盐穗木金属硫蛋白HcMT的功能活性分析及转基因植物的研究

发布时间：2018-09-10 17:23

【摘要】：在植物界广泛存在的金属硫蛋白(Metallothioneins,MTs)是一类可与金属离子结合的、富含半胱氨酸残基(10-30%)的小分子蛋白,可以被众多非生物因素诱导产生,其所具有的强烈的清除自由基和结合重金属离子的能力使其成为植物研究的热点。盐穗木(Halostachys caspica)金属硫蛋白HcMT含有78个氨基酸,其中半胱氨酸数量为14个,理论分子量为7.70 k D,理论等电点为5.05。生物信息学分析结果显示,HcMT符合典型的植物Type 2型MT序列特征,通过MEGA 4.0系统进化树分析与其它一些Type 2型MT进化起源表明,其和海蓬子(Salicornia brachiata)的亲缘关系较近,氨基酸相似性达93%。为研究HcMT的功能活性,将HcMT基因亚克隆至p GEX-6p-2原核表达载体上,在大肠杆菌BL21中表达,分离纯化融白蛋白GST-HcMT,通过原子吸收分光光度法测定GST-HcMT结合金属离子的量以判断其与金属离子的结合能力。结果显示,诱导表达的GST-HcMT分子量在34 k D左右,与预期一致,且通过Western blot进一步得到验证;诱导的同时添加金属离子,分离纯化GST-HcMT与金属离子复合物后,原子吸收结果表明其具有结合Cu~(2+)、Zn~(2+)、Pb~(2+)、Cd~(2+)的能力,结合量分别是对照的25倍、10倍、4倍和2倍,结合能力大小依次是:Cu~(2+)Cd~(2+)Zn~(2+)Pb~(2+)。同时发现,工程菌pGEX-6p-2-HcMT/BL21具有耐受H_2O_2、NaHCO_3、Na_2CO_3的能力。将其亚克隆至pET32a原核表达载体,E.coli BL21表达后分离纯化融合蛋白Trx A-HcMT,Western blot鉴定其活性。采取与GST-HcMT相同的研究策略,发现Trx A-HcMT与GST-HcMT相比可以特异性的结合更多的金属离子,如Cd~(2+)、Co~(2+)、Cu~(2+)、Fe~(3+)、Mg~(2+)、Pb~(2+)、Zn~(2+)、Hg~(2+)。在Cd~(2+)、Cu~(2+)、Zn~(2+)胁迫下,含pET32a-HcMT/BL21的工程菌繁殖速度明显优于含pET32a/BL21的工程菌。GST-HcMT与Trx A-HcMT的这种差异,可能与标签蛋白中半胱氨酸残基含量相关。为初步探索HcMT工业化生产的可行性,通过单因素实验及20 L发酵罐放大培养优化了影响转盐穗木金属硫蛋白大肠杆菌发酵培养基的成分及其它因素。优化培养基为:每900 m L培养基含蔗糖4 g,酵母浸粉24 g,蛋白胨8 g;每100 m L磷酸缓冲液含KH_2PO_4 2.31g、K_2HPO_4?3H_2O 12.54 g,两部分分别灭菌后混合。蛋白表达条件为:乳糖6 mmol/L,诱导4 h。通过20 L发酵罐发酵培养,可使菌体量达到2.68 g/L,比摇瓶培养提高了31%,每克干重菌体结合的铜离子达到315.3 mg,比摇瓶培养提高了69%,适宜放大培养。为进一步研究HcMT基因的功能活性及其应用于植物修复的可行性,将HcMT构建至p CAMBIA1301植物表达载体,通过EHA105农杆菌介导浸染烟草。基因组PCR鉴定筛选到转p CAMBIA1301-HcMT烟草阳性植株,转HcMT基因烟草的各种非生物胁迫耐受性以及对不同金属离子的富集情况有待于进一步的研究。
[Abstract]:Metallothionein (Metallothioneins,MTs), widely present in the plant world, is a class of small molecular proteins that bind to metal ions and are rich in cysteine residues (10-30%), which can be induced by many abiotic factors. Because of its strong ability of scavenging free radicals and binding heavy metal ions, it has become a hot spot in plant research. (Halostachys caspica) metallothionein HcMT contains 78 amino acids, including 14 cysteine, 7.70kD theoretical molecular weight and 5.05 theoretical isoelectric point. Bioinformatics analysis showed that HcMT conformed to the typical plant Type 2 type MT sequence. The phylogenetic analysis of MEGA 4. 0 phylogenetic tree showed that HcMT was closely related to (Salicornia brachiata). The amino acid similarity is 93%. In order to study the functional activity of HcMT, HcMT gene was subcloned into prokaryotic expression vector of p GEX-6p-2 and expressed in Escherichia coli BL21. The amount of metal ions bound by GST-HcMT was determined by atomic absorption spectrophotometry to determine the binding ability of albumin GST-HcMT, to metal ions. The results showed that the molecular weight of the induced GST-HcMT was about 34kD, which was consistent with the expectation, and was further verified by Western blot, and the metal ions were added at the same time to separate and purify the complex of GST-HcMT and metal ions. The results of atomic absorption showed that it had the ability of binding to Cu~ (2) Zn ~ (2) Pb2 ~ (2) O ~ (2) CD ~ (2), the binding amount was 25 times as much as that of the control, and the binding capacity was 4 times and 2 times as much as that of the control, respectively, and the binding capacity was respectively: Cu2 (2) Cd~ (2) Zn~ (2) Pb~ (2). At the same time, it was found that the engineering bacterium pGEX-6p-2-HcMT/BL21 had the ability to tolerate Hs / S _ 2O _ 2 / NaHCOC _ 3. It was subcloned into pET32a prokaryotic expression vector E. coli BL21 and purified by Western blot. The fusion protein Trx A-HcMTO was identified by Western blot. Using the same research strategy as GST-HcMT, it was found that Trx A-HcMT could specifically bind more metal ions than GST-HcMT, such as Cd~ (2) Co2 + Cu2 (2) Fe3 + Mg2 (2) Pb2 + Zn2 + Hg2. Under Cd~ (2) Cu2 + Zn2 stress, the reproductive rate of engineering bacteria containing pET32a-HcMT/BL21 was significantly higher than that of pET32a/BL21. GST-HcMT and Trx A-HcMT, which might be related to the content of cysteine residues in labeled proteins. In order to explore the feasibility of industrial production of HcMT, the composition and other factors affecting the fermentation medium of Metallothionein Escherichia coli were optimized by single factor experiment and 20L fermenter amplification. The optimized medium was as follows: sucrose 4 g per 900ml medium, yeast extract 24 g, peptone 8 g, and KH_2PO_4 2.31 g / 100ml phosphate buffer containing KH_2PO_4 2.31g / L K2HPO-2HPO-2H2O12.54g. the two parts were sterilized and mixed respectively. The protein expression conditions were as follows: lactose 6 mmol/L, induced for 4 h. By fermentation in 20L fermenter, the biomass reached 2.68g / L, which was 31g / L higher than that of shaking flask culture. The copper ion binding per gram of dry cell was 315.3 mg, higher than that of shaking flask culture, which was suitable for magnifying culture. In order to further study the functional activity of HcMT gene and the feasibility of its application in phytoremediation, HcMT was constructed into p CAMBIA1301 plant expression vector and infected with EHA105 Agrobacterium tumefaciens. The positive plants were screened by genomic PCR. The tolerance to abiotic stress and the enrichment of different metal ions in transgenic tobacco with HcMT gene need to be further studied.
【学位授予单位】：新疆大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2;Q946

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