NaCl诱导表达的盐藻果糖-1,6-二磷酸醛缩酶基因克隆及原核表达

Cloning and Prokaryotic Expression of the Fructose1,6-diphosphate Aldolase Full-length cDNA of Dunaliella salina Induced by NaCl

通过RACE技术克隆到盐藻(DunaliellasalinaTeod.)果糖 1,6 二磷酸醛缩酶(DsALDP)的全长cDNA.对其序列分析及在GeneBank中进行同源搜索,发现DsALDP基因属于果糖 1,6 二磷酸醛缩酶基因家族,其与植物叶绿体果糖 1,6 二磷酸醛缩酶(AldP)基因的一致性为73%～66%.Northern杂交结果及醛缩酶活性分析均表明它确为在盐诱导下特异表达.将DsALDPcDNA构建到pET32a载体上,转入E.coliBL21进行表达分析.IPTG诱导后可以检测到一62ku基因产物大量表达.经不同浓度NaCl处理,表达DsALDP基因产物的细菌耐盐性明显高于对照组.

The fulllength cDNA of fructose1, 6diphosphate aldolase in Dunaliella salina was cloned by rapid amplification of cDNA ends (RACE). It was shown from sequence analysis and BlastP search in GeneBank that DsALDP belonged to fructose1, 6diphosphate aldolase family, and the identities of DsALDP to AldP of other plants were 73%66%. It was confirmed by northern blot and aldolase activity analysis that DsALDP was specially expressed under NaCl stress. After insertion of DsALDP to vector pET32a, the recombined plasmid was transferred into E. coli BL21 to analyze its expression. After adding IPTG, a Large amount of 62 ku fusion gene product was detected. The bacteria were cultured in media with different NaCl concentration. As a result, the bacteria expressing DsALDP exhibited a higher salt tolerance with increasing NaCl concentration than bacteria with no DsALDP expression.