CN108486081A - 一种植物巯基丙酮酸硫转移酶及其应用 - Google Patents
一种植物巯基丙酮酸硫转移酶及其应用 Download PDFInfo
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Abstract
本发明公开了一种植物巯基丙酮酸硫转移酶(AtMST)及其应用。本发明以拟南芥cDNA为模板,克隆巯基丙酮酸硫转移酶基因AtMST,构建重组体pET28a‑AtMST,并将其转化进入大肠杆菌BL21(DE3)中,经IPTG诱导表达、Ni柱纯化,证明了MST蛋白以巯基丙酮酸钠为底物,具有催化H2S产生的活性。结合H2S信号在植物体内广泛而重要的功能,在实际生产中,可以通过转基因技术,从而建立对生物胁迫和非生物胁迫具有更高抵抗能力的转基因植物。
Description
技术领域
本发明涉及一种植物巯基丙酮酸硫转移酶及其应用,属于分子生物学领域。
背景技术
H2S是继一氧化氮(NO)和一氧化碳(CO)以来的第三种气体信号分子。在植物中,H2S信号参与植物的各个生理过程,如促进种子萌发、根形态建成、增强叶片的光合作用、调节气孔运动、延缓植物衰老;H2S还通过调控基因表达,与植物激素协同等方式参与到植物抵抗各种生物胁迫和非生物胁迫的过程中,如水分胁迫、温度胁迫、重金属胁迫等。
在植物体内,H2S主要通过酶催化反应降解生成。目前已经被鉴定的催化植物体内源H2S产生的酶见下表,它们均以半胱氨酸(Cys)为底物发挥作用,目前尚未有人报道植物体内有其他底物可以产生H2S。
表1 植物体内催化产生H2S的酶
MST是植物体内氰化物去毒化过程中重要的酶,该酶可将底物巯基丙酮酸钠上的硫烷硫原子转移到氰离子上,生成无毒的硫氰酸盐和丙酮酸,但尚未有报道显示该蛋白与植物体内的H2S生成有关。
发明内容
本发明提供了植物巯基丙酮酸硫转移酶及其应用,包括以下步骤:
(1)以拟南芥cDNA为模板,克隆巯基丙酮酸硫转移酶基因AtMST,可操作性地连接至T7启动子(T7:: AtMST)。
(2)转化大肠杆菌BL21(DE3)株系,加入IPTG诱导表达基因AtMST;
(3)将所述大肠杆菌经25-37℃培养3-5 h,至OD600达到0.8-1.0,加0.05-0.5mM的IPTG,16-37℃诱导培养12-22h,收集大肠杆菌细胞,超声破碎,提取总蛋白;
(4)Ni琼脂糖凝胶纯化柱纯化目的蛋白。
上述方法中,巯基丙酮酸硫转移酶基因AtMST为拟南芥基因,细胞为大肠杆菌。
上述方法中,所述酶的编码基因登录号为At1g79230,可操作性地连接至T7启动子T7:: AtMST。
上述方法中,所述构建体为原核表达构建体pET28a。
本发明提供的方法包括获得目的蛋白的方法,与未诱导表达的总蛋白进行比较时,催化H2S产生的速率显著增加。
本发明的有益效果:
通过原核表达和蛋白体外纯化,获得目的蛋白AtMST具有显著的催化H2S产生的活性,该蛋白可作为H2S的产生酶发挥作用。结合H2S作为气体信号分子在植物体内广泛而重要的功能,在实际生产中,可以通过转基因技术,从而建立对生物胁迫和非生物胁迫具有更高抵抗能力的转基因植物。
附图说明
图1为拟南芥AtMST基因克隆以及T7:: AtMST转化大肠杆菌菌液PCR鉴定,显示目的条带的结果。
图2 为T7:: AtMST重组质粒转化大肠杆菌,并在IPTG诱导条件下与无IPTG诱导的大肠杆菌的对比示意图。
图3 为IPTG诱导并经过纯化后转入T7:: AtMST重组质粒的目的蛋白与其他处理相比,催化H2S产生的速率极显著升高的示意图。
具体实施方式
下面通过实施例来进一步说明本发明,但不局限于以下实施例。
实验材料:拟南芥(Arabidopsis thaliana)叶片,大肠杆菌BL21(DE3)株系。
实施例1.拟南芥AtMST基因的克隆。
