CN109943571A - 一种金属硫蛋白基因mt20、其编码得到的金属硫蛋白及其表达和应用 - Google Patents
一种金属硫蛋白基因mt20、其编码得到的金属硫蛋白及其表达和应用 Download PDFInfo
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Abstract
本发明公开了一种金属硫蛋白基因MT20，其碱基序列如SEQ ID NO：1所示，来源于土壤微生物，该金属硫蛋白基因重金属耐受性好，在1.0M浓度镉溶液中仍然可以存活生长。并且对镉金属富集效应明显，因而在重金属污染治理等行业具有较大的应用潜力。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种金属硫蛋白基因、其编码得到的金属硫蛋白及其表达和应用。
背景技术
金属硫蛋白(metallothionein，简称MT)是一类普遍存在于生物体内的金属结合蛋白。金属硫蛋白是具有结合金属能力和高诱导特性的低分子量蛋白质。富含半胱氨酸的短肽，对多种重金属有高度亲和性。它是分子质量较低，半胱氨酸残基和金属含量极高的蛋白质。与其结合的金属主要是镉、铜和锌，广泛地存在于从微生物到人类各种生物中，其结构高度保守。
重金属生物修复是利用生物材料进行重金属治理的技术，而重金属抗性基因是生物修复的重要资源。基于基因改造的生物材料可以突破自然生物材料的性状限制，获得生物量、生长速度和修复效率的优化组合，来自微生物的重金属抗性基因已经在高效修复生物材料的开发方面显示出巨大的潜力。早在1998年，Nature Biotechnology(Rugh et al.,1998)就报道了稳定表达细菌汞还原基因merA的黄杨(yellow poplar，又译作鹅掌楸)，该转基因植物展示了一种生态友好的植物修复途径。来自沼泽红假单胞菌(Rhodopseudomonas palustris)的砷甲基化基因arsM最近被用于了构建基因工程菌，该工程菌展示了高效修复砷污染土壤的潜力(Chen et al.,2014)。少数研究也展示了基因改造生物材料在镉污染修复上的潜力。例如，Shim等人将酵母的镉抗性基因YCF1转入杨树，促进了该植株在镉污染土壤中的生长，也增强了该植株的镉富集能力(Shim et al.,2013)；Kang等人将三个微生物镉抗性基因SpPCS、GshI和MntA转入大肠杆菌，使转基因菌株的镉富集能力提高了25倍(Kang et al.,2007)。
现阶段对于重金属耐受/富集效果的金属硫蛋白来源往往来自于动物、植物，对微生物来源的金属硫蛋白研究较少。
发明内容
本发明的目的在于获得一种重金属耐受/富集效果的金属硫蛋白基因，从而得到由其编码的金属硫蛋白，进而将其应用于重金属污染治理领域中。
本发明第一方面是提供一种，金属硫蛋白基因，其碱基序列如SEQ ID NO：1所示，由该基因编码得到的氨基酸序列如SEQ ID NO：2所示。本发明通过以下步骤分离得到上述金属硫蛋白基因：
S1.用铜溶液污染土壤，避光恒温25度培养14天
S2.使用商用DNA提取试剂盒提取土壤全基因组
S3.使用二代Illumia测序平台HiSeq对基因组进行全测序
S4.通过通用生物信息学方法对测序序列进行质检、过滤、组装
S5.从公开基因数据库UniProt和GenBank中搜索所有有关的金属硫蛋白基因序列
S6.手动检查所述金属硫蛋白基因序列可靠性，编号后将可靠金属硫蛋白基因序列汇总，经软件翻译形成金属硫蛋白氨基酸序列数据库，所述金属硫蛋白氨基酸序列数据库见SEQ ID NO：3～SEQ ID NO：54；
S7.利用生物信息学检索方法BLAST，在测序组装的土壤基因组中搜索可能的金属硫蛋白基因序列，以所述金属硫蛋白氨基酸序列数据库作为检索模板
S8.获得的所述可能的金属硫蛋白基因序列进行手动检查，核对序列的核苷酸偏好性、金属结合位点等信息，进一步确定高可靠性的金属硫蛋白基因序列
S9.功能验证
9.1将所述高可靠性的金属硫蛋白基因序列通过化学合成获得高可靠性疑似金属硫蛋白序列，所述高可靠性疑似金属硫蛋白序列直接连接至pET28a(+)质粒，然后转化大肠杆菌BL21(DE3)，得到转化菌株；
9.2通过DropAssay试验测定所述大肠杆菌BL21(DE3)及所述转化菌株的镉抗性，使用所述大肠杆菌BL21(DE3)作为对照，当最小抑制浓度下该转化菌株成活，认为所述高可靠性的金属硫蛋白基因序列为有功能/活性的金属硫蛋白基因；
9.3通过测定生长管内600nm处吸光值获得所述大肠杆菌BL21(DE3)的生长曲线；
9.4收获生长曲线测定中的所述大肠杆菌BL21(DE3)；
9.5通过强酸消解所述大肠杆菌BL21(DE3)，使用原子吸收测定溶液镉浓度并计算所述大肠杆菌BL21(DE3)内镉吸收量。
本发明的优点和有益效果为：
本发明公开的金属硫蛋白基因，来源于土壤微生物，该金属硫蛋白基因重金属耐受性好，在1.0M浓度镉溶液中仍然可以存活生长。并且对镉金属富集效应明显，因而在重金属污染治理等行业具有较大的应用潜力。
附图说明
图1是本发明公开的大肠杆菌在不同浓度镉溶液中的生长情况。
图2是本发明公开的大肠杆菌生长曲线。
对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，可以根据以上附图获得其他的相关附图。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案，下面结合具体实施例进一步说明本发明的技术方案。
实施例一
金属硫蛋白基因筛选获得的过程：
S1.用铜溶液污染土壤，避光恒温25度培养14天；
S2.使用商用DNA提取试剂盒(DNeasy PowerMax Soil Kit)按照其说明书规定流程进行操作提取土壤全基因组，凝胶电泳测定DNA质量；
S3.提交商业测序，使用二代Illumia测序平台HiSeq对基因组进行全测序
S4.通过通用生物信息学方法对测序序列进行质检、过滤、组装
S5.从公开基因数据库UniProt和GenBank中搜索所有有关的金属硫蛋白基因序列
S6.手动检查所述金属硫蛋白基因序列可靠性，编号后将可靠金属硫蛋白基因序列汇总，经过软件翻译形成金属硫蛋白氨基酸序列数据库，所述金属硫蛋白氨基酸序列数据库见SEQ ID NO：3～SEQ ID NO：54；
S7.利用生物信息学检索方法BLAST，在测序组装的土壤基因组中搜索可能的金属硫蛋白基因序列，以所述金属硫蛋白氨基酸序列数据库作为检索模板，
S8.获得的所述可能的金属硫蛋白基因序列进行手动检查，核对序列的核苷酸偏好性、金属结合位点等信息，进一步确定高可靠性的金属硫蛋白基因序列，所述可靠性的金属硫蛋白基因序列碱基序列如SEQ ID NO：1所示，编号为MT20。
实施例二
9.1将所述高可靠性的金属硫蛋白基因序列MT20通过化学合成获得高可靠性疑似金属硫蛋白序列如SEQ ID NO：2所示(通过商业公司GENEWIZ,Suzhou,完成)，所述高可靠性疑似金属硫蛋白序列直接连接至pET28a(+)质粒，质粒通过电转化转入宿主大肠杆菌Escherichia coli(E.coli)BL21(DE3)，得到转化菌株；
实施例三
功能验证的详细过程。
9.2最小抑制浓度(MIC)，通过测量不同Cd浓度培养基中细菌细胞的生长能力来评估Cd的MIC值。首先，含有转化菌株的MT20及其相应的空白对照(空pET28a(+))在LauriaBertani(LB)-琼脂平板上于29℃生长过夜。然后，将含有细菌菌落的重组质粒扩散到含有各种浓度(0-16mM)Cd(包括卡那霉素(50mg/L))和诱导剂(IPTG-Isopropyl-B-D-1-thiogalactopyranoside，100mg/L)的LB-plate平板中。)测定他们的MIC。
测定空白对照大肠杆菌Escherichia coli(E.coli)BL21(DE3)，Cd最小抑制浓度，DropAssay试验测定所述大肠杆菌BL21(DE3)；见图1，在Cd浓度为0.8mmol/L时，空白对照大肠杆菌Escherichia coli(E.coli)BL21(DE3)，图中标号为pET28a无法存活，其最小抑制浓度为Cd 0.8mmol/L；而转化菌株，图中标号为MT20，在Cd浓度为0.8mmol/L、0.9mmol/L时均可存活，其最小抑制浓度为Cd1.0mmol/L证明该转化菌株镉耐受性好。
9.3用于筛选不同Cd溶液中更耐受菌株的生长模式(生长曲线)。
对于该测定，首先，将储存的细菌细胞转移到含有卡那霉素和IPTG的LB-琼脂平板中，并在37℃下孵育过夜以进行活化。然后将单个细菌菌落转移到含有抗生素卡那霉素(50μg/mL)，IPTG(100μg/mL)和1.0mM Cd的100ml LB液体培养基中。将细菌样品在37℃，160-180rpm连续摇动。摇动12-14小时后，通过nanodrop分光光度计每隔1小时检查细菌样品在600nm处的吸光值(光密度OD600)，持续24小时。将所有读数绘制成图2，以获得1.0mM Cd中每种细菌的生长曲线。
如图2所示，其中PET1～PET3均为空白对照大肠杆菌Escherichia coli(E.coli)BL21(DE3)，转化菌株图中标号为MT20，在生长过程中，转化菌株的吸光值(菌群数量)均高于三个空白对照大肠杆菌样品，并且维持较高增长，在第13个小时达到最高峰，转换菌株的菌群数量比空白对照大肠杆菌的菌群数量高约50％，证明其在Cd环境下耐受性能好，寿命长。
9.4、9.5Cd结合试验
检查OD600后，使用高速离心机收集细菌细胞。将收集的生物质在65℃温育24-48小时以进行干燥。然后将干燥的生物质加入管中。通过使用硝酸(浓度65％)过夜消解生物质。完全消化后，加热以除去液体。然后向每个管中加入2ml3％硝酸。14-16小时后，将溶液过滤并转移到干净且干燥的试管中。完成酸消解后，使用原子吸收分光光度计(TAS-990)测定所有样品中的镉浓度。通过检测空白对照大肠杆菌样品的镉吸附值为93.63g/kg，转化菌株的镉吸附值为159.02g/kg，该转化菌株的镉吸附能力强于空白对照大肠杆菌样品。
SEQUENCE LISTING
<110> 中国科学院遗传与发育生物学研究所农业资源研究中心
<120> 一种金属硫蛋白基因MT20、其编码得到的金属硫蛋白及其表达和应用
<130> 1
<160> 54
<170> PatentIn version 3.5
<210> 1
<211> 261
<212> DNA
<213> Unknown
<220>
<223> unknown
<400> 1
atggccgaat gcgcgtggtg ccacaacgag tacgacaaaa cgttttctgt gacgtacgga 60
gatcagtcgt acgtctttga ttgcttcgaa tgcgcgatca cgatgctggc gccaacgtgc 120
gctcactgtg gctgtcgcat catcggacac ggcgatgagg tggacgggcg cttctactgc 180
tgcgcttcct gtgtaagggc cgccgaaccg gtggatctgc acgaccgcgc gactgctggc 240
cacgtgggtg gcacacgcta g 261
<210> 2
<211> 86
<212> PRT
<213> Unknown
<220>
<223> unknown
<400> 2
Met Ala Glu Cys Ala Trp Cys His Asn Glu Tyr Asp Lys Thr Phe Ser
1 5 10 15
Val Thr Tyr Gly Asp Gln Ser Tyr Val Phe Asp Cys Phe Glu Cys Ala
20 25 30
Ile Thr Met Leu Ala Pro Thr Cys Ala His Cys Gly Cys Arg Ile Ile
35 40 45
Gly His Gly Asp Glu Val Asp Gly Arg Phe Tyr Cys Cys Ala Ser Cys
50 55 60
Val Arg Ala Ala Glu Pro Val Asp Leu His Asp Arg Ala Thr Ala Gly
65 70 75 80
His Val Gly Gly Thr Arg
85
<210> 3
<211> 57
<212> PRT
<213> Thermosynechococcus vulcanus
<400> 3
Met Thr Thr Val Thr Gln Met Lys Cys Ala Cys Pro His Cys Leu Cys
1 5 10 15
Ile Val Ser Leu Asn Asp Ala Ile Met Val Asp Gly Lys Pro Tyr Cys
20 25 30
Ser Glu Val Cys Ala Asn Gly Thr Cys Lys Glu Asn Ser Gly Cys Gly
35 40 45
His Ala Gly Cys Gly Cys Gly Ser Ala
50 55
<210> 4
<211> 54
<212> PRT
<213> Microcystis aeruginosa
<400> 4
Met Ile Ala Val Thr Thr Met Lys Cys Ala Cys Gly Ser Cys Thr Cys
1 5 10 15
Gln Val Ser Ile Ala Asp Ala Ile Lys Lys Asn Asp Gln Tyr Tyr Cys
20 25 30
Cys Gln Ala Cys Ala Asn Gly His Val Lys Glu Lys Gly Cys Gly His
35 40 45
Pro Gly Cys Val Cys Gly
50
<210> 5
<211> 57
<212> PRT
<213> Thermosynechococcus sp.
<400> 5
Met Thr Thr Val Thr Gln Met Lys Cys Ala Cys Pro His Cys Leu Cys
1 5 10 15
Ile Val Ser Leu Ser Asp Ala Ile Met Val Asp Gly Lys Pro Tyr Cys
20 25 30
Ser Glu Val Cys Ala Asn Gly Thr Cys Lys Glu Ser Asn Gly Cys Gly
35 40 45
His Ser Gly Cys Gly Cys Gly Ser Ala
50 55
<210> 6
<211> 57
<212> PRT
<213> Methylobacterium radiotolerans
<400> 6
Met Ala Ser Val Asp Val Glu Met Val Lys Cys Ala Cys Gln Asp Cys
1 5 10 15
Val Cys Val Ile Pro Val Ala Lys Ala Val Ser Arg Asp Gly Lys Ala
20 25 30
Tyr Cys Cys Asp Asp Cys Ala Asp Gly His Lys Asp His Ala Gly Cys
35 40 45
Glu His Ala Gly Cys Ala Cys His Gly
50 55
<210> 7
<211> 56
<212> PRT
<213> Microcystis aeruginosa
<400> 7
Met Ile Ala Val Thr Met Met Lys Cys Ala Cys Glu Ser Cys Leu Cys
1 5 10 15
Val Val Leu Ile Ala Asp Ala Ile Lys Lys Asn Asp Gln Tyr Tyr Cys
20 25 30
Ser Gln Ala Cys Ala Asn Gly His Val Asn Glu Asn Glu Lys Gly Cys
35 40 45
Gly His Gln Gly Cys Gly Cys Val
50 55
<210> 8
<211> 56
<212> PRT
<213> Microcystis aeruginosa
<400> 8
Met Ile Ala Val Thr Met Met Lys Cys Ala Cys Lys Pro Cys Leu Cys
1 5 10 15
Val Val Ser Ile Ala Asp Ala Ile Lys Glu Asn Asp Lys Tyr Tyr Cys
20 25 30
Ser Gln Ala Cys Ala Asn Gly His Val Asn Glu Asn Glu Lys Gly Cys
35 40 45
Gly His Gln Gly Cys Gly Cys Val
50 55
<210> 9
<211> 56
<212> PRT
<213> Synechococcus
<400> 9
Met Thr Ser Thr Thr Leu Val Lys Cys Ala Cys Glu Pro Cys Leu Cys
1 5 10 15
Asn Val Asp Pro Ser Lys Ala Ile Asp Arg Asn Gly Leu Tyr Tyr Cys
20 25 30
Ser Glu Ala Cys Ala Asp Gly His Thr Gly Gly Ser Lys Gly Cys Gly
35 40 45
His Thr Gly Cys Asn Cys His Gly
50 55
<210> 10
<211> 53
<212> PRT
<213> Mycobacterium tuberculosis
<400> 10
Met Arg Val Ile Arg Met Thr Asn Tyr Glu Ala Gly Thr Leu Leu Thr
1 5 10 15
Cys Ser His Glu Gly Cys Gly Cys Arg Val Arg Ile Glu Val Pro Cys
20 25 30
His Cys Ala Gly Ala Gly Asp Ala Tyr Arg Cys Thr Cys Gly Asp Glu
35 40 45
Leu Ala Pro Val Lys
50
<210> 11
<211> 48
<212> PRT
<213> Mycobacterium caprae
<400> 11
Met Thr Asn Tyr Glu Ala Gly Thr Leu Leu Thr Cys Ser His Glu Gly
1 5 10 15
Cys Gly Cys Arg Val Arg Ile Glu Val Pro Cys His Cys Ala Gly Ala
20 25 30
Gly Asp Ala Tyr Arg Cys Thr Cys Gly Asp Glu Leu Ala Pro Val Lys
35 40 45
<210> 12
<211> 53
<212> PRT
<213> Mycobacterium bovis
<400> 12
Met Arg Val Ile Arg Met Thr Asn Tyr Glu Ala Gly Thr Leu Leu Thr
1 5 10 15
Cys Ser His Glu Gly Cys Gly Cys Arg Val Arg Ile Glu Val Pro Cys
20 25 30
His Cys Ala Gly Ala Gly Asp Ala Tyr Arg Cys Thr Cys Gly Asp Glu
35 40 45
Leu Ala Pro Val Lys
50
<210> 13
<211> 55
<212> PRT
<213> Leptolyngbya sp.
<400> 13
Met Ala Thr Val Thr Gln Met Lys Cys Ala Cys Glu Pro Cys Leu Cys
1 5 10 15
Ile Val Asp Ile Ser Lys Ala Ile Gln Lys Asp Gly Gln Tyr Tyr Cys
20 25 30
Ser Glu Gly Cys Ala Ser Gly His Gly Asp Asn Ser Lys Gly Cys Gly
35 40 45
His Thr Gly Cys Asn Cys His
50 55
<210> 14
<211> 54
<212> PRT
<213> filamentous cyanobacterium
<400> 14
Met Ala Thr Val Thr Gln Met Lys Cys Ala Cys Glu Ser Cys Leu Cys
1 5 10 15
Val Val Asp Ile Ser Lys Ala Ile Glu Lys Glu Gly Gln Tyr Tyr Cys
20 25 30
Gly Glu Ala Cys Ala Asn Gly His Ser Glu Gly Ser Thr Gly Cys Gly
35 40 45
His Pro Gly Cys Asn Cys
50
<210> 15
<211> 54
<212> PRT
<213> Halomicronema hongdechloris
<400> 15
Met Thr Thr Val Thr Gln Met Lys Cys Ala Cys Asp Ser Cys Leu Cys
1 5 10 15
Ile Val Asp Thr Ser Lys Ala Val Glu Lys Glu Gly His Tyr Tyr Cys
20 25 30
Ser Glu Ala Cys Ala Asn Gly His Pro Glu Gly Ser Gly Cys Gly His
35 40 45
Thr Gly Cys Thr Cys His
50
<210> 16
<211> 55
<212> PRT
<213> Geitlerinema sp.
<400> 16
Met Thr Thr Val Thr Gln Met Lys Cys Ala Cys Glu Ser Cys Leu Cys
1 5 10 15
Val Val Asp Thr Asp Lys Ala Val Glu Lys Asp Gly Gln Tyr Tyr Cys
20 25 30
Ser Glu Ala Cys Ala Asn Gly His Pro Asp Gly Ser Gly Cys Gly His
35 40 45
Gln Gly Cys Thr Cys His Ala
50 55
<210> 17
<211> 55
<212> PRT
<213> Phormidium tenue
<400> 17
Met Thr Thr Val Thr Gln Met Lys Cys Ala Cys Asp Ser Cys Leu Cys
1 5 10 15
Val Val Asp Thr Ser Gln Ala Val Glu Lys Asp Gly His Tyr Phe Cys
20 25 30
Ser Glu Ala Cys Ala Asn Gly His Pro Glu Gly Ser Ala Gly Cys Gly
35 40 45
His Pro Gly Cys Gly Cys Asn
50 55
<210> 18
<211> 54
<212> PRT
<213> Lyngbya confervoides
<400> 18
Met Thr Thr Val Thr Gln Met Lys Cys Ala Cys Asp Ser Cys Leu Cys
1 5 10 15
Ile Val Asn Thr Ser Lys Ala Val Glu Lys Glu Gly His Tyr Tyr Cys
20 25 30
Ser Asp Ala Cys Ala Asn Gly His Pro Glu Gly Ser Gly Cys Gly His
35 40 45
Thr Gly Cys Thr Cys His
50
<210> 19
<211> 54
<212> PRT
<213> Kamptonema
<400> 19
Met Thr Thr Val Thr Gln Met Lys Cys Ala Cys Ser Ser Cys Leu Cys
1 5 10 15
Val Val Ser Leu Thr Glu Ala Ile Glu Lys Asn Gly Gln Tyr Tyr Cys
20 25 30
Ser Asn Ala Cys Ala Asp Gly His Pro Asn Gly Thr Gly Cys Gly His
35 40 45
Ala Gly Cys Gly Cys His
50
<210> 20
<211> 48
<212> PRT
<213> Spirulina subsalsa
<400> 20
Val Lys Cys Ala Cys Ser Thr Cys Glu Cys Met Val Ser Pro Asp Lys
1 5 10 15
Ala Ile Glu Lys Asp Gly Lys Tyr Tyr Cys Gly Glu Ala Cys Ala Asn
20 25 30
Gly His Thr Asp Gly Ser His Gly Cys Gly His Pro Gly Cys Asn Cys
35 40 45
<210> 21
<211> 54
<212> PRT
<213> Acaryochloris marina
<400> 21
Met Ala Thr Val Thr Gln Met Lys Cys Ala Cys Glu Ser Cys Leu Cys
1 5 10 15
Ile Val Thr Ile Ser Asp Ala Ile Gln Lys Gly Gly Gln Tyr Phe Cys
20 25 30
Gly Gln Ala Cys Ala Asp Gly His Pro Ser Gly Ser Gly Cys Gly His
35 40 45
Thr Gly Cys Gly Cys His
50
<210> 22
<211> 54
<212> PRT
<213> Acaryochloris marina
<400> 22
Met Ala Thr Val Thr Gln Met Lys Cys Ala Cys Glu Ser Cys Leu Cys
1 5 10 15
Ile Val Thr Ile Ser Asp Ala Ile Gln Lys Gly Gly Gln Tyr Phe Cys
20 25 30
Gly Gln Ala Cys Ala Asp Gly His Pro Ser Gly Ser Gly Cys Gly His
35 40 45
Thr Gly Cys Gly Cys His
50
<210> 23
<211> 53
<212> PRT
<213> Tolypothrix campylonemoides
<400> 23
Met Thr Asn Val Thr Gln Leu Lys Cys Ala Cys Glu Pro Cys Leu Cys
1 5 10 15
Val Val Ser Leu Glu Asp Ala Ile Gln Lys Asp Gly Lys Tyr Tyr Cys
20 25 30
Ser Glu Ala Cys Ala Glu Gly His Gln Thr Met Gln Gly Cys Gly His
35 40 45
Ser Gly Cys Gly Cys
50
<210> 24
<211> 53
<212> PRT
<213> Tolypothrix campylonemoides
<400> 24
Met Thr Asn Val Thr Gln Leu Lys Cys Ala Cys Glu Pro Cys Leu Cys
1 5 10 15
Val Val Ser Leu Glu Asp Ala Ile Gln Lys Asp Gly Lys Tyr Tyr Cys
20 25 30
Ser Glu Ala Cys Ala Glu Gly His Gln Thr Met Gln Gly Cys Gly His
35 40 45
Ser Gly Cys Gly Cys
50
<210> 25
<211> 53
<212> PRT
<213> Calothrix sp.
<400> 25
Met Thr Thr Val Thr Met Met Lys Cys Ala Cys Glu Arg Cys Leu Cys
1 5 10 15
Val Val Ser Thr Ala Asp Ala Ile Glu Lys Glu Gly Lys Tyr Tyr Cys
20 25 30
Ser Gln Ala Cys Ala Asp Gly His Lys Asp Glu Lys Gly Cys Ala His
35 40 45
Ser Gly Cys Gly Cys
50
<210> 26
<211> 52
<212> PRT
<213> Cyanobacterium aponinum
<400> 26
Thr Thr Val Thr Gln Met Lys Cys Ala Cys Pro Ser Cys Leu Cys Ile
1 5 10 15
Ile Asp Ile Ser Gln Ala Ile Ser Arg Asp Gly His Tyr Tyr Cys Ser
20 25 30
Thr Ala Cys Ala Glu Gly His Lys Glu Gly Glu Gly Cys Gly His Ser
35 40 45
Gly Cys Gly Cys
50
<210> 27
<211> 53
<212> PRT
<213> Scytonema tolypothrichoides
<400> 27
Met Thr Ser Val Thr Gln Met Lys Cys Ala Cys Glu Pro Cys Leu Cys
1 5 10 15
Ile Val Ser Leu Glu Asn Ala Ile Gln Lys Asp Glu Lys Tyr Tyr Cys
20 25 30
Ser Glu Ala Cys Ala Glu Gly His Lys Thr Met Lys Gly Cys Gly His
35 40 45
Asn Gly Cys Gly Cys
50
<210> 28
<211> 53
<212> PRT
<213> Limnoraphis robusta
<400> 28
Met Thr Ser Val Thr Gln Met Lys Cys Ala Cys Glu Ser Cys Leu Cys
1 5 10 15
Val Val Ser Leu Glu Ser Ala Ile Lys Lys Asp Gly Lys Pro Tyr Cys
20 25 30
Ser Glu Ala Cys Ala Asn Gly His Ser Asn Gly Ser Gly Cys Gly His
35 40 45
Thr Gly Cys Thr Cys
50
<210> 29
<211> 54
<212> PRT
<213> Oscillatoria brevis
<400> 29
Met Thr Thr Val Thr Gln Ile Lys Cys Ala Cys Pro Ser Cys Leu Cys
1 5 10 15
Val Val Ser Leu Thr Glu Ala Ile Glu Lys Ser Gly Lys Ser Tyr Cys
20 25 30
Ser Ser Ala Cys Ala Asp Gly His Pro Asn Gly Thr Gly Cys Gly His
35 40 45
Thr Gly Cys Glu Cys His
50
<210> 30
<211> 53
<212> PRT
<213> Fischerella
<400> 30
Met Thr Thr Val Thr Gln Met Lys Cys Ala Cys Glu Ser Cys Leu Cys
1 5 10 15
Ile Val Ser Ile Glu Asp Ala Ile Gln Lys Asp Asn Lys Tyr Tyr Cys
20 25 30
Ser Glu Ala Cys Ala Asp Gly His Gln Thr Thr Lys Gly Cys Gly His
35 40 45
Ser Gly Cys Gly Cys
50
<210> 31
<211> 54
<212> PRT
<213> Leptolyngbya ohadii
<400> 31
Met Thr Thr Val Thr Gln Met Lys Cys Ala Cys Ser Asp Cys Leu Cys
1 5 10 15
Ile Val Asn Leu Asn Asp Ala Ile Met Lys Asp Gly Lys Ala Tyr Cys
20 25 30
Gly Asp Ala Cys Ala Asn Gly His Thr Gly Gly Ser Gly Cys Gly His
35 40 45
Thr Gly Cys Gly Cys His
50
<210> 32
<211> 53
<212> PRT
<213> Acaryochloris marina
<400> 32
Met Ala Thr Val Thr Gln Met Lys Cys Ala Cys Glu Ser Cys Leu Cys
1 5 10 15
Ile Val Thr Ile Ser Asp Ala Ile Gln Lys Gly Gly Gln Tyr Phe Cys
20 25 30
Gly Gln Ala Cys Ala Asp Gly His Pro Ser Gly Ser Gly Cys Gly His
35 40 45
Thr Gly Cys Arg Cys
50
<210> 33
<211> 54
<212> PRT
<213> Phormidesmis priestleyi
<400> 33
Met Thr Ala Val Thr Gln Met Lys Cys Ala Cys Glu Pro Cys Leu Cys
1 5 10 15
Ile Val Thr Thr Glu Gly Ala Val Gln Lys Asp Gly Lys Leu Tyr Cys
20 25 30
Ser Glu Val Cys Ala Asp Gly His Pro Asn Gly His Gly Asp Cys Gly
35 40 45
His Lys Gly Cys Thr Cys
50
<210> 34
<211> 54
<212> PRT
<213> Microcoleus vaginatus
<400> 34
Met Thr Thr Ala Thr Gln Thr Lys Cys Ala Cys Pro Ser Cys Ser Cys
1 5 10 15
Val Val Asn Val Ser Glu Ala Ile Glu Lys Asp Gly Lys Thr Tyr Cys
20 25 30
Ser Ser Ala Cys Ala Asp Gly His Pro Asn Gly Ser Gly Cys Gly His
35 40 45
Thr Gly Cys Glu Cys His
50
<210> 35
<211> 54
<212> PRT
<213> Geminocystis herdmanii
<400> 35
Thr Val Thr Gln Met Lys Cys Ala Cys Pro Ser Cys Leu Cys Ile Val
1 5 10 15
Asp Ile Ala Ser Ala Ile Gln Lys Asp Asn Gln Tyr Phe Cys Ser Asp
20 25 30
Ala Cys Ala Asn Gly His Lys Glu Gly Thr Thr Gly Cys Ser His Ser
35 40 45
Gly Cys Gly Cys His Gly
50
<210> 36
<211> 54
<212> PRT
<213> Geitlerinema sp.
<400> 36
Met Thr Thr Val Thr Gln Met Lys Cys Ala Cys Glu Ser Cys Leu Cys
1 5 10 15
Val Val Asn Leu Ser Asp Ala Val His Lys Asp Glu Lys Tyr Tyr Cys
20 25 30
Cys Glu Ala Cys Ala Asn Gly His Gln Ser Gly Asp Gly Cys Gly His
35 40 45
Ser Gly Cys Gly Cys His
50
<210> 37
<211> 53
<212> PRT
<213> Mastigocladopsis repens
<400> 37
Met Thr Ser Val Thr Gln Met Lys Cys Ala Cys Glu Pro Cys Leu Cys
1 5 10 15
Ile Val Ser Leu Glu Asp Ala Ile Gln Lys Asp Asp Lys Tyr Tyr Cys
20 25 30
Ser Glu Ala Cys Ala Glu Gly His Gln Thr Met Lys Gly Cys Gly His
35 40 45
Asn Gly Cys Gly Cys
50
<210> 38
<211> 52
<212> PRT
<213> Cyanothece sp.
<400> 38
Thr Val Thr Gln Met Lys Cys Ala Cys Ser Ser Cys Val Cys Ile Val
1 5 10 15
Asp Leu Ser Asp Ala Ile Gln Lys Asp Gly Lys Tyr Tyr Cys Ser Asp
20 25 30
Ala Cys Ala Asn Gly His Pro Asp Gly Ala Gly Cys Ser His His Gly
35 40 45
Cys Glu Cys His
50
<210> 39
<211> 53
<212> PRT
<213> Mastigocoleus testarum
<400> 39
Met Ala Asp Val Thr Ser Met Lys Cys Ala Cys Ala Asp Cys Leu Cys
1 5 10 15
Ile Val Ser Leu Lys Asp Ala Ile Ala Lys Asn Gly Gln Tyr Tyr Cys
20 25 30
Ser Glu Val Cys Ala Asn Gly His Val Asp Gly Ser Gly Cys Gly His
35 40 45
Thr Gly Cys Lys Cys
50
<210> 40
<211> 53
<212> PRT
<213> Calothrix sp.
<400> 40
Met Thr Thr Val Thr Gln Met Lys Cys Ala Cys Glu Ser Cys Leu Cys
1 5 10 15
Ile Val Ser Leu Ser Ser Ala Val Met Lys Glu Gly Lys Pro Tyr Cys
20 25 30
Gly Glu Ala Cys Ala Asn Gly His Ala Asp Gly Lys Gly Cys Gly His
35 40 45
Thr Gly Cys Glu Cys
50
<210> 41
<211> 56
<212> PRT
<213> Pseudanabaena sp.
<400> 41
Met Ala Ser Ala Thr Leu Val Lys Cys Ala Cys Ser Lys Cys Leu Cys
1 5 10 15
Val Ile Asp Pro Ser Asp Ala Ile Glu Ala Asn Gly Lys Tyr Tyr Cys
20 25 30
Cys Lys Ala Cys Ala Ser Gly His Val Asp Gly Thr Asn Asp Ser His
35 40 45
Cys Ser Asp Val Gly Cys Glu Cys
50 55
<210> 42
<211> 58
<212> PRT
<213> Geminocystis sp.
<400> 42
Met Thr Thr Ala Thr Ile Thr Gln Met Lys Cys Ala Cys Pro Ser Cys
1 5 10 15
Leu Cys Ile Val Asp Ile Gly Thr Ala Leu Gln Lys Glu Gly Lys Tyr
20 25 30
Phe Cys Ser Thr Ala Cys Ala Glu Gly His Lys Glu Gly Thr Thr Gly
35 40 45
Cys Ser His Thr Gly Cys Gly Cys Asn Gly
50 55
<210> 43
<211> 53
<212> PRT
<213> Calothrix sp.
<400> 43
Met Thr Thr Val Thr Gln Met Lys Cys Ala Cys Glu Ser Cys Leu Cys
1 5 10 15
Ile Val Ser Leu Ser Ser Ala Val Met Lys Glu Gly Lys Pro Tyr Cys
20 25 30
Gly Glu Ala Cys Ala Asn Gly His Gln Asp Gly Lys Gly Cys Gly His
35 40 45
Thr Gly Cys Gly Cys
50
<210> 44
<211> 53
<212> PRT
<213> Rivularia sp.
<400> 44
Met Ala Ala Val Asp Leu Met Lys Cys Ala Cys Asp Lys Cys Leu Cys
1 5 10 15
Ile Val Lys Val Glu Thr Ala Ile Asp Arg Asp Gly Lys His Tyr Cys
20 25 30
Ser Glu Ala Cys Ala Glu Gly His Lys Thr Ile Thr Gly Cys Gly His
35 40 45
Ser Gly Cys Gly Cys
50
<210> 45
<211> 55
<212> PRT
<213> Coleofasciculus chthonoplastes
<400> 45
Met Thr Thr Ala Thr Gln Thr Gln Cys Ala Cys Asp Ser Cys Ala Cys
1 5 10 15
Met Val Ser Thr Asp Ser Ala Val Gln Lys Asp Gly Lys Tyr Tyr Cys
20 25 30
Ser Asp Ala Cys Ala Asn Gly His Pro Asn Gly Ala Gly Cys Gly His
35 40 45
Ser Gly Cys Glu Cys His Ala
50 55
<210> 46
<211> 53
<212> PRT
<213> Stanieria cyanosphaera
<400> 46
Met Ser Thr Val Thr Ser Met Lys Cys Ala Cys Asp Arg Cys Leu Cys
1 5 10 15
Val Val Ser Leu Glu Asp Ala Val Lys Lys Asp Gly Lys Tyr Tyr Cys
20 25 30
Cys Glu Ala Cys Ala Asn Gly His Thr Asp Gly Ser Gly Cys Gly His
35 40 45
Gln Gly Cys Gly Cys
50
<210> 47
<211> 54
<212> PRT
<213> Hydrocoleum sp.
<400> 47
Met Thr Thr Val Thr Gln Met Lys Cys Ala Cys Glu Ser Cys Leu Cys
1 5 10 15
Ile Val Ser Ile Glu Ser Ala Val Lys Lys Asn Gly Gln Asn Tyr Cys
20 25 30
Ser Glu Ala Cys Ala Asn Asn His Pro Asp Gly Ala Gly Cys Gly His
35 40 45
Glu Gly Cys Glu Cys Asn
50
<210> 48
<211> 53
<212> PRT
<213> Chamaesiphon minutus
<400> 48
Met Ser Thr Val Thr Gln Met Lys Cys Ala Cys Glu Ser Cys Leu Cys
1 5 10 15
Ile Val Ser Leu Ser Asp Ala Ile Val Lys Asp Gly Lys His Tyr Cys
20 25 30
Gly Asp Ala Cys Ala Asn Gly His Pro Ala Gly Gln Gly Cys Gly His
35 40 45
Thr Gly Cys Gly Cys
50
<210> 49
<211> 55
<212> PRT
<213> Tolypothrix bouteillei
<400> 49
Met Thr Thr Val Ser Gln Met Lys Cys Ala Cys Lys Ser Cys Leu Cys
1 5 10 15
Val Val Ser Leu Ser Asp Ala Leu Met Lys Asp Gly Lys Ala Tyr Cys
20 25 30
Gly Glu Ala Cys Ala Asn Gly His Thr Asn Gly Glu Cys Cys Gly His
35 40 45
Thr Gly Cys Asp Cys His Ala
50 55
<210> 50
<211> 54
<212> PRT
<213> Hapalosiphonaceae
<400> 50
Met Thr Thr Val Thr Gln Met Lys Cys Ala Cys Glu Ser Cys Leu Cys
1 5 10 15
Val Val Ser Leu Thr Asp Ala Val Ile Lys Asp Gly Lys Pro Tyr Cys
20 25 30
Gly Glu Ala Cys Ala Asn Gly His Pro Asn Gly Glu Gly Cys Gly His
35 40 45
Gln Gly Cys Gly Cys His
50
<210> 51
<211> 53
<212> PRT
<213> Aliterella atlantica
<400> 51
Met Thr Thr Ala Thr Gln Thr Gln Cys Ala Cys Glu Ser Cys His Cys
1 5 10 15
Pro Val Ser Glu Thr Glu Ala Val Gln Lys Asp Gly Lys Thr Tyr Cys
20 25 30
Ser Glu Ala Cys Ala Gln Gly His Pro Asp Gly Lys Gly Cys Gly His
35 40 45
Ala Gly Cys Asp Cys
50
<210> 52
<211> 53
<212> PRT
<213> Aliterella atlantica
<400> 52
Met Thr Thr Ala Thr Gln Thr Gln Cys Ala Cys Glu Ser Cys His Cys
1 5 10 15
Pro Val Ser Glu Thr Glu Ala Val Gln Lys Asp Gly Lys Thr Tyr Cys
20 25 30
Ser Glu Ala Cys Ala Gln Gly His Pro Asp Gly Lys Gly Cys Gly His
35 40 45
Ala Gly Cys Asp Cys
50
<210> 53
<211> 53
<212> PRT
<213> Cyanobacterium stanieri
<400> 53
Met Thr Thr Val Thr Gln Met Lys Cys Ala Cys Pro Ser Cys Leu Cys
1 5 10 15
Ile Val Asn Leu Ser Asp Ala Ile Gln Lys Asn Asp His Tyr Tyr Cys
20 25 30
Cys Gln Ala Cys Ala Asp Gly His Pro Asn Gly Ser Gly Cys Gly His
35 40 45
Thr Gly Cys Gly Cys
50
<210> 54
<211> 53
<212> PRT
<213> Xenococcus sp.
<400> 54
Thr Val Thr Gln Met Lys Cys Ala Cys Pro Ser Cys Leu Cys Ile Val
1 5 10 15
Asn Val Ser Asp Ala Ile Ser Lys Glu Gly Lys Tyr Tyr Cys Ser Asp
20 25 30
Ala Cys Ala Lys Gly His Ser Glu Gly Ala Gly Cys Ser His Ala Gly
35 40 45
Cys Gly Cys His Ala
50
Claims (9)
1.一种金属硫蛋白基因，其特征在于，其碱基序列如SEQ ID NO：1所示。
2.一种金属硫蛋白，其特征在于，所述金属硫蛋白由权利要求1所述的基因编码得到，其氨基酸序列如SEQ ID NO：2所示。
3.包含权利要求1所述的金属硫蛋白基因的重组载体。
4.根据权利要求4所述的重组载体，其特征在于，所述载体为大肠杆菌表达载体。
5.包含权利要求1所述的金属硫蛋白基因的重组菌株。
6.根据权利要求5所述的重组菌株，其特征在于，所述菌株为大肠杆菌。
7.一种根据权利要求1所述的金属硫蛋白基因、权利要求2所述的金属硫蛋白或权利要求5/6所述的重组菌株在重金属污染治理中的应用。
8.根据权利要求7所述的应用，其特征在于，所述应用为在废水重金属污染治理和/或在土壤重金属污染治理中的应用。
9.根据权利要求8所述的应用，其特征在于，所述重金属为铜、钴、铅和/或镉。
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| CN110386671A (zh) * | 2019-07-29 | 2019-10-29 | 中国科学院地理科学与资源研究所 | 一种转基因植物强化河道湖泊污染水体原位修复的方法 |
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