利用高通量转录组测序(RNA-Seq)技术对不同盐浓度胁迫下的发菜样本比对分析,从发菜盐胁迫差异表达基因中筛选蔗糖合成酶S1基因,以发菜基因组为模板,克隆蔗糖合成酶S1基因完整片段并进行生物信息学分析,将目的基因导入大肠杆菌初步表达,以此进一步优化目的蛋白表达条件。该研究通过设计特异性引物成功克隆出片段大小为2 409 bp的蔗糖合成酶S1基因。生物信息学分析表明,蔗糖合成酶S1蛋白平均分子质量为92.85 kDa,为亲水性蛋白且保守性高,理论等电点为5.52,不含跨膜区。该蛋白氨基酸序列中含有37个Ser磷酸化位点、13个Thr磷酸化位点、8个Tyr磷酸化位点,其二级结构主要为α螺旋和随机卷曲。纯化回收体外扩增得到的目的基因,连接质粒pETsumo,构建表达载体pETsumo-S1,转化至大肠杆菌BL21(DE3)中表达,获得预期大小的外源蛋白。优化表达条件可知当菌液OD600值达到0.8时添加终浓度为0.05 mmol/L的诱导剂异丙基-β-D-硫代半乳糖苷(isopropyl-beta-D-thiogalactopyranoside,IPTG),37 ℃、200 r/min条件下诱导6 h目的蛋白表达量最高。研究结果丰富了发菜盐胁迫响应基因的研究,为发菜基因簇、基因调控位点等研究奠定基础,对于从分子层面阐明发菜响应盐胁迫的调控机制具有重要意义。
Using high-throughput transcriptome sequence (RNA-Seq) to analyze Nostoc flagelliforme samples under different salt concentrations, we screened sucrose synthase S1 gene from the differentially expressed genes of N. flagelliforme under salt stress. Using the N. flagelliforme genome as a template, we cloned the complete fragment of sucrose synthase S1 gene and analyzed by bioinformatics, and introduced the target gene into Escherichia coli for preliminary expression, so as to further optimize the expression conditions of the target protein. In this study, we cloned the sucrose synthase S1 gene with a fragment size of 2 409 bp successfully by designing specific primers. Bioinformatics analysis showed that the average molecular weight of sucrose synthase S1 protein is 92.85 kDa, which is a hydrophilic protein and highly conserved. Its theoretical isoelectric point is 5.52 and does not contain a transmembrane region. The protein amino acid sequence contains 37 Ser phosphorylation sites, 13 Thr phosphorylation sites, and 8 Tyr phosphorylation sites. Its secondary structure was mainly α-helix and random coil. Purify and recover the target gene amplified in vitro, connect the plasmid pETsumo to construct the expression vector pETsumo-S1, and transform it into E. coli BL21 (DE3) for expression to obtain the foreign protein of the expected size. Optimizing the expression conditions showed that when the OD600 value of the bacterial solution reached 0.8, the final concentration of the inducer IPTG was added at a final concentration of 0.05 mmol/L, then induce the bacterial solution for 6 h under 37 ℃ and 200 r/min ,and the target protein expression reaches the highest. The results enriched the research on the salt stress response genes of N. flagelliforme, laying a foundation for the research of N. flagelliforme gene clusters and gene regulatory loci, and are of great significance for elucidating the regulation mechanism of the salt stress response of N. flagelliforme from the molecular level.
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