酶两个保守的功能域。运用蛋白三维结构预测软件分析显示,PtSS3编码的蛋白共含有37个α-螺旋、27个β-折叠和62个无规卷曲结构,Prot Scale工具分析结果表明该蛋白属于亲水性蛋白。进化分析显示该基因家族在进化过程中分化为单子叶组、双子叶组、单双子叶1组和单双子叶2组4个分支,PtSS3蛋白属于单双子叶1组。进一步的qRT-PCR分析结果表明,PtSS3在毛白杨根、茎、叶及雌雄花芽组织中均呈现组成型表达模式。根据对同源蛋白的功能研究推测,PtSS3可能与在源库组织的韧皮部装载过程中和卸载后蔗糖代谢的能量提供关系密切,并且可能在光合作用的碳分配过程中同样发挥着重要的作用;PtSS3蛋白与其他家族成员保持保守性的同时,又有不可代替性。

To investigate the expression pattern of PtSS3 gene,as well as the structure and fuction of PtSS3 protein,the full-length sequence of Populus tomentosa PtSS3 is obtained after amplifying the c DNA from P.tomentosa RNA by PCR based on RT-PCR.The results show that the whole length of gene is 2 474 bp,including a complete open reading frame,coding 823 amino acids.With blast searching in NCBI and Bioediter software analysis,PtSS3 has 85%–98% and 87%–98% homologies of nucleic acid and amino acid respectively with Populus trichocarpa,Populus euphratica,Hevea brasiliensis and Jatropha carcas.The amino acid sequence includes two conserved functional domains,that is sucrose synthesis domain and glyctosyl transfer domain.The three-dimensional structure prediction shows that PtSS3 protein 37 α-helixes,27 β-folds and 62 coils.PtSS3 is also considered to be a hydrophilic protein by Prot Scale tool online.Phylogenetic analysis indicates that sucrose synthase gene family is divided into 4 branches in the evolution process,they are monocots group,dicots group,MD 1 group and MD 2 group.PtSS3 protein belongs to MD 1 group.The result of q RT-PCR analysis suggests PtSS3 presented constitutive expression in root,stem,leaf and male and female floral tissues.Based on the functional studies of homologous proteins,PtSS3 may be closely related to the energy supply of sucrose metabolism during the process of phloem loading and unloading in the source sink,and may also play an important role in the process of carbon distribution of photosynthesis.PtSS3 retains conservation with other Pt SS gene family members,meanwhile,has its irreplaceability.