中文摘要：

目的：克隆杜氏盐藻硝酸盐还原酶（NR）基因5’上游序列，并对其功能进行分析。方法：利用BamHI、EcoRI、HindIII、PstI、SalI、Xbal6种限制性内切酶分别酶切盐藻基因组DNA，并与接头连接，构建成盐藻基因组步行文库。采用LA—PCR方法，从上述盐藻步行基因组文库中扩增NR基因5’上游序列，测序并进行分析。为检测其表达特性，构建了该片段与GUS嵌合基因的表达载体pNR-GUS，通过电击法将所构建的重组表达载体转化盐藻，组织化学染色法观察GUS的表达。结果：从盐藻基因组步行文库中扩增出约1200bp特异片段，序列分析表明5’上游序列含有启动子的特征性序列。GUS瞬时表达染色结果显示，该DNA片段具有硝酸盐诱导和铵抑制的启动子活性。结论：所克隆的盐藻的5’上游序列可能是一种具有“开关”活性的可控性启动子。

英文摘要：

Aim： Clone and characterize of the 5＇- flanking region of the nitrate reductase （NR） gene derived from Dunaliella salina （D. salina ）. Methods ： The genomic DNA from D. salina was respectively digested with BamHI, EcoRI, HindIII, Pst I, Sal I and Xba I. A genomic walking ends of the digested DNA fragments, and then genomic walking libraries comprising cassette was ligated to the BL, EL, HL, PL, SL and XL were constructed. The 5＇- flanking region of the NR gene from genomic walking libraries of D. salina was amplified by LA-PCR. The DNA sequences were analyzed with the software - Promoter Predictions. Isolated 5＇- flanking regions fused to the GUS gene were tested for transient expression in the alga. Results： A single specific PCR product of about 1200bp in length from the HL library was generated. Also, several conserved motifs, such as CAAT-box, GAGA-box were found, which are related to regulation of transcription, and the putative binding sites of transcriptional factors such as EBP, EFII, NF-E1 and LV. BLAST showed that the DNA sequences shared high homology with 5＇-upstream region of the NR gene from Dunaliella viridis. The isolated 5＇-flanking regions were able to strongly drive GUS reporter gene expression, suggesting that it contains the promoter elements necessary for the transcription of the NR gene. The expression pattern of the GUS gene and the NR gene were similar, both ware induced by nitrate and repressed by ammonium. Conclusion： The cloned 5＇- sequences of NR gene derived from D. salina might expression of the heterologous gene in transgenic D. flanking be a specific promoter with the ability to＂ switch on or off＂ an salina.