鹽穗木金屬硫蛋白HcMT的功能活性分析及轉(zhuǎn)基因植物的研究
發(fā)布時間：2018-09-10 17:23
【摘要】：在植物界廣泛存在的金屬硫蛋白(Metallothioneins,MTs)是一類可與金屬離子結(jié)合的、富含半胱氨酸殘基(10-30%)的小分子蛋白,可以被眾多非生物因素誘導(dǎo)產(chǎn)生,其所具有的強烈的清除自由基和結(jié)合重金屬離子的能力使其成為植物研究的熱點。鹽穗木(Halostachys caspica)金屬硫蛋白HcMT含有78個氨基酸,其中半胱氨酸數(shù)量為14個,理論分子量為7.70 k D,理論等電點為5.05。生物信息學(xué)分析結(jié)果顯示,HcMT符合典型的植物Type 2型MT序列特征,通過MEGA 4.0系統(tǒng)進化樹分析與其它一些Type 2型MT進化起源表明,其和海蓬子(Salicornia brachiata)的親緣關(guān)系較近,氨基酸相似性達93%。為研究HcMT的功能活性,將HcMT基因亞克隆至p GEX-6p-2原核表達載體上,在大腸桿菌BL21中表達,分離純化融白蛋白GST-HcMT,通過原子吸收分光光度法測定GST-HcMT結(jié)合金屬離子的量以判斷其與金屬離子的結(jié)合能力。結(jié)果顯示,誘導(dǎo)表達的GST-HcMT分子量在34 k D左右,與預(yù)期一致,且通過Western blot進一步得到驗證;誘導(dǎo)的同時添加金屬離子,分離純化GST-HcMT與金屬離子復(fù)合物后,原子吸收結(jié)果表明其具有結(jié)合Cu~(2+)、Zn~(2+)、Pb~(2+)、Cd~(2+)的能力,結(jié)合量分別是對照的25倍、10倍、4倍和2倍,結(jié)合能力大小依次是:Cu~(2+)Cd~(2+)Zn~(2+)Pb~(2+)。同時發(fā)現(xiàn),工程菌pGEX-6p-2-HcMT/BL21具有耐受H_2O_2、NaHCO_3、Na_2CO_3的能力。將其亞克隆至pET32a原核表達載體,E.coli BL21表達后分離純化融合蛋白Trx A-HcMT,Western blot鑒定其活性。采取與GST-HcMT相同的研究策略,發(fā)現(xiàn)Trx A-HcMT與GST-HcMT相比可以特異性的結(jié)合更多的金屬離子,如Cd~(2+)、Co~(2+)、Cu~(2+)、Fe~(3+)、Mg~(2+)、Pb~(2+)、Zn~(2+)、Hg~(2+)。在Cd~(2+)、Cu~(2+)、Zn~(2+)脅迫下,含pET32a-HcMT/BL21的工程菌繁殖速度明顯優(yōu)于含pET32a/BL21的工程菌。GST-HcMT與Trx A-HcMT的這種差異,可能與標(biāo)簽蛋白中半胱氨酸殘基含量相關(guān)。為初步探索HcMT工業(yè)化生產(chǎn)的可行性,通過單因素實驗及20 L發(fā)酵罐放大培養(yǎng)優(yōu)化了影響轉(zhuǎn)鹽穗木金屬硫蛋白大腸桿菌發(fā)酵培養(yǎng)基的成分及其它因素。優(yōu)化培養(yǎng)基為:每900 m L培養(yǎng)基含蔗糖4 g,酵母浸粉24 g,蛋白胨8 g;每100 m L磷酸緩沖液含KH_2PO_4 2.31g、K_2HPO_4?3H_2O 12.54 g,兩部分分別滅菌后混合。蛋白表達條件為:乳糖6 mmol/L,誘導(dǎo)4 h。通過20 L發(fā)酵罐發(fā)酵培養(yǎng),可使菌體量達到2.68 g/L,比搖瓶培養(yǎng)提高了31%,每克干重菌體結(jié)合的銅離子達到315.3 mg,比搖瓶培養(yǎng)提高了69%,適宜放大培養(yǎng)。為進一步研究HcMT基因的功能活性及其應(yīng)用于植物修復(fù)的可行性,將HcMT構(gòu)建至p CAMBIA1301植物表達載體,通過EHA105農(nóng)桿菌介導(dǎo)浸染煙草�；蚪MPCR鑒定篩選到轉(zhuǎn)p CAMBIA1301-HcMT煙草陽性植株,轉(zhuǎn)HcMT基因煙草的各種非生物脅迫耐受性以及對不同金屬離子的富集情況有待于進一步的研究。
[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.
【學(xué)位授予單位】：新疆大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q943.2;Q946
本文編號：2235113
[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.
【學(xué)位授予單位】：新疆大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q943.2;Q946
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