雨生红球藻sir基因克隆及在硫代谢中的功能分析

Cloning of the sir Gene of Haematococcus pluvialis and Its Function in Sulfur Metabolism

亚硫酸还原酶是硫代谢的关键酶。本文首次从雨生红球藻(Haematococcus pluvialis)中克隆了亚硫酸还原酶基因(sir),发现该sir的DNA序列由3885个核苷酸组成,包括9个内含子,cDNA序列全长为2079 bp,编码692个氨基酸,属于NiR/SiR铁氧还蛋白超家族。系统发育分析表明,雨生红球藻中的sir基因与其他淡水绿藻的亲缘关系最近。在0~50 mmol/L Na_(2)SO_(3)浓度范围内,sir基因转录水平与Na_(2)SO_(3)浓度呈正相关,说明雨生红球藻中sir基因与亚硫酸盐代谢直接相关。对不同硫浓度条件下sir的转录水平分析表明,雨生红球藻sir的转录水平会随培养时间呈周期性变化,0.5 g/L MgSO_(4)·7H_(2)O组sir基因的转录水平在前4天明显上调,显著高于其他各组。培养基中不同硫浓度对雨生红球藻生长的影响表明,0.5 g/L MgSO4·7H_(2)O组表现出明显的生长优势,在第10天表现出最高的细胞密度。因此,0.5 g/L MgSO_(4)·7H_(2)O更适宜作为雨生红球藻培养中的硫酸盐添加量。

Sulfite reductase is a key enzyme of sulfur metabolism.The sulfite reductase gene(sir)was first cloned from Haematococcus pluvialis,which consists of 3885 nucleotides and includes 9 introns.The total length of the cDNA is 2079 bp,encoding a protein of 692 amino acid,which belongs to the NiR/SiR ferredoxin superfamily.Phylogenetic analysis showed that the sir gene in H.pluvialis had the highest affinity with other freshwater green algae.Under different Na_(2)SO_(3)concentrations(0~50 mmol/L),the transcription level of sir gene was positively correlated with the concentration of Na_(2)SO_(3),indicating that the sir gene cloned from H.pluvialis is directly related to the sulfite metabolism pathway.The real-time PCR results showed that the transcriptional level of sir in H.pluvialis changed periodically with culture time,and the transcriptional level of sir gene in 0.5 g/L MgSO_(4)·7H_(2)O group was significantly higher than those of other groups in the first four days.The effect of different sulfur concentrations in the culture medium on the growth of H.pluvialis showed that 0.5 g/L MgSO_(4)·7H_(2)O group showed obvious growth advantages and the highest cell density appeared on the 10th day.It indicates that 0.5 g/L MgSO_(4)·7H_(2)O is more suitable for sulfate addition in the culture of H.pluvialis.This research provides an experimental basis for understanding of sulfur metabolism pathway in H.pluvialis and also provides theoretical guidance for optimization of culture medium for H.pluvialis.