CN114990044B - 一种降解高氯酸盐的抗辐射细菌的制备及其应用 - Google Patents
一种降解高氯酸盐的抗辐射细菌的制备及其应用 Download PDFInfo
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Abstract
本发明公开了一种降解高氯酸盐的抗辐射细菌的制备及其应用。将源自Dechloromonas agitata CKB的降解高氯酸盐的基因cld,pcrA, pcrB,pcrC,pcrD融入耐辐射奇球菌Deinococcus radiodurans R1基因组,构建出新的基因工程菌DR‑agitata。所得菌株兼具辐射抗性和高氯酸盐降解能力,对于改良含有高浓度高氯酸盐的高辐射区和火星表面等土壤具有重要的应用前景。
Description
技术领域
本发明属于基因工程领域,涉及一种降解高氯酸盐的抗辐射细菌的制备及其应用。
背景技术
高氯酸盐可用作火箭燃料、烟火中的氧化剂和安全气囊中的***物,是自然环境中的主要无机污染物之一,广泛存在于地表水、地下水和土壤中,在许多食物和饮用水中均有发现高氯酸盐的存在。高氯酸盐作为一种内分泌干扰物,由于高氯酸根离子半径及电荷与碘离子非常接近,当其进入人体后,会与碘离子竞争进入人体的甲状腺,干扰甲状腺对碘的吸收,影响甲状腺激素T3及T4的合成和释放,导致甲状腺功能退化,影响人体发育,尤其对胚胎、孕妇、哺乳期妇女和少年儿童的影响更为显著。由于高氯酸根的正四面体结构,使其具有高度的化学稳定性和良好的迁移性,这些因素决定了其去除的困难性。
目前降解高氯酸盐的工艺包括物理法(吸附、离子交换和膜分离等技术)、化学法(金属纳米颗粒催化的氧化还原反应和电化学还原等技术)和生物法(植物修复和微生物修复等技术),其中微生物修复是目前最具发展潜力的高氯酸盐降解手段。高氯酸盐还原菌广泛存在于自然环境中,主要菌属有梭菌属(Clostridia)、偶氮螺旋体属(Azospira)[或称脱氯螺体属(Dechlorosoma)]、脱氯单胞菌属(Dechloromonas)等。高氯酸盐还原菌降解高氯酸盐的途径为:通过高氯酸盐还原酶(Pcr)将高氯酸盐首先降解为氯酸盐,然后进一步降解为亚氯酸盐,最后通过亚氯酸盐歧化酶(Cld)将亚氯酸盐降解为氯离子并产生氧气。其中高氯酸盐还原酶由操纵子pcrABCD编码,pcrA与pcrB产生的PcrAB蛋白复合体为高氯酸盐还原酶的有效催化部分,pcrC产生的PcrC蛋白负责电子传递,pcrD产生的PcrD蛋白负责PcrAB蛋白复合体的组装与修饰,而亚氯酸盐歧化酶由基因cld编码。
火星探测器在火星表面土壤检测出约0.6 %的高氯酸盐,高浓度的高氯酸盐对生物存在很大的毒害作用,这对于生命体在火星上生存是个极大的挑战。由于火星的环境极端,存在极强的辐射,而绝大多数的高氯酸盐还原菌的抗辐射性能较差,难以在火星生存,因此无法对于未来火星环境的改良提供直接有效的帮助。
发明内容
针对目前能降解高氯酸盐的抗辐射细菌的缺少,以及未来对于这种细菌可能存在的需求,本发明深入挖掘高氯酸盐还原菌和抗辐射细菌的特性,提供了一种降解高氯酸盐的抗辐射细菌的制备及其应用。
一种降解高氯酸盐的抗辐射细菌的制备方法,
1)提取D. agitata CKB的基因组;
2)以D. agitata CKB的基因组DNA为模板,参考pRADK载体的序列,设计出一对用于同源重组的引物,并利用该引物对扩增得到含cld、pcrA、pcrB、pcrC和pcrD的目标脱氯基因;
3)对pRADK载体进行线性化;
4)将目标脱氯基因重组至线性化后的pRADK载体上;
5)将启动子P1(如SEQ ID NO.6所示)***至基因cld前;将启动子P2(如SEQ IDNO.7所示)***至基因cld与pcrA之间;将启动子P3(如SEQ ID NO.8所示)***至基因pcrA与pcrB之间;将启动子P4(如SEQ ID NO.9所示)***至基因pcrB与pcrC之间;将启动子P5(如SEQ ID NO.10所示)***至基因pcrC与pcrD之间;
6)获得质粒pRADP1-P2-P3-P4-P5-agitata,并转入耐辐射奇球菌D. radioduransR1的感受态,得到一种降解高氯酸盐的抗辐射细菌称为DR-agitata。
一种降解高氯酸盐的抗辐射细菌,耐辐射奇球菌D. radiodurans R1的感受态通过吸收***了脱氯基因和各个启动子的质粒pRADP1-P2-P3-P4-P5-agitata,从而同时具有降解高氯酸盐和抗辐射能力。