取生长4周的莲座叶,提取总RNA(TRIzol提取试剂盒,TaKaRa公司),反转录为cDNA(去基因组反转录试剂盒,abm公司),以该cDNA为模板,用AtMST特异性引物进行PCR,克隆得到AtMST基因。如图1显示出,在以拟南芥cDNA为模板进行高保真PCR后,得到大小为1140 bp的片段为目的片段;阴性对照是以ddH2O为模板,没有得到任何条带;第4泳道为重组pET28a-AtMST构建体进行菌液PCR检测所得片段,说明重组构建体构建成功。
实施例2. T7:: AtMST重组质粒转化大肠杆菌,在IPTG诱导条件下与无IPTG诱导的大肠杆菌的对比实验(制备目的蛋白的方法以及空白对照实验):
实验材料的准备:取拟南芥叶片提取总RNA,反转录为cDNA,克隆AtMST基因(At1g79230),构建T7:: AtMST重组体,转化大肠杆菌BL21(DE3)感受态细胞,卡那霉素筛选得到阳性菌。挑取单菌落,PCR鉴定,得到转化成功的大肠杆菌,用于实验。
将上述两种大肠杆菌各分为两组,一组加入IPTG(终浓度为0.05-0.5 mM),另一组加入等量双蒸水,于25-37℃条件下振荡培养12-20 h,10000 rpm,4℃离心集菌,加入10 mLPBS,超声破碎;12000 rpm,4℃离心,取上清,进行聚丙烯酰胺凝胶电泳。转入T7:: AtMST表达载体的大肠杆菌在IPTG诱导下表达出特异的蛋白条带,纯化得到目的蛋白(见图2)。
图2显示出,在T7:: AtMST重组质粒转化大肠杆菌,并在IPTG诱导条件下可以产生重组MST条带,纯化后得到单一的重组MST蛋白条带。而无IPTG诱导的大肠杆菌蛋白中无产生的MST蛋白条带,说明本发明成功表达并纯化出目的蛋白。
实施例3:H2S产率的测定实验。
按照以下配方将反应体系混合:1mL混合体系中加入150 μmol·L-1巯基丙酮酸钠,100μg蛋白,其余用pH=7的PBS缓冲液补充,将混合液加入到40 mL带盖锥形瓶中;剪去1.5mL离心管管盖,向其中加入500μL质量分数1% 的醋酸锌溶液;将离心管放入锥形瓶中,盖好锥形瓶盖。37℃,115 rpm震荡,充分反应15 min后,分别加入100μL 20 mM DPD和30 mM FeCl3溶液终止反应,黑暗静置15 min,测定OD670。根据标准曲线计算出H2S的产率。
图3结果显示,在IPTG诱导时,AtMST基因表达产生的巯基丙酮酸硫转移酶,可以以巯基丙酮酸钠为底物,催化H2S产生,从而增加内源性H2S的含量;相比之下,在未加入IPTG进行诱导的对照组蛋白中,未见其有催化H2S产生的活性。说明本发明所得目的蛋白MST具有催化H2S产生的新功能。
在上述实施例中,对本发明的实施方式做了描述,很显然,在本发明的发明构思下,仍可作出很多变化的应用。如将AtMST蛋白进行真核表达,改变测定H2S产率反应体系的底物(如将巯基丙酮酸钠改为巯基丙酮酸)等。因此,在本发明构思下做出的任何改变都将属于本发明的保护范围。
序列表
<110> 山西大学
<120> 一种植物巯基丙酮酸硫转移酶及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1140
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 1
atggcctcga cccttttctc cagaactttc ttggctgcta gtcaccgact gattactcct 60
tctcttccgc aaaagatctt taatccagcc acctttctca gtaggtcact ccactctcag 120
ttaggctccg cttctacagc ctataaatca actacttggg ctcgtcgagc tatggcttct 180
actggagttg agacaaaagc cggttactcc acatcatccg tatcaaccag tgaacctgtt 240
gtttctgttg attggcttca tgctaatctt agagagcctg atttgaagat tttggatgct 300
tcatggtata tgccggatga gcagagaaat ccgatccaag aatatcaggt tgctcatatt 360
ccccgcgctc tcttctttga tttggatgga atatcagatc gaaaaactag tttgccacat 420
atgttgccca ctgaggaagc ttttgctgct ggttgttctg ctcttggaat tgataacaaa 480
gatgaagtgg ttgtctatga tggaaagggg atctttagtg cagcccgtgt atggtggatg 540
ttccgagttt ttggacatga aaaagtttgg gtgctcgatg gaggtctacc aagatggcgt 600
gcatcaggtt atgatgttga atctagtgca tcaggtgatg ctattctgaa agccagtgcc 660
gcaagtgagg ctatagagaa aatttatcaa ggacaaaccg tcagtccgat aacctttcag 720
actaagttcc agccacatct agtgtggaca cttgatcagg tcaagaacaa tatggaggat 780
ccgacttatc aacacataga cgctcgttcc aaagccaggt ttgatggtac tgctccagaa 840
ccccgtaagg gaataagaag cggtcatata cctggaagca aatgtatccc ttttcctcag 900
atgtttgatt cttgtaacac attgttacca gcagaggagc tgaagaaacg atttgaccaa 960
gaagatatct cactggacaa gcctattatg gcctcgtgtg ggactggtgt aacagcttgc 1020
atcttggcaa tggggcttca ccggctgggg aaaaccgacg tgccgatcta tgatgggtcg 1080
tggactgaat gggcgacaca accagacttg cccatagaga gtgttgaatc ttcttcatga 1140
Claims (6)
1.一种植物巯基丙酮酸硫转移酶,其特征在于:由以下步骤制备得到:以拟南芥cDNA为模板,克隆基因AtMST,构建pET28a-AtMST重组体,并转化大肠杆菌BL21(DE3)进行原核表达纯化,得到AtMST蛋白。
2.根据权利要求1所述的植物巯基丙酮酸硫转移酶,其特征在于:包括以下步骤:
(1)以拟南芥cDNA为模板,克隆巯基丙酮酸硫转移酶基因AtMST,构建AtMST的重组构建体;
(2)转化大肠杆菌BL21(DE3)株系,加入IPTG诱导表达巯基丙酮酸硫转移酶基因;
(3)将所述大肠杆菌经37℃培养3 h,至OD600达到0.8-1.0,加0.05-0.5mM的IPTG,16-37℃诱导培养20h,收集大肠杆菌细胞,超声破碎,提取总蛋白;
(4)Ni琼脂糖凝胶纯化柱纯化目的蛋白。
3.根据权利要求2所述的植物巯基丙酮酸硫转移酶,其特征在于:所述酶的编码基因为At1g79230,可操作性地连接至T7启动子T7:: AtMST。
4.根据权利要求2所述的植物巯基丙酮酸硫转移酶,其特征在于:所述构建体为原核表达构建体pET-28a。
5.一种权利要求1~4任一项所述的植物巯基丙酮酸硫转移酶在催化硫化氢产生中的应用。
6.根据权利要求5所述的应用,其特征在于:应用过程中,取1mL混合体系中加入150 μmol·L-1巯基丙酮酸钠,100μg蛋白,其余用pH=7的PBS缓冲液补充,将混合液加入到40 mL带盖锥形瓶中;剪去1.5mL 离心管管盖,向其中加入500μL质量分数1% 的醋酸锌溶液;将离心管放入锥形瓶中,盖好锥形瓶盖;37℃,115 rpm震荡,充分反应15 min后,分别加入100μL20 mM DPD和30 mM FeCl3溶液终止反应,黑暗静置15 min,测定OD670。
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