杜氏盐藻硝酸盐还原酶缺陷型突变株的筛选与鉴定  

Isolation and characterization of nitrate reductase-deficient mutants of Dunaliella salina

作　　者：李慎柯[1] 贾岩龙[1] 刘红涛[1] 李杰 鲁照明[1] 王建民[1] 薛乐勋[1] 

机构地区：[1]郑州大学细胞生物学研究室

出　　处：《郑州大学学报（医学版）》2006年第3期429-432,共4页Journal of Zhengzhou University(Medical Sciences)

基　　金：国家自然科学基金资助项目30270031;河南省医学科技创新人才工程项目2001006

摘　　要：目的:制备杜氏盐藻氯酸盐抗性突变株库并从中筛选出硝酸盐还原酶(NR)缺陷型突变株,测定其NR cDNA全长序列,分析其突变位点。方法:①用乙基亚硝基脲(ENU)对杜氏盐藻进行诱变,得到突变株库。然后用氯酸盐抗性筛选方法获得氯酸盐抗性藻株,经复筛和不同氮源生长试验,获得了氯酸盐抗性突变株库。再用96孔板法进行单藻培养,获得氯酸盐抗性突变株单藻群。用磺胺比色法测定各个单藻群的NR活性,挑选出NR活性完全丧失的突变株。然后用氯酸盐抗性实验和不同氮源生长实验验证其NR突变的稳定性。②根据已知的盐藻NR全长序列,设计3对PCR引物,提取突变株盐藻细胞mRNA,反转录成cDNA,分3段扩增其NRcDNA的全长序列,产物与T载体连接后转化至大肠杆菌JM109中,经筛选后测序,测序结果与野生型盐藻NRcDNA序列比较,分析其突变位点。结果:序列分析表明,3株突变株共有11处相同的突变,其中的3处点突变引起了氨基酸性质的改变。它们均在近3′端有一段相同的移码突变,造成19个氨基酸的完全改变。此外,2株还分别由于在1235和1639的位点突变而产生了一个终止密码子。结论:筛选出了3株杜氏盐藻NR完全缺陷型突变株。Aim： To isolate and characterize a mutant strain of Dunaliella salina（ D. salina）with deficiency of nitrate reductase. Methods：Wild-type cells of D. salina treated with Ethynetrosourea （ENU） were respectively cultured in three kinds of media containing nitrite, urea and ammonium as N-source. The mutant library was obtained after chlorate-resistance selections. The final selection was performed on 96-well plate for isolating single-cell colonies . The nitrate reduetase （NR） activities of all the single-cell colonies were examined by sulfanilamide eolormetry and three of them had no NR activity. Subsequently, chlorate-resistant experiment and growth experiment were used to verify the stability of their NR deficiency. DNA sequencing and amino acid homology analysis were performed to determine their mutations at the molecular level. Results： Sequencing analysis showed that ll same point mutations were found in the three mutant strains and three of them caused changes in properties of the amino acids. There was an identical frame-shift mutation resulting in changes of 19 amino acids near the 3＇-terminal domain of the 3 strains each. Interestingly, stop codons were respectively found at nucleotides 1 235 and 1 639 in the 2 strains. Conclusion ： Three strains of NR-deficient mutants of D. salina have been obtained.

关 键 词：杜氏盐藻 硝酸盐还原酶 突变株 

分 类 号：Q754[生物学—分子生物学]