所述的抗辐射细菌的应用,用于高氯酸盐降解的体系为:5 mg/L~100 mg/L ClO4 -,50 mg/L~1000 mg/L乙酸钠,40 μg/mL氯高铁血红素。
所述的应用,高氯酸盐降解体系的温度范围为16 ℃~42 ℃。
最适反应温度为30 ℃。
所述的抗辐射细菌,用于降解高辐射区的高浓度高氯酸盐。
所述的抗辐射细菌,未来可用于改良火星表面土壤。
与现有技术相比,本发明具有如下有益效果:
(1)绝大多数高氯酸盐还原菌的扩增需要严格的厌氧条件,且扩增时间较长,而本发明所制备的一种能降解高氯酸盐的抗辐射细菌只需要常规需氧培养,且扩增时间明显快于现有的绝大多数高氯酸盐还原菌。
(2)绝大多数高氯酸盐还原菌不抗辐射,无法在辐射环境下生存和降解高氯酸盐,而本发明所制备的一种能降解高氯酸盐的抗辐射细菌能够在辐射环境下完成对高氯酸盐的降解,对未来改良含有高浓度高氯酸盐的高辐射区和火星表面等土壤提供了一种可能的选择。
附图说明
图1是脱氯基因在Dechloromonas agitata CKB基因组里的位置分布示意图。
图2是P1,P2,P3,P4,P5启动子在脱氯基因间的***位置示意图。
图3是RT-qPCR的琼脂糖凝胶电泳结果,泳道1~7依次为marker、cld、pcrA、pcrB、 pcrC、pcrD、DR_1343。
图4是脱氯基因相较于Dr_1343的相对表达量。
图5是高氯酸盐与亚甲基蓝反应形成的络合物在波长655 nm处的吸光值与ClO4 -浓度关系的标准曲线图。
图6是经过降解体系处理后ClO4 -的剩余含量。
图7是Deinococcus radiodurans R1与DR-agitata的抗UV辐照表型。
具体实施方式
以下结合附图和具体实施例对本发明做进一步的阐述。
实施例1:一种含有降解高氯酸盐基因的穿梭表达载体构建
(1)利用R2A液体培养基活化Dechloromonas agitata CKB(购自德国菌种库DSMZ,编号:DSM 13637),然后使用天根生化公司的细菌基因组DNA提取试剂盒(DP 302-02)提取D. agitata CKB的基因组,并使用Thermo Fisher公司的NanoDrop 1000测定其浓度和纯度。
(2)由于NCBI数据库中缺少D. agitata CKB的基因组序列文件,而仅有cld、pcrA、 pcrB、pcrC(部分)和pcrD的基因序列(以下将上述五个基因统称为“脱氯基因”),因此根据脱氯基因的序列及其在D. agitata CKB基因组里的位置分布示意图(如图1所示),以及pRADK载体的序列,设计出一对可用于同源重组的引物,上游引物agitata-F为:5’-GACGGGTCCAGGGAGAGTGTatgcgcaagtctacaggattgc-3’(SEQ ID NO.11,大写字母为pRADK载体上的同源片段),下游引物agitata-R为:5’-CTGCAGGTCGAATCGGATCCttattctcctagctcttctttcaattctcttaaattagc-3’(SEQ ID NO.12,大写字母为pRADK载体上的同源片段),以D. agitata CKB的基因组DNA为模板,使用TaKaRa公司的PrimeSTAR® HS DNA Polymerasewith GC Buffer对目的片段进行扩增,PCR扩增体系及条件按照该试剂说明书设置,经琼脂糖凝胶电泳检测条带单一后,使用Promega公司的Wizard® SV Gel and PCR Clean-UpSystem试剂盒对PCR产物进行纯化回收。
(3)利用PCR扩增的方法对pRADK载体进行线性化,设计出一对线性化的引物,上游引物save-pRADK-F为:ACACTCTCCCTGGACCCGTCACTAG(SEQ ID NO.13),下游引物save-pRADK-R为:GGATCCGATTCGACCTGCAGGCATG(SEQ ID NO.14),以pRADK质粒DNA为模板,使用TaKaRa公司的PrimeSTAR® HS DNA Polymerase with GC Buffer对目的片段进行扩增,PCR扩增体系及条件按照该试剂说明书设置,经琼脂糖凝胶电泳检测条带单一后,使用Promega公司的Wizard® SV Gel and PCR Clean-Up System试剂盒对PCR产物进行纯化回收。
(4)使用诺唯赞公司的ClonExpress II One Step Cloning重组酶***将脱氯基因的片段重组至线性化后的pRADK载体上,重组体系及条件按照该试剂说明书设置。
(5)将重组的产物转化至大肠杆菌DH5α感受态细胞(全式金公司)中,涂布在含有100 μg/mL氨苄青霉素的固体LB培养基上,37 ℃倒置培养过夜。
(6)挑选若干单菌落于5 ml含100 μg/mL氨苄青霉素的液体LB培养基中,37 ℃220 rpm震荡培养10 h左右。使用Axygen公司的AxyPrep质粒DNA小量试剂盒抽提质粒DNA,并交由生工公司的KBseq进行全质粒测序,经过BLAST序列比对正确后,将质粒放至-20 ℃保存。
实施例2:耐辐射奇球菌通用强启动子的***
(1)设计引物将构建成功的pRADK-agitata载体线性化,上游引物save-pRADK-agitata-F为:AAGCTTCTAGAATTCGAGCTCCCGGGTAC(SEQ ID NO.15),下游引物save-pRADK-agitata-R为:ATGCGCAAGTCTACAGGATTGCTGCTAAC(SEQ ID NO.16),以pRADK-agitata质粒DNA为模板,使用TaKaRa公司的PrimeSTAR® HS DNA Polymerase with GC Buffer对目的片段进行扩增,PCR扩增体系及条件按照该试剂说明书设置,经琼脂糖凝胶电泳检测条带单一后,使用Promega公司的Wizard® SV Gel and PCR Clean-Up System试剂盒对PCR产物进行纯化回收。
(2)根据启动子P1的序列,设计出一对能够与pRADK-agitata载体进行同源重组的引物,上游引物P1-F为:AGCTCGAATTCTAGAAGCTTccgcccccagatcctgaccg(SEQ ID NO.17,大写字母为pRADK-agitata载体上的同源片段),下游引物P1-R为:AATCCTGTAGACTTGCGCATacactctccctggacccgtcactagaac(SEQ ID NO.18,大写字母为pRADK-agitata载体上的同源片段),以启动子P1的PCR产物为模板,使用TaKaRa公司的PrimeSTAR® HS DNA Polymerasewith GC Buffer对目的片段进行扩增,PCR扩增体系及条件按照该试剂说明书设置,经琼脂糖凝胶电泳检测条带单一后,使用Promega公司的Wizard® SV Gel and PCR Clean-UpSystem试剂盒对PCR产物进行纯化回收。
(3)使用诺唯赞公司的ClonExpress II One Step Cloning重组酶***将脱氯基因的片段重组至线性化后的pRADK载体上,重组体系及条件按照该试剂说明书设置。
(4)将重组的产物转化至大肠杆菌DH5α感受态细胞(全式金公司)中,涂布在含有100 μg/mL氨苄青霉素的固体LB培养基上,37 ℃倒置培养过夜。
(5)挑选若干单菌落于5 ml含100 μg/mL氨苄青霉素的液体LB培养基中,37 ℃220 rpm震荡培养10 h。使用Axygen公司的AxyPrep质粒DNA小量试剂盒抽提质粒DNA,并交由擎科生物公司进行测序,经过序列比对正确后,将质粒放至-20 ℃保存。
(6)效仿上述(1)~(5)的方法,将启动子P2***至基因cld与pcrA之间,载体线性化的上游引物save-pRADP1-F为:TTAGCGTCCCATGGACAACGTATTCACG(SEQ ID NO.19),载体线性化的下游引物save-pRADP1-R为:ATGGCACGACTGAGTCGCAGAG(SEQ ID NO.20),目的片段P2的上游引物P2-F为:CGTTGTCCATGGGACGCTAAagaccatcggtgtggacggc(SEQ ID NO.21,大写字母为载体上的同源片段),目的片段P2的下游引物P2-R为:CTGCGACTCAGTCGTGCCATgcttggtacctcctgcattgaagtctg(SEQ ID NO.22,大写字母为载体上的同源片段)。
(7)效仿上述(1)~(5)的方法,将启动子P3***至基因pcrA与pcrB之间,载体线性化的上游引物save-pRADP1-P2-F为:CTAGAGACTGATCGGAGTTGCGCCGATATAGC(SEQ IDNO.23),载体线性化的下游引物save-pRADP1-P2-R为:ATGTCAAATATGACGAAGTCGCCTAAACGCCAATTG(SEQ ID NO.24),目的片段P3的上游引物P3-F为:CAACTCCGATCAGTCTCTAGctaaggaaagcctccacatgcc(SEQ ID NO.25,大写字母为载体上的同源片段),目的片段P3的下游引物P3-R为:GACTTCGTCATATTTGACATcagaatagtgataatccaacccatgttcg(SEQ ID NO.26,大写字母为载体上的同源片段)。
(8)效仿上述(1)~(5)的方法,将启动子P4***至基因pcrB与pcrC之间,载体线性化的上游引物save-pRADP1-P2-P3-F为:TCAGGTCATAGGCGAAATCATCATGTCCGC(SEQ IDNO.27),载体线性化的下游引物save-pRADP1-P2-P3-R为:ATGAAAAGAATCGTTGCTCTGTTAAGCACGTTGC(SEQ ID NO.28),目的片段P4的上游引物P4-F为:TGATTTCGCCTATGACCTGAagcccggcataaacctcaccg(SEQ ID NO.29,大写字母为载体上的同源片段),目的片段P4的下游引物P4-R为:AGAGCAACGATTCTTTTCATgtcaaacctcctgtgattgaaatggaccttcagg(SEQ ID NO.30,大写字母为载体上的同源片段)。
(9)效仿上述(1)~(5)的方法,将启动子P5***至基因pcrC与pcrD之间,载体线性化的上游引物save-pRADP1-P2-P3-P4-F为:TTACTCCGGTTCGGGCGCGTCTTC(SEQ ID NO.31),载体线性化的下游引物save-pRADP1-P2-P3-P4-R为:ATGAATAGTATTACCGACGATCGACTCGTCTTGGC(SEQ ID NO.32),目的片段P5的上游引物P5-F为:ACGCGCCCGAACCGGAGTAAaacgctcaggcctgcacccttg(SEQ ID NO.33,大写字母为载体上的同源片段),目的片段P5的下游引物P5-R为:TCGTCGGTAATACTATTCATgatcccatgatgcaccatcccagagaaac(SEQ ID NO.34,大写字母为载体上的同源片段)。
(10)最终获得质粒pRADP1-P2-P3-P4-P5-agitata(各个启动子在脱氯基因间的***位置示意图如图2所示),并转入耐辐射奇球菌D. radiodurans R1(购自美国模式菌种保藏中心,编号:ATCC 13939)的感受态,涂布在含有4 μg/ mL 氯霉素的固体TGY培养基上,30℃倒置培养5 d左右,长出粉红色单菌落。挑选若干单菌落于5 ml含4 μg/ mL 氯霉素的液体TGY培养基中,30 ℃ 200 rpm震荡培养2 d左右,将菌液与50%甘油混合,放至-80 ℃保存。改造后的耐辐射奇球菌称为DR-agitata。
实施例3:DR-agitata中脱氯基因的相对表达量的测定
(1)DR-agitata总RNA的提取:将DR-agitata于5 ml含4 μg/ mL 氯霉素的液体TGY培养基中,30 ℃ 200 rpm震荡培养至OD600 2.0左右,使用全式金公司的TransZol Up PlusRNA Kit试剂盒提取DR-agitata的总RNA,将RNA至于-80 ℃保存。
(2)RT-PCR的引物设计:根据以往相关文献的经验,Dr_1343能够稳定表达,且表达量高,所以选择Dr_1343作为内参基因,相关引物设计如下:RT-Dr_1343-F:ccaccggcatcttcaccagcc(SEQ ID NO.35),RT-Dr_1343-R:ggtcatgatggccttctcgatgccg(SEQ ID NO.36);RT-cld-F:cacctacttcgaaaccgccgacctc(SEQ ID NO.37),RT-cld-R:ttagcgtcccatggacaacgtattcacg(SEQ ID NO.38);RT-pcrA-F:atggcacgactgagtcgcagag(SEQ ID NO.39),RT-pcrA-R:cagttgatcaggtgcgctccgc(SEQ ID NO.40);RT-pcrB-F:atgtcaaatatgacgaagtcