CN102604901A - 一种重金属汞抗性相关蛋白DbsMerA及其编码基因和应用 - Google Patents
一种重金属汞抗性相关蛋白DbsMerA及其编码基因和应用 Download PDFInfo
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- CN102604901A CN102604901A CN2012100798328A CN201210079832A CN102604901A CN 102604901 A CN102604901 A CN 102604901A CN 2012100798328 A CN2012100798328 A CN 2012100798328A CN 201210079832 A CN201210079832 A CN 201210079832A CN 102604901 A CN102604901 A CN 102604901A
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Abstract
本发明公开了一种金属汞抗性相关蛋白DbsCzcA及其编码基因和应用。该汞抗性相关蛋白DbsCzcA氨基酸序列如SEQIDNO:1所示，其编码基因如SEQIDNO:2所示。通过转化大肠杆菌实验证明该基因在大肠杆菌中的表达可以提高其对重金属汞的抗性。本发明的DbsCzcA编码基因可用于提高微生物和植物对重金属汞的抗性，进一步用于清除环境重金属污染，为生物修复提供性能优良的生物资源。
Description
技术领域
本发明属于微生物基因工程，和生物修复领域。具体地说，本发明提供了生活在酸性矿山废水(acid mining drainage，AMD)的一种未知微生物所含有的一种对重金属汞抗性的基因即汞还原酶基因(DbsMerA)的序列，以及该序列推测编码蛋白质分子的氨基酸序列。本发明在提高微生物和植物对重金属汞的抗性方面有着重大的应用价值。
背景技术
随着矿产资源的开发利用、工业发展，重金属对环境造成的污染日趋严重，土壤重金属污染已经成为一个危害全球环境质量的问题。土壤重金属会影响植物的生长发育，降低农作物的产量和质量，带来了严重的经济损失。此外，受土壤重金属污染的作物在植物体中积累，并通过食物链富集到人体和动物体中，危害人畜健康，引发癌症和其他疾病等。治理重金属污染刻不容缓，各种修复技术和措施正在研究和应用中。各国政府和科学家着力通过两个途径解决这一问题：一为利用物理的，化学的方法试图清除土壤或水体的重金属污染：二为利用现代生物技术清除污染。自从20世纪80年代以来，生物修复技术因其具有处理费用低、对环境影响小、效率高等优点，越来越受到广大科技人员的广泛关注。生物修复一般分为植物修复、动物修复和微生物修复三种类型，其中植物修复和微生物修复是研究的热点。微生物修复就是利用微生物将环境中的污染物降解或转化为其他无害物质的过程。近年来，基于微生物对重金属的作用机理，以修复有毒有害金属污染或回收有经济价值重金属为目的的生物处理技术日趋成熟。植物修复指利用植物去治理水体、土壤和底泥等介质中的污染的技术。然而用于重金属污染修复的生物往往会受到重金属的毒害，生长缓慢、生物量小，甚至不能生存，所以重金属对植物和微生物的毒害作用是生物修复的主要限制因素。
解决生物修复中重金属对生物的毒害作用的根本途径在于研究耐受重金属的分子生物学机制，克隆对重金属耐受的关键基因，通过基因工程手段获得用于生物修复中性能优良的转基因工程生物。
由于技术上的原因，直到最近，对微生物基因资源的利用主要局限于可培养微生物。然而，已培养微生物仅占自然界中微生物的不到1％，因此各种生境中的微生物宏基因组是一个巨大而未发掘的基因资源库。极端环境具有丰富的微生物资源，当中许多与逆境和关键生命过程相关的基因在长期的适应进化中获得了更强的耐性潜能，发掘这些抗性基因已成为国际重要的研究热点。酸性矿山废水(AMD)是极端生境微生物学研究的重要***。AMD来源于采矿活动使含硫矿物(主要为黄铁矿，FeS2)暴露于空气和水中，在微生物催化作用下迅速氧化产酸所致，其pH值一般在1-4左右，而且富含硫酸盐以及Pb、Zn、Cu、Cd和Ni等重金属，是采矿业面临的最严重环境问题之一。在AMD中生存的原核微生物在长期的进化过程中逐渐形成了一些独特机制，以应对低pH值、高盐度以及高重金属等多种极端环境协迫。因此，AMD生境成为极具特色和丰富的抗逆基因库。
研究表明与运输汞有关的基因是由几个mer基因merA，merB，merC，merD，merT，merP，merR组成的mer操纵子。mer操纵子在不同的细菌中存在于不同的遗传单元上，如在Shigella flexneri中存在于转座子Tn21，在Stathylociccus aureus中存在于质粒pDU1358等。有机汞裂解酶、汞还原酶和其它转运***组分及相关调控因子共同组成一个操纵子。merR的结合位点，位于转录起始点上游-35和-10序列之间的OP位点，编码调节蛋白，merT和merC编码有关吸收的内膜蛋白(分子量为15.1扔和14kD)，merP编码位于周质空间结合Hg2+的蛋白(12kD)，merA编码汞还原酶，该酶常以二聚体(66kD)形式在细胞质内与内膜呈疏松的结合，汞还原酶与谷胱甘肽还原酶、硫辛酰胺脱氢酶有很高的相似性。merB编码有机汞裂解酶，该酶难以分离纯化，定位问题尚未确定。merD是G-菌中除merR以外的第二个调节基因。转录调节蛋白merD和merR一样，有螺旋-转角-螺旋构型，结合于操纵子的OP位点，来调节mer操纵子基因。当Hg2+存在时，Hg2+与merR结合，Hg2+与merR复合物并不离开DNA，而是在OP处使DNA发生扭曲、折叠，使RNA聚合酶与DNA结合，激活merT，merP和merc及merA、merB转录，merT、merP、merC三个蛋白组成。Hg2+吸收、结合转运***，将Hg2+运至merA(汞还原酶)作用部位，从而高效、快速地将Hg2+还原为Hg0，降低Hg2+对细胞的毒性。
发明内容
本发明的目的在于提供AMD微生物中的汞还原酶，以及编码该蛋白的新基因，为今后开发基因工程产品奠定物质基础。
在本发明的一个方面，提供了一种重金属汞抗性相关蛋白，它为如下蛋白分子(i)或(ii)：
(i)具有如SEQ ID NO：1所述的氨基酸序列；
(ii)在如SEQ ID NO：1限定的氨基酸序列经过取代，缺失或叠加一个或几个氨基酸衍生的蛋白质与(i)的蛋白质具有相同的功能。
在本发明的另一个方面，提供了一种DNA分子，它包括：编码所述的汞还原酶的核苷酸序列(如SEQ ID NO：2)。
发明发现DbsMerA蛋白具有金属汞抗性作用，可以在治理环境汞污染中应用。
发明同时保护了该蛋白的编码基因，具有如SEQ ID NO：2所示的序列；以及该基因在治理环境汞污染中的应用。
发明用表达DbsMerA的基因工程菌作为试验对象，发现在汞胁迫下，其汞耐受性大大高于非基因工程菌。在汞离子浓度为50μM以下时，尤其是30μM以下时，仍有较高的存活率。
与现有技术相比，本发明具有以下有益效果：
本发明从AMD微生物中克隆了一个新的汞还原酶基因，命名为DbsMerA，并对其对重金属汞的抗性进行了功能分析。通过大肠杆菌转化实验证明该基因可以提高大肠杆菌中对汞的抗性。
应用本发明的基因序列或氨基酸序列进行转基因开发基因工程产品面具有重大的应用价值，具体来说可用于培育高生物量的重金属超富集植物或微生物，用于重金属污染土壤和水体的生态修复。
附图说明
附图1为DbsMerA在大肠杆菌中的表达时相；其中：
M：蛋白分子量标准
1：携带空载体pET28a的BL21(DE3)菌株
2-9：携带pET28a-DbsMerA的BL21(DE3)菌株诱导表达分别0、1、2、3、4、5、6、7小时。
附图2为表达DbsMerA的大肠杆菌在不同汞浓度下培养12小时后的生长情况；
附图3为表达DbsMerA的大肠杆菌在30μM汞胁迫下的生长曲线。
具体实施方式
实施例1DbsMerA基因全序列的克隆
野外微生物样品采集：
在云浮铅/锌矿选取不同酸化阶段(以pH为标准)的AMD，使用0.22μm的滤膜收集20LAMD里面的细胞。为了保持核酸的完整保存，保存样品冻于液氮中，24小时内带回实验室，并放于-70℃冰箱长期保存。
核酸的提取：
DNA提取采用SET方法，过程如下：向SET buffer中加入溶菌酶及蛋白酶K，消化30min后，15,000rpm离心15min后用氯仿抽提2次，用异丙醇沉淀过夜后，75％乙醇清洗2次，最后溶于灭菌水中。用Qiagen tip-100柱纯化回收基因组DNA，用核酸蛋白分析仪检测DNA质量及浓度。
基因组测序：
用Roche公司的GS FLX Titanium General Library Preparation Kit制备上机样品。使用Roche 454Genome Seqencer FLX测序仪，通过进行测序，用Pyrobayer软件获取碱基序列。
基因组序列分析：
去除低质量的测序结果后，对基因组进行如下分析：
序列拼接：使用Euler-SR及454公司的GS De Novo Assembler Software进行序列拼接。用N50指数评价拼接的效果。
基因组注释：将拼接好的微生物的全基因组序列用IMG和SEED***进行基因组的注释，发现新的基因。
DbsMerA基因片段的克隆：
通过PCR方法：以含目的基因的克隆质粒为模板，按基因序列设计一对引物(在上游和下游引物分别引入不同的酶切位点)，PCR获得一条1.7kb大小的条带，并克隆到PCR2.1(购自invitrogen公司)载体中，序列委托invitrogen公司测定。
DbsMerA基因序列分析：
测序结果表明DbsMerA基因大小为1653bp(见SEQ ID NO：2)，编码550个氨基酸(见SEQ ID NO：1)。根据PSORT分析，DbsMerA基因表达产物很可能是胞质溶胶蛋白。
实施例2DbsMerA基因的功能分析
本实施例中利用大肠杆菌转化实验分析DbsMerA基因的功能。
构建重组表达载体：
设计以下一对引物，在DbsMerA基因5’引入BamHI酶切位点，3’引入XhoI酶切位点。
DbsMerA-F(SEQ ID NO：3)：
5’CGCGGATCCATGAATAAAGCGATGATGTTGGGC3’
DbsMerA-R(SEQ ID NO：4)：
5’CCGCTCGAGTCCCGCGCAGCAGGATAAT3’
以PCR2.1-DbsMerA载体为模板进行PCR。分别将PCR产物和酵母穿梭表达载体pET28a用BamHI和XhoI进行酶切，将酶切产物回收、连接、并转化大肠杆菌DH5α。经测序并酶切鉴定，得到了pET28a-DbsMerA重组子。
蛋白表达：
将重组子pET28a-DbsMerA转化大肠杆菌表达菌株BL21(DE3)，通过PCR验证筛选阳性克隆。挑取含重组质粒的菌体单斑至10ml LB(含Kan50μg/ml)中37℃过夜培养。将1ml菌液加入到含100ml LB培养基(含Kan50μg/ml)，37℃震荡培养至OD600约为0.4-1.0(最好0.6，大约需2hr)。
加入IPTG至终浓度为1mM进行诱导，每隔1小时收集1ml菌液，离心12000g×60s收获沉淀，用80μl ddH2O重悬，加入20μl 5×SDS-PAGE上样缓冲液，混匀，沸水浴10min。取上清作为样品做SDS-PAGE分析(见附图1)。
转化子对重金属汞抗性的测定：
挑取含重组质粒的菌体单斑至10ml LB(含Kan 50μg/ml)中37℃过夜培养。将100ul菌加入到10ml Hg2+浓度分别为0μM、10μM、20μM、30μM、40μM、50μM的LB培养基(含Kan 50μg/ml、IPTG 1mM)中，37℃、200rpm震荡培养12小时，分别测定OD600值。
根据实验结果，选取Hg2+浓度为30μM作为胁迫条件，进行了生长曲线的测定。挑取含重组质粒的菌体单斑至10ml LB(含Kan 50μg/ml)中37℃过夜培养。将100ul菌加入到10ml Hg2+浓度为30μM的LB培养基(含Kan 50μg/ml、IPTG 1mM)中，37℃、200rpm震荡培养，每隔2小时测定OD600值。
实验结果表明，表达DbsMerA基因的BL21(DE3)在0-50μM Hg2+浓度下都可以正常生长，而空载体对照在Hg2+浓度超过30μM时便不能生长(见附图2)。在30μM Hg2+浓度下，表达DbsMerA基因的BL21(DE3)在培养过程中都可以生长，而空载体对照的生长一直受到抑制(见附图3)。实验结果说明，DbsMerA对重金属汞的防御起着重要作用。
SEQ ID NO：1
MNKAMMLGITGMTCNHCALMVENALCAVPGVALAQVAFPKGQAQIEVSEPVSMEVLAAAINKAGYGVSLPENAGTPVAMEAKNAHATGLHVAIIGSGS SAFAAAIRVTEGGGRVTMIESGTLGGTCVNVGCVPSKIMIRAAQMAQWQRAHPFRGLSHSVPDVDRAVLVQQQQDRVAELREAKYDHVLATHPDITFVQGHARFVDATTLVVRTADGSEQRLQADRFLIAAGAHAHCPDIEGLAQTPYWTSTEALVASELPRHLLVVGGAVVAVELAQAFRRLGCAVTLLARSTLLSKDDPALGVGLEAVFQEEGIRVLKHTVPTSVHYEKDQFLVSIGAETIHADRLLVATGRTPNTADLGLEQIGVITDKSGAILVDAHMQSSVPHIYAAGDCTTHPQFVYVAAAGGTRAAINLLGGDAILDLRVLPVVVFTDPQVATVGLDERTAAQQGFTTDSRTLTLDNVPRALANFDTRGFIKLIADK HT G K LL G A QIL A A EG GE IIQ A A TL AIR G G L G IQ D L ADQLFPYLTMVEGLKLCAQTFTKDVSQLSCCAG
SEQ ID NO：2
ATGAATAAAGCGATGATGTTGGGCATTACGGGCATGACCTGTAATCACTGCGCCCTGATGGTAGAAAATGCGTTGTGCGCGGTTCCTGGCGTCGCGCTGGCACAGGTGGCTTTTCCGAAAGGGCAGGCACAGATCGAAGTTTCCGAACCGGTATCCATGGAAGTGCTTGCTGCCGCCATCAACAAAGCGGGCTACGGTGTTTCACTGCCTGAAAATGCCGGCACTCCGGTGGCAATGGAGGCAAAAAATGCTCATGCTACCGGCCTTCATGTCGCCATCATTGGTTCTGGATCAAGCGCCTTTGCTGCCGCCATCCGAGTGACGGAAGGAGGCGGCCGAGTGACCATGATTGAATCAGGTACGCTGGGCGGCACCTGCGTCAATGTCGGCTGCGTGCCTTCCAAGATCATGATCCGTGCCGCGCAGATGGCCCAGTGGCAGCGTGCCCATCCTTTTCGTGGTTTAAGTCATAGCGTACCCGACGTGGACCGCGCCGTCCTGGTCCAGCAACAACAGGATCGCGTGGCTGAACTGCGTGAAGCCAAGTATGATCATGTGCTGGCTACGCATCCGGATATCACCTTTGTGCAGGGTCACGCCCGCTTTGTCGATGCCACAACCCTCGTGGTGCGCACTGCCGACGGATCGGAACAACGGCTCCAAGCGGATCGTTTTCTGATTGCGGCGGGCGCCCATGCGCACTGCCCGGACATTGAGGGTCTGGCGCAAACGCCTTACTGGACGTCAACGGAAGCCTTGGTAGCGTCCGAATTGCCGCGTCATCTCCTCGTTGTGGGGGGGGCAGTAGTCGCGGTGGAACTGGCGCAGGCCTTTCGCAGGTTGGGCTGCGCAGTCACTCTGCTGGCGCGCAGCACACTCCTGTCCAAAGATGATCCGGCACTGGGCGTGGGGCTGGAGGCCGTATTTCAGGAAGAAGGGATTCGTGTGCTCAAGCACACCGTCCCCACGTCGGTGCATTACGAGAAGGACCAATTTTTGGTCAGCATAGGCGCAGAGACAATCCACGCGGATCGCTTGCTGGTGGCAACCGGGCGCACGCCCAACACGGCGGATCTTGGATTGGAGCAGATCGGGGTAATCACCGACAAATCCGGGGCCATTTTGGTGGATGCGCATATGCAAAGCAGTGTGCCCCACATTTATGCTGCCGGTGATTGCACGACCCATCCTCAATTCGTCTATGTCGCAGCGGCGGGCGGCACCCGGGCGGCTATCAATCTGCTGGGCGGCGATGCCATCCTGGATTTACGGGTACTTCCCGTCGTCGTGTTTACCGATCCCCAAGTCGCTACAGTGGGGCTGGACGAGCGGACAGCGGCGCAGCAAGGATTCACCACCGACAGCCGCACTCTGACCCTCGATAACGTACCCCGCGCACTGGCGAACTTTGATACACGGGGTTTTATTAAACTGATCGCGGACAAGCATACCGGCAAACTGTTGGGTGCGCAGATTCTCGCCGCAGAGGGCGGAGAAATCATCCAGGCTGCCACACTGGCCATACGTGGCGGTCTCGGCATTCAAGATCTTGCCGATCAGCTTTTCCCCTATTTG A C C AT G G T G G A G G GA T T G A A A C T C T G T G C CCAAACCTTCACCAAAGATGTCTCGCAATTATCCTGCTGCGCGGGATAA。
SEQUENCE LISTING
<110> 中山大学
<120> 一种重金属汞抗性相关蛋白DbsMerA及其编码基因和应用
<130>
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<170> PatentIn version 3.2
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515 520 525
Glu Gly Leu Lys Leu Cys Ala Gln Thr Phe Thr Lys Asp Val Ser Gln
530 535 540
Leu Ser Cys Cys Ala Gly
545 550
<210> 2
<211> 1653
<212> DNA
<213> 人工序列
<400> 2
atgaataaag cgatgatgtt gggcattacg ggcatgacct gtaatcactg cgccctgatg 60
gtagaaaatg cgttgtgcgc ggttcctggc gtcgcgctgg cacaggtggc ttttccgaaa 120
gggcaggcac agatcgaagt ttccgaaccg gtatccatgg aagtgcttgc tgccgccatc 180
aacaaagcgg gctacggtgt ttcactgcct gaaaatgccg gcactccggt ggcaatggag 240
gcaaaaaatg ctcatgctac cggccttcat gtcgccatca ttggttctgg atcaagcgcc 300
tttgctgccg ccatccgagt gacggaagga ggcggccgag tgaccatgat tgaatcaggt 360
acgctgggcg gcacctgcgt caatgtcggc tgcgtgcctt ccaagatcat gatccgtgcc 420
gcgcagatgg cccagtggca gcgtgcccat ccttttcgtg gtttaagtca tagcgtaccc 480
gacgtggacc gcgccgtcct ggtccagcaa caacaggatc gcgtggctga actgcgtgaa 540
gccaagtatg atcatgtgct ggctacgcat ccggatatca cctttgtgca gggtcacgcc 600
cgctttgtcg atgccacaac cctcgtggtg cgcactgccg acggatcgga acaacggctc 660
caagcggatc gttttctgat tgcggcgggc gcccatgcgc actgcccgga cattgagggt 720
ctggcgcaaa cgccttactg gacgtcaacg gaagccttgg tagcgtccga attgccgcgt 780
catctcctcg ttgtgggggg ggcagtagtc gcggtggaac tggcgcaggc ctttcgcagg 840
ttgggctgcg cagtcactct gctggcgcgc agcacactcc tgtccaaaga tgatccggca 900
ctgggcgtgg ggctggaggc cgtatttcag gaagaaggga ttcgtgtgct caagcacacc 960
gtccccacgt cggtgcatta cgagaaggac caatttttgg tcagcatagg cgcagagaca 1020
atccacgcgg atcgcttgct ggtggcaacc gggcgcacgc ccaacacggc ggatcttgga 1080
ttggagcaga tcggggtaat caccgacaaa tccggggcca ttttggtgga tgcgcatatg 1140
caaagcagtg tgccccacat ttatgctgcc ggtgattgca cgacccatcc tcaattcgtc 1200
tatgtcgcag cggcgggcgg cacccgggcg gctatcaatc tgctgggcgg cgatgccatc 1260
ctggatttac gggtacttcc cgtcgtcgtg tttaccgatc cccaagtcgc tacagtgggg 1320
ctggacgagc ggacagcggc gcagcaagga ttcaccaccg acagccgcac tctgaccctc 1380
gataacgtac cccgcgcact ggcgaacttt gatacacggg gttttattaa actgatcgcg 1440
gacaagcata ccggcaaact gttgggtgcg cagattctcg ccgcagaggg cggagaaatc 1500
atccaggctg ccacactggc catacgtggc ggtctcggca ttcaagatct tgccgatcag 1560
cttttcccct atttgaccat ggtggaggga ttgaaactct gtgcccaaac cttcaccaaa 1620
gatgtctcgc aattatcctg ctgcgcggga taa 1653
<210> 3
<211> 33
<212> DNA
<213> 人工序列
<400> 3
cgcggatcca tgaataaagc gatgatgttg ggc 33
<210> 4
<211> 28
<212> DNA
<213> 人工序列
<400> 4
ccgctcgagt cccgcgcagc aggataat 28
Claims (9)
1. 一种金属汞抗性相关蛋白，其特征在于氨基酸序列如SEQ ID NO:1所示。
2. 权利要求1所述金属汞抗性相关蛋白在治理环境汞污染中的应用。
3. 权利要求1所述金属汞抗性相关蛋白的编码基因，其特征在于核苷酸序列如SEQ ID NO:2所示。
4. 权利要求3所述金属汞抗性相关蛋白的编码基因在治理环境汞污染中的应用。
5. 含有权利要求3所述金属汞抗性相关蛋白的编码基因的表达载体。
6. 根据权利要求5所述载体，其特征在于出发载体为pET28a。
7. 一种基因工程菌，其特征在于是由权利要求6所述的表达载体转染大肠杆菌得到的。
8. 权利要求7所述基因工程菌在治理环境汞污染中的应用。
9. 根据权利要求8所述的应用，其特征在于环境汞污染中的汞离子浓度为50μM以下。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103951089A (zh) * | 2014-05-12 | 2014-07-30 | 江南大学 | 酿酒酵母基因缺失菌株在清除环境水质中重金属的应用 |
| CN106964647A (zh) * | 2017-06-06 | 2017-07-21 | 临沂大学 | 一种利用复合菌剂修复铅污染土壤的方法 |
| CN114686411A (zh) * | 2020-12-29 | 2022-07-01 | 上海科技大学 | 一种工程菌的混合物、用于感应和吸附重金属污染物的试剂盒和处理方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5965796A (en) * | 1995-04-21 | 1999-10-12 | University Of Georgia Research Foundation Inc. | Metal resistance sequences and transgenic plants |
| WO2002048335A2 (en) * | 2000-12-13 | 2002-06-20 | University Of Georgia Research Foundation, Inc. | Metal resistant plants and use for phytoremediation |
| CN1614024A (zh) * | 2003-11-06 | 2005-05-11 | 中国科学院上海生命科学研究院 | 降解有机汞污染的转基因烟草 |
| CN101921724A (zh) * | 2010-05-07 | 2010-12-22 | 南京师范大学 | 一种用于监测环境中Hg2+污染的重组工程菌及其应用 |
-
2012
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5965796A (en) * | 1995-04-21 | 1999-10-12 | University Of Georgia Research Foundation Inc. | Metal resistance sequences and transgenic plants |
| WO2002048335A2 (en) * | 2000-12-13 | 2002-06-20 | University Of Georgia Research Foundation, Inc. | Metal resistant plants and use for phytoremediation |
| CN1614024A (zh) * | 2003-11-06 | 2005-05-11 | 中国科学院上海生命科学研究院 | 降解有机汞污染的转基因烟草 |
| CN101921724A (zh) * | 2010-05-07 | 2010-12-22 | 南京师范大学 | 一种用于监测环境中Hg2+污染的重组工程菌及其应用 |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103951089A (zh) * | 2014-05-12 | 2014-07-30 | 江南大学 | 酿酒酵母基因缺失菌株在清除环境水质中重金属的应用 |
| CN106964647A (zh) * | 2017-06-06 | 2017-07-21 | 临沂大学 | 一种利用复合菌剂修复铅污染土壤的方法 |
| CN114686411A (zh) * | 2020-12-29 | 2022-07-01 | 上海科技大学 | 一种工程菌的混合物、用于感应和吸附重金属污染物的试剂盒和处理方法 |
| CN114686411B (zh) * | 2020-12-29 | 2023-05-05 | 上海科技大学 | 一种工程菌的混合物、用于感应和吸附重金属污染物的试剂盒和处理方法 |
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| CN102604901B (zh) | 2013-06-12 |
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