坛紫菜细胞质型果糖1,6-二磷酸酶基因的克隆及表达分析  

Cloning and expression analysis of the cytosolic fructose-1,6-bisphosphatase gene from Pyropia haitanensis

作　　者：曲玲[1] 徐燕[1] 纪德华[1] 陈昌生[1] 谢潮添[1] 

机构地区：[1]集美大学水产学院,福建厦门361021

出　　处：《应用海洋学学报》2015年第3期402-410,共9页Journal of Applied Oceanography

基　　金：国家"863"计划资助项目(2012AA10A411);国家自然科学基金资助项目(41176151;41276177);福建省种业创新与产业化工程资助项目(2014S1477-10)

摘　　要：细胞质型果糖1,6-二磷酸酶(c FBPase)是糖异生途径的主要限速酶,它除了主要参与蔗糖合成途径的调控外,对其他碳水化合物的合成与分配也产生一定影响.本文以坛紫菜(Pyropia haitanensis)转录组测序获得的unigene序列为基础,采用c DNA末端快速扩增(RACE)技术克隆了坛紫菜c FBPase基因序列,命名为Phc FBPase(Gen Bank收录号:KM434064).序列分析结果表明,Phc FBPase序列全长1 406 bp,包含一个1 101 bp的开放阅读框,所编码的多肽包含366个氨基酸,分子量为39.1 k Da,等电点为5.42.基因表达水平的定量分析结果表明Phc FBPase基因在坛紫菜叶状体世代的表达水平显著高于丝状体世代,意味着坛紫菜生活史两个不同世代的光合固碳机制存在差异性;对坛紫菜叶状体进行高温胁迫不同时间水平,该基因的表达水平在胁迫初期表现为先上调表达,随着胁迫时间延长又开始下调表达,即在短时间的高温胁迫下,Phc FBPase基因的表达有一个应激上调的过程,但长时间的高温胁迫则会抑制Phc FBPase基因的表达;不同失水胁迫条件下,Phc FBPase基因的表达不受低水平的失水胁迫影响,但可被高水平(>45%)的失水胁迫所抑制,并可在复水后迅速回升至正常水平.由此说明Phc FBPase基因在应答环境逆境胁迫时,没有发挥明显的主动拮抗作用,而只是被动的适应.Cytosolic fructose-1,6-bisphosphatase（ c FBPase）,the key enzyme in the sucrose biosynthetic pathway,play important roles in carbon assimilation and metabolism. In this study,based on unigene sequences from whole transcriptome sequencing of Pyropia haitanensis,the full length of c FBPase,obtained by rapid amplification of c DNA ends（ RACE） technology,was named as Phc FBPase（ Gen Bank accession： KM434064）. The full-length c DNA of the Phc FBPase gene comprised 1 406 nucleotides and contained an open reading frame of 1 101 bp,encoding a protein of366 amino acid putative peptides residues with the predicted molecular weight of 39. 1 KDa and theoretical isoelectric point of 5. 42. The expression patterns of the Phc FBPase gene,as measured by real-time quantitative PCR,revealed that the expression of Phc FBPase gene in the thallus phase was significantly higher than in the conchocelis phase,which suggested that the different mechanisms of inorganic carbon utilization exist among the different life phase of P.haitanensis. During high-temperature stress,the expression levels of Phc FBPase significantly upregulated firstly and then decreased. However,the expression levels of Phc FBPase had no significantly change when the water loss was ≤45% during desiccation,but as the desiccation continue,the expression levels of Phc FBPase were significantly downregulated. These results suggested that the Phc FBPase had no evident roles in response to high-temperature stress and extreme desiccation stress.

关 键 词：海洋生物学 坛紫菜 果糖1 6-二磷酸酶 基因克隆 c DNA末端快速扩增(RACE) 荧光定量PCR 

分 类 号：P735[天文地球—海洋生物学]