前期通过高通量转录组测序对不同浓度盐胁迫下发菜的差异表达基因进行了分析,结果发现钾离子通道蛋白TrkA-N在 0.3 mol/L盐浓度下转录水平比无盐时明显下调。为更好研究该基因及其编码蛋白,通过分子生物学手段、以发菜基因组为模板,克隆钾通道蛋白编码基因TrkA-N序列,成功获得长度为696 bp的核酸序列。进行生物学信息分析结果表明,钾离子通道蛋白TrkA-N平均分子质量为31.41 kDa,理论等电点为5.82,该蛋白不含跨膜区,是亲水性蛋白。蛋白氨基酸序列中分别有10个Ser位点、6个Thr位点、2个Tyr位点,二级结构主要为α螺旋和β折叠。随后,将TrkA-N编码基因与诱导表达质粒pET28a进行重组并转化至大肠杆菌BL21(DE3)中进行表达,最佳表达条件为培养10 h后待菌液OD600值达到0.8时,添加终浓度为1 mmol/L 的诱导剂异丙基-β-D-硫代半乳糖苷(isopropyl β-D-1-thiogalactopyranoside,IPTG),继续诱导20 h,最终在大肠杆菌中成功表达出分子质量为31.41 kDa的蛋白。该研究为进一步阐明发菜响应盐胁迫的机制奠定一定的理论基础。
In previous experiments, high-throughput transcriptome sequencing was used to analyze the differentially expressed genes in Nostoc flagelliforme under salt stress of different concentrations. The results showed that the transcription level of potassium channel protein TrkA-N was significantly down-regulated at 0.3 mol /L salt concentration compared with that of the control group (without salt). In order to further study this gene and its coding protein, the TrkA-N sequence encoding the potassium channel protein was cloned by molecular biology methods using the genome of Nostoc flagelliforme as the template, and the nucleic acid sequence with a length of 696 bp was successfully obtained. By analyzing the biological information of the protein encoded by the nucleic acid sequence, the following results were obtained: the average molecular weight of potassium ion channel protein TrkA-N was 31.41 kDa, and the theoretical isoelectric point was 5.82. This protein did not contain transmembrane region and was a hydrophilic protein. There were 10 Ser residues, 6 Thr residues and 2 Tyr residues in the amino acid sequence of this protein, and the secondary structure of this protein was mainly α-helix and β-sheet. The TrkA-N coding gene was cloned into pET28a and transformed into E. coli BL21(DE3) for expression. The optimal expression condition was when OD600 reached 0.8 after 10 h of cultivation, adding 1 mmol/L of IPTG and induced for 20 h. Finally, the 31.41 kDa protein was successfully expressed in E. coli. This study provides theoretical basis to further clarify the mechanism of response to salt stress.
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