苏云金杆菌毒素调控基因plcR的特性与表达

Characterization and Expression of plcR Gene of Bacillus thuringiensis

根据蜡状芽胞杆菌plcR基因和papR基因序列设计特异引物,对6个Bt菌株(WB1、WB7、WB9、HD98、8010、8311)及5个Bc菌株(6A1、6A2、6A3、6A4、6S1)进行了PCR检测。结果显示,3个Bt菌株及4个Bc菌株含有plcR-papR基因。克隆了Bt8010、Bc6A2和6A3的plcR、papR基因,核苷酸序列分析表明,三个菌株的plcR、papR基因与NCBI数据库中的Bt、Bc及Ba相应序列都有很高的相似性。Bt8010的plcR基因编码框由846个核苷酸组成,可编码282个氨基酸;papR基因的编码框由144个核苷酸组成,可编码48个氨基酸。推导的氨基酸序列分析表明,Bt8010的PapR有21个氨基酸的信号肽序列,PlcR没有信号肽序列。与Bc6A2、6A3和Bc 569相比,Bt8010的PlcR和PapR在氨基酸序列上与Bc相应序列存在相对较大的差异。将plcR-papR基因连接到表达载体pHT304中,并转化至大肠杆菌JM109中成功进行了表达,为研究Bt plcR基因的功能奠定了基础。

The plcR and papR gene specific primers were designed according to the published gene sequences of Bc, 6 Bt strains（i, e. ,WB1, WB7, WB9, HD98, 8010 and 8311 ） and 5 Bc strains（ i. e. , 6A1, 6A2, 6A3, 6A4 and 6S1 ） were detected by PCR. The results showed that 3 Bt strains and 4 Bc strains harbored plcR-papR genes. The ORFs of plcR and papR genes from Bt 8010, Bc 6A2 and Bc 6A3 were cloned and sequenced. Gene sequence analysis indicated that the plcR and papR genes from 8010 had a significant similarity with the relevant genes from Bc 6A2 and Bc 6A3, as well as the data in NCBI. The ORF of plcR gene of Bt 8010 was 846 bp, encoding 282 amino acids. The ORF ofpapR gene of Bt 8010 was 144 bp, encoding 48 amino acids. The deduced amino acid sequences showed that the PapR of Bt 8010 was a secreted protein with a signal peptide of 21 amino acids while no signal peptide sequence was found in PIER. Compared with Bc 6A2, Bc 6A3 and Bc 569, there were significant differences in the PlcR and PapR of Bt 8010. For the function analysis on the plcR and papR genes, the plcR and papR gene sequences from Bt 8010 was inserted in the expression vector pHT304. The plcR gene was successfully expressed in Escherichia coli JM109.