浒苔中MnSOD和CAT基因克隆和表达分析

Gene cloning and expression analysis of Mn SOD and CAT from Ulva prolifera

实验以大型绿藻浒苔为材料,克隆了其抗氧化系统关键酶锰超氧化物歧化酶(manganese superoxide dismutase,Mn SOD)和过氧化氢酶(catalase,CAT)基因的部分片段,利用荧光定量PCR技术研究了Mn SOD和CAT对温度和水杨酸作用的响应规律。结果获得浒苔318 bp的Mn SOD基因,该Mn SOD推测的氨基酸序列与裂片石莼Mn SOD相似性最高为89%;获得了229 bp的CAT基因,该浒苔CAT编码氨基酸序列与裂片石莼和雨生红球藻CAT序列相似性分别为99%和93%。在利用M n SOD和CAT氨基酸序列构建的系统树中,浒苔均先与裂片石莼聚类,再与其他绿藻聚在一起。不同温度对浒苔Mn SOD和CAT基因表达影响表明,35℃高温培养对浒苔Mn SOD和CAT基因表达影响最显著,其表达量分别为25℃培养的2.18倍和2.05倍;5℃低温培养次之;而15℃与25℃培养对M n SOD和CAT基因表达影响差异不显著(P>0.05)。在高温35℃下,添加0.1 mmol/L水杨酸后Mn SOD和CAT基因表达量分别为各自对照的2.08倍和5.30倍,而0.01 mmol/L水杨酸添加后基因表达与对照差异不显著(P>0.05)。研究表明,Mn SOD和CAT基因表达的增强不仅是浒苔对高温胁迫的响应,而且也是水杨酸缓解高温对浒苔不利影响的方式之一。

Manganese superoxide dismutase （MnSOD）and catalase （CAT）are key enzymes in the antioxidant system and important indicators in the resistance study of plants. In this paper, partial cDNA fragments of MnSOD and CAT genes were cloned, and their expression patterns in response to different temperatures and salicylic acid concentrations were investigated by real-time quantitative PCR technique in macroalga Ulva prolifera（Chlorophyta）. The cloned MnSOD cDNA was 318 bp,which encoded 105 amino acid residues. The putative MnSOD amino acid sequence of U. prolifera shared 89% identity with that of U. fasciata. Total 229 bp CAT cDNA was obtained,which showed 99% identity with that of U. fasciata,and 93% identity with that of Haematococcus pluvialis by Blast analysis. The phylogenetic trees suggested that U. prolifera clustered with that of U. fasciata in the tree constructed with MnSOD amino acid sequences ; and U. prolifera also clustered with U. fasciata firstly, and then clustered with H. pluvialis and Chlamydomonas reintmrdtii in the tree constructed with CAT amino acid sequences. The expression of MnSOD and CAT genes varied in response to different temperatures, of which high temperature showed the most significant effect on the two genes＇ expression. The expression of MnSOD and CAT was 2. 18-fold and 2.05-fold in 35℃ culture condition compared with that at 25 ℃ ,respectively. The expression of MnSOD and CAT was 1. 47-fold and 1.31-fold up-regulated at 5 ℃ compared with that at 25℃, respectively. However, the expression levels of the two genes in 15℃ and 25 ℃ culture conditions showed no significant difference. Salicylic acid is one of plant hormones, which not only played an important role in the growth and development in plants,but also showed key roles in the biotic and abiotic stress resistance. In this macroalga U. prolifera cultured at 35℃ ,0.1 mmol/L salicylic acid promoted expression of MnSOD and CAT mRNA to 2.08 times and 5. 30 times that of the control groups. But the expression levels of MnSOD and CAT mRNAs showed no significant difference from the controls when 0.01 mmol/L salicylic acid was added. In conclusion,the expression levels of MnSOD and CAT mRNAs enhanced in response to high or low temperatures. And the expression enhancement of the two genes was also one way of salicylic acid to alleviate the adverse effect caused by high temperature stress in U. prolifera.