【摘要】：在植物界廣泛存在的金屬硫蛋白(Metallothioneins,MTs)是一類(lèi)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(qiáng)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(diǎn)。鹽穗木(Halostachys caspica)金屬硫蛋白HcMT含有78個(gè)氨基酸,其中半胱氨酸數(shù)量為14個(gè),理論分子量為7.70 k D,理論等電點(diǎn)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(guò)MEGA 4.0系統(tǒng)進(jìn)化樹(shù)分析與其它一些Type 2型MT進(jìn)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(dá)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(dá)載體上,在大腸桿菌BL21中表達(dá),分離純化融白蛋白GST-HcMT,通過(guò)原子吸收分光光度法測(cè)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(dá)的GST-HcMT分子量在34 k D左右,與預(yù)期一致,且通過(guò)Western blot進(jìn)一步得到驗(yàn)證;誘導(dǎo)的同時(shí)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(duì)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(shí)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(dá)載體,E.coli BL21表達(dá)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(guò)單因素實(shí)驗(yàn)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(dá)條件為:乳糖6 mmol/L,誘導(dǎo)4 h。通過(guò)20 L發(fā)酵罐發(fā)酵培養(yǎng),可使菌體量達(dá)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(dá)到315.3 mg,比搖瓶培養(yǎng)提高了69%,適宜放大培養(yǎng)。為進(jìn)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(dá)載體,通過(guò)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(yáng)性植株,轉(zhuǎn)HcMT基因煙草的各種非生物脅迫耐受性以及對(duì)不同金屬離子的富集情況有待于進(jìn)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(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q943.2;Q946

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王穎;徐炳政;王欣卉;李晶晶;張東杰;張桂芳;;酵母源金屬硫蛋白對(duì)慢性鉛中毒小鼠排鉛及肝臟保護(hù)作用[J];現(xiàn)代食品科技;2015年08期

2 南桂仙;喬坤;高野哲夫;柳參奎;;蒙古柳金屬硫蛋白的擬南芥遺傳轉(zhuǎn)化及抗性分析[J];基因組學(xué)與應(yīng)用生物學(xué);2015年01期

3 陳詩(shī)翔;張春雷;馬川;;金屬硫蛋白(MT)在激光誘導(dǎo)的皮膚血管損傷修復(fù)中的作用[J];中國(guó)激光醫(yī)學(xué)雜志;2014年05期

4 鄒學(xué)敏;朱樂(lè)玫;肖滿(mǎn)紅;;金屬硫蛋白對(duì)鎳鉻聯(lián)合染毒小鼠腎臟損傷的修復(fù)作用[J];環(huán)境與健康雜志;2014年06期

5 孫偉霞;尹霞;傅耀文;許鐘鎬;;金屬硫蛋白對(duì)慢性間斷缺氧所致腎臟損傷的保護(hù)作用[J];中華腎臟病雜志;2014年05期

6 鄒學(xué)敏;肖滿(mǎn)紅;朱樂(lè)玫;吳成秋;;金屬硫蛋白對(duì)鎳鉻染毒小鼠睪丸損傷保護(hù)作用[J];中國(guó)公共衛(wèi)生;2014年10期

7 于立博;劉繼文;;鷹嘴豆-金屬硫蛋白拮抗小鼠骨髓干細(xì)胞鉛毒性的機(jī)制[J];中國(guó)老年學(xué)雜志;2013年23期

8 陳潔;韓亮;李國(guó)躍;吳怡;林華慶;;金屬硫蛋白的分離純化技術(shù)及功能研究進(jìn)展[J];廣東藥學(xué)院學(xué)報(bào);2013年05期

9 史曉偉;沈勇偉;周學(xué)蘭;;金屬硫蛋白與運(yùn)動(dòng)[J];體育科技文獻(xiàn)通報(bào);2013年09期

10 王廷璞;馬偉超;李一婧;鄒亞麗;安建平;李小東;;不同重金屬脅迫蔬菜產(chǎn)生金屬硫蛋白的同源性檢測(cè)[J];食品安全質(zhì)量檢測(cè)學(xué)報(bào);2013年04期

相關(guān)碩士學(xué)位論文 前1條

1 陳虹;檉柳金屬硫蛋白基因（MT）的功能驗(yàn)證及轉(zhuǎn)MT基因山新楊的獲得[D];新疆農(nóng)業(yè)大學(xué);2007年

，

本文編號(hào)：2235113