条斑紫菜锰超氧化物歧化酶基因克隆与序列分析

为研究紫菜抗逆抗病的分子机制,以本实验室建立的条斑紫菜表达序列标签(EST)和全长cDNA富集文库,结合PCR技术,克隆了条斑紫菜编码锰超氧化物歧化酶(Mn-SOD)的cDNA及基因组DNA全长序列,并进行了序列特性相关分析.研究结果表明:该基因cDNA序列全长为958个核苷酸,包含一个完整Mn-SOD基因的ORF,编码224个氨基酸和终止密码子;该基因在基因组中序列长度为1416bp,包含4个外显子和3个内含子,这既不同于高等植物的6个外显子和5个内含子,也不同于人的5个外显子和4个内含子;该基因编码区密码子的平均GC含量为60.9%,第三位密码子的GC含量高达84.9%;序列中包含4个与Mn2 的结合位点及一个保守的金属结合结构域,预测分子量为24469.09Da,等电点为5.99;条斑紫菜Mn-SOD与人的相关蛋白具有类似的空间结构,都有6个跨膜α螺旋结构域;由cDNA序列推导的氨基酸序列与莱茵衣藻相似性为57.7%,系统发生分析表明条斑紫菜Mn-SOD与绿藻莱茵衣藻和硅藻海链藻的亲缘关系较近.这是该基因在红藻门中的首次报道.

Cloning and sequence analysing of Mn-SOD Gene from Porphyra yezoensis Udea

In order to understand the mechanisms of anti-stress of Porphyra yezoensis, cDNA and its genomic sequence of manganese superoxide dismutase (Mn-SOD) was cloned by the technique of polymerase chain reaction (PCR) based on the express sequence tags and full-length cDNA enriched library constructed by the authors' laboratory. The result shows that the full-length cDNA of Mn-Sod consists of 958bp including an ORF encoding for 224 amino acids and a terminate codon, while the length of genomic sequence is 1416bp including 4 exons and 3 introns, which is different from the gene of human (5 exons and 4 introns) and that of higher plant (6 exons and 5 introns). The average GC content of the coding region is 60.9%, while GC content of the third position of this gene is as high as 84.9%. Four Mn 2+ binding sites and a conservative metal binding region are also found in the gene. The predicted molecular mass of the deduced peptide is 24469.09Da and the pI is 5.99. The 3D structure of Mn-SOD of P. yezoensis is similar to that of human, in which 6 helixes can be observed. The sequence similarity of deduced amino acid between P. yezoensis and Chlamydomonas is 57.7%. Phylogenetic analysis indicates the close relationship of Mn-SOD among the P. yezoensis, C. reindardtii and Thalasiosira. This is the first report on Mn-SOD from a species of Rhodophyta.