四种cry基因启动子在spoIIID基因突变株中的活性比较

Activity of four cry gene promoters in spoIIID mutant of Bacillus thuringiensis

【目的】分析spoIIID基因突变对苏云金芽胞杆菌cry1、cry3、cry4和cry8基因启动子Pcry1Ac、Pcry3A、Pcry4A和Pcry8E的影响,比较以上启动子在无芽胞spoIIID基因突变体(HD-△SpoIIID)中的转录活性。【方法】分别构建了Pcry1Ac、Pcry3A、Pcry4A和Pcry8E与lacZ基因融合的转录分析载体,并导入HD-73野生型菌株和HD-△SpoIIID突变株中测定β-半乳糖苷酶活性;通过高温诱变方法在HD-△SpoIIID基础上筛选出缺失cry1Ac基因的HD--△SpoIIID突变体;构建了4种启动子与cry1Ac基因融合表达载体,分别将它们转入HD-ΔSpoIIID和HD--ΔSpoIIID中,分析Cry1Ac蛋白表达量及其生物活性。【结果】HD-73和HD-ΔSpoIIID菌株中四个启动子转录活性由高到低分别为:Pcry8E>Pcry1Ac>Pcry4A>Pcry3A;spoIIID基因的缺失未影响Pcry1Ac和Pcry8E转录活性,Pcry3A在HD-ΔSpoIIID菌株中转录活性略有升高,Pcry4A在HD-ΔSpoIIID菌株中转录活性在T5到T10略有降低。从翻译水平来看在HD-ΔSpoIIID中cry8E启动子略低于cry1Ac启动子,并高于Pcry4Aa,Pcry3A指导的Cry1Ac蛋白产量,生物活性测定结果与蛋白表达量相符。【结论】cry8E基因启动子Pcry8E在spoIIID突变体中在转录水平活性是最高的启动子,而cry1Ac启动子指导自身基因cry1Ac表达时,在翻译水平上略高于cry8E启动子指导的Cry1Ac产量。

[Objective] We studied the influence of spoIIID gene deletion on the activity of cry1Ac,cry3A,cry4A and cry8E gene promoters in Bacillus thuringiensis and compared the activity among these promoters in spoIIID mutant（HD-△SpoIIID）.[Methods] We constructed 4 promoter fusions with lacZ gene and transformed them into wild-type strain HD-73 and HD-△SpoIIID to analyze their transcriptional activity.We constructed a spoIIID gene mutant（HD——△SpoIIID） with deletion of the cry1Ac-harboring native plasmid based on HD-△SpoIIID strain.We constructed four promoter fusions with cry1Ac gene and transformed them into HD-ΔSpoIIID and HD——ΔSpoIIID to perform Cry protein quantization and bioassay.[Results]By Beta-galactosidase assay we found that the activities of the four promoters were,in decreasing order,Pcry8EPcry1APcry4APcry3A in both HD-73 and HD-ΔSpoIIID strains.The deletion of spoIIID had no effect on transcriptional activity of Pcry1Ac and Pcry8E.The transcriptional activity of Pcry3A in HD-ΔSpoIIID was slightly higher than that in HD-73.The transcriptional activity of Pcry4A in HD-ΔSpoIIID was decreased compared to HD-73.The Cry1Ac protein production directed by Pcry1Ac was as much as Pcry8E in HD-ΔSpoIIID and higher than that by Pcry4A and Pcry3A in accordance with the bioassay result.[Conclusion] The cry8E gene promoter is the strongest promoter among four promoters in spoIIID gene mutant at transcriptional level.The Cry1Ac protein production directed by Pcry1Ac is almost equal to that by Pcry8E.