CN108795913B - 一种植物中能催化h2s产生的酶及其应用 - Google Patents
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Abstract
本发明公开了一种植物中能催化H2S产生的酶及其应用。本发明以拟南芥叶片cDNA为模板,克隆胱硫醚β‑裂解酶(CBL)基因(AtCBL,At3g57050),体外构建其原核表达载体;将载体导入大肠杆菌BL21菌株中,加入异丙基‑β‑D‑硫代半乳糖苷(IPTG)诱导蛋白表达。再以L‑半胱氨酸为底物,测定其催化H2S产生的速率,从而证明CBL蛋白具有催化H2S产生的活性,调控植物内源H2S的产生。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中能催化H2S产生的酶及其应用,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信号与众多生理过程相关,如促进种子萌发、根形态建成、增强叶片的光合作用,延缓植物衰老;H2S还可以通过调控基因表达,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,如盐胁迫、冷或热胁迫、重金属胁迫等。
在植物体内,H2S主要是通过酶催化反应降解半胱氨酸(Cys)生成的。目前已经被鉴定的催化植物体内源H2S产生的酶见下表:
表1 植物体内催化产生H2S的酶
CBL是植物硫代谢过程中一种重要的酶,在植物合成甲硫氨酸(Met)的过程中,CBL可以催化胱硫醚生成同型半胱氨酸,之后经甲硫氨酸合酶催化生成Met。但是,CBL在植物中是否具有催化产生H2S的活性尚未有报道。
发明内容
本发明旨在提供一种植物中能催化H2S产生的酶及其应用。
实现本发明的方法包括提取大肠杆菌总蛋白的方法,其包括:以拟南芥cDNA为模板,克隆胱硫醚β-裂解酶基因AtCBL,构建AtCBL的重组构建体;转化大肠杆菌BL21株系,加入IPTG诱导表达所述基因。本发明通过体外构建pET-28a-AtCBL原核表达载体,并将其转化进入大肠杆菌BL21中,经IPTG诱导、表达,证明了CBL具有催化H2S产生的活性。
本发明提供了一种植物中具有催化内源性H2S产生的酶的合成方法,包括:以拟南芥叶片cDNA为模板,克隆胱硫醚β-裂解酶(CBL)基因(AtCBL, At3g57050),体外构建其原核表达载体;将该载体导入大肠杆菌BL21菌株中,加入异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白表达。
上述方法具体包括以下步骤:
(1)以拟南芥叶片cDNA为模板,克隆胱硫醚β-裂解酶(CBL)基因(AtCBL,At3g57050),体外构建其原核表达载体;
所述酶的编码基因为At3g57050,可操作性地连接至T7启动子;
(2)在大肠杆菌细胞中引入AtCBL基因的重组构建体,
(3)表达所述基因以调节细胞中H2S合成的水平;
(4)将大肠杆菌经20-40℃培养3-5 h,0.05-0.5mM的IPTG、16-24℃诱导培养16-24h,收集大肠杆菌细胞,超声破碎,提取总蛋白。
上述方法中,所述β-裂解酶基因AtCBL为拟南芥基因,细胞为大肠杆菌。
上述方法中,所述构建体为原核表达构建体pET-28a。
上述方法中产生的总蛋白,与未诱导的大肠杆菌总蛋白相比时,H2S产率显著增加。
本发明的有益效果:采用本发明方法,经IPTG诱导表达后,转入T7:: AtCBL的大肠杆菌总蛋白,分别与大肠杆菌原菌种、转入pET28a质粒的大肠杆菌总蛋白进行比较时,催化H2S产生的速率显著增加。
附图说明
图1为拟南芥AtCBL基因克隆以及T7:: AtCBL转化大肠杆菌菌液PCR鉴定,显示目的条带的结果。
图2为不同大肠杆菌总蛋白表达情况图。
图3不同大肠杆菌总蛋白催化H2S产生的产率对比图。
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。
实验材料的准备:取拟南芥叶片提取总RNA,反转录为cDNA,克隆AtCBL基因(At3g57050),构建T7:: AtCBL载体,转化大肠杆菌DH5α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;
实施例1:拟南芥AtCBL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AtCBL特异性引物进行PCR,克隆到AtCBL基因。
图1示出了拟南芥AtCBL基因克隆以及T7:: AtCBL转化大肠杆菌菌液PCR鉴定的琼脂糖凝胶电泳图。泳道3和4显示出目的条带。
实施例2:大肠杆菌总蛋白的提取:
用平板划线法将低温保藏的大肠杆菌BL21菌株进行活化,挑取单菌落于LB液体培养基中,37℃培养14-20 h,集菌,制备大肠杆菌感受态细胞;分别用pET28a质粒、T7:: AtCBL表达载体转化大肠杆菌感受态细胞,PCR鉴定;分别将大肠杆菌原种、转入pET28a质粒的大肠杆菌、转入T7:: AtCBL表达载体的大肠杆菌单菌落挑取至LB液体培养基(Kan+)中,37℃,200 rpm振荡培养至OD600= 0.8-1.0,将上述三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16-24℃条件下振荡培养24h,10000rpm,4℃离心集菌,加入10 mL PBS,超声破碎;10000 rpm,4℃离心,取上清,进行聚丙烯酰胺凝胶电泳。转入T7:: AtCBL表达载体的大肠杆菌在IPTG诱导下表达出特异的蛋白条带。
图2为不同大肠杆菌总蛋白表达情况图。图2说明:实施例2中,在T7:: AtCBL重组质粒转化大肠杆菌,并在IPTG诱导条件下可以产生重组CBL条带(泳道6),无IPTG诱导(泳道1,3,5),或者未转入T7:: AtCBL重组质粒的大肠杆菌(泳道2,4)中未见CBL产生。
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:蒸馏水615 μL ,100 mM L-Cys 80 μL,500 mMDTT 5 μL,500 mM pH 9.0 Tris-HCl 200 μL,将混合液加入到20 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,将大肠杆菌总蛋白100 μL加入混合液,盖好锥形瓶盖。37℃,120 rpm震荡,充分反应30 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示出了不同大肠杆菌总蛋白催化H2S产生的产率对比图。
实施例3中,在IPTG诱导下转入T7:: AtCBL重组质粒的大肠杆菌总蛋白,分别与IPTG诱导下转入pET28a质粒的大肠杆菌总蛋白、IPTG诱导下大肠杆菌BL21总蛋白以及未用IPTG诱导的转入T7:: AtCBL重组质粒的大肠杆菌总蛋白、未用IPTG诱导的转入pET28a质粒的大肠杆菌总蛋白和未用IPTG诱导的大肠杆菌BL21总蛋白相比,催化H2S产生的产率极显著升高。
由以上结果可知,在IPTG诱导时,CBL基因表达产生的重组胱硫醚-β裂解酶,可以以L-半胱氨酸为底物,催化H2S产生,从而增加内源性H2S的含量;相比之下,在无T7:: AtCBL的大肠杆菌中,即使加入了IPTG进行诱导,也未见其有催化H2S产生的活性,
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AtCBL蛋白进行真核表达,改变测定H2S产率反应体系的底物(如将L-半胱氨酸改为D-半胱氨酸)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。
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<110> 山西大学
<120> 一种植物中能催化H2S产生的酶及其应用
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<400> 1
atgacatctt ctctgtcact tcactcctcc ttcgtacctt ccttcgctga tctctccgac 60
cgaggtttga tcagtaagaa ctctccgacc agtgtttcca tttccaaggt tccaacttgg 120
gagaagaagc agatctcaaa tcggaactct ttcaagctga attgcgtgat ggagaaaagt 180
gtcgatggtc aaactcattc taccgttaac aataccactg atagcttaaa tactatgaat 240
atcaaagaag aagctagcgt ctctacatta ttggtgaact tggataataa atttgatccc 300
tttgatgcaa tgagcactcc gctttaccaa actgctactt ttaagcagcc ttctgctatt 360
gaaaatggac cttatgatta tacaagaagt ggcaatccta cacgggatgc attggaaagc 420
ctccttgcga agcttgacaa ggcagataga gcattttgct ttactagcgg aatggctgct 480
cttagtgctg ttacacatct tatcaaaaat ggcgaagaaa ttgttgctgg agatgatgta 540
tatggtggct ctgacagatt actatcccaa gttgttccaa gatctggcgt tgtggtaaaa 600
cgagtaaaca caactaagtt agacgaggtt gctgctgcaa ttggtcccca aacaaagctt 660
gtgtggcttg agtctccaac aaacccaaga caacaaattt ctgatatacg aaaaatatct 720
gagatggctc atgctcaagg tgctcttgtg ttggtggaca acagtattat gtcaccagtg 780
ctctctcggc cattagaact tggagctgac atcgtgatgc actcggctac taagtttata 840
gccggacaca gtgacgtgat ggcaggtgtg cttgctgtaa aaggcgaaaa attggcaaag 900
gaggtgtatt tcctccaaaa ctcagaaggt tctggattag ctcctttcga ctgttggctt 960
tgccttcgag gaatcaagac aatggcttta cggatagaaa agcaacagga aaacgcacgg 1020
aaaattgcaa tgtacttgtc ttctcatcca agagtaaaga aagtgtacta tgctggtcta 1080
ccagatcatc ctggtcacca tctccacttc tctcaggcga agggtgcagg atcagttttt 1140
agctttataa ctggatcagt tgcgctttca aagcatctcg tagaaaccac caaatacttc 1200
agcatagctg tcagttttgg gagtgttaag tcactgataa gcatgccatg cttcatgtca 1260
catgcaagca tacctgcaga agttcgtgag gccagaggtt tgacggaaga tcttgtccgt 1320
atatctgcag gaattgaaga tgttgatgat ttgatatctg atcttgacat tgccttcaaa 1380
accttccctc tctag 1395
Claims (2)
1. 拟南芥胱硫醚β-裂解酶基因AtCBL在催化H2S产生中的应用,其特征在于:核苷酸序列如SEQ ID No:1所示。
2.根据权利要求1所述的应用,其特征在于:应用过程中,取蒸馏水615 μL ,100 mM L-Cys 80 μL,500 mM DTT 5 μL,500 mM pH 9.0 Tris-HCl 200 μL,混合,将混合液加入到20mL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,将重组表达AtCBL的大肠杆菌总蛋白100 μL加入混合液,盖好锥形瓶盖;37℃,120 rpm震荡,充分反应30 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。
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