铁皮石斛可溶性酸性转化酶基因克隆与表达分析

【目的】克隆铁皮石斛Dendrobium officinale可溶性酸性转化酶基因SAI,分析该基因生物学信息及时空表达特异性。【方法】基于同源序列克隆铁皮石斛SAI基因c DNA全长,采用生物信息学方法进行序列分析,并采用qRTPCR进行组织特异性表达分析。【结果】铁皮石斛SAI基因全长为1 595 bp,c DNA编码区1 368 bp,Genbank登录号为KU598852。该基因编码455个氨基酸,蛋白相对分子质量为50 700。NCBI BLASTx分析表明,该基因氨基酸序列与柑橘Citrus unshiu酸性转化酶基因、甜橙C.sinensis酸性β-呋喃果糖苷酶基因序列相似性最高,达77%,与其他植物酸性转化酶基因的相似性均高于68%。聚类分析表明,铁皮石斛SAI基因与玉米Zea mays液泡转化酶基因、甘蔗Saccharum officinarum SAI基因亲缘关系最近。该基因编码的蛋白质属于非跨膜结构的亲水性热稳定蛋白,定位于液泡中。SAI基因在2年生铁皮石斛的茎中表达量最高,3年生的叶中表达量最低。【结论】成功克隆铁皮石斛液泡酸性转化酶基因,该基因在铁皮石斛不同组织不同生育时期的表达量差异较大。

Cloning and expression analysis of soluble acid invertase gene from Dendrobium officinale

Objective] To clone soluble acid invertase ( SAI) gene in Dendrobium officinale, and perform bioinformatics and spatio-temporal expression analysis .Method]The full length cDNA of SAI gene in D. officinale was cloned based on homologous sequences , the sequences were analyzed using bioinformatics tools, and the tissue-specific expressions were analyzed using qRT-PCR.Result] The full length SAI gene in D.officinale is 1 595 bp, the full length cDNA is 1 368 bp, and the Genbank accession number is KU598852.The SAI gene encodes 455 amino acids, and the protein molecular mass is 50 700.NCBI BLASTx results show that the D.officinale acid invertase gene has 77%similarity with Citrus unshiu acid invertase gene and C.sinensis acid beta-fructofuranosidase gene , and has above 68%similarity with acid invertase genes from other plants .Cluster analysis shows that D.officinale acid invertase gene has the closest genetic relationship with Zea mays vacuolar invertase gene and sugarcane ( Saccharum officina-rum) SAI gene.The encoded protein of D.officinale SAI gene is positioned in vacuole , and is a hydro-philic , non-transmembrane and heat-stable protein .RT-PCR results showed that the expression level of SAI gene was the highest in 2-year-old D.officinale stems, and the lowest in 3-year-old D.officinale leav-es.Conclusion] The cloned gene is identified as the D.officinale vacuolar SAI gene.Its expression has great differences among different tissues and at different developmental stages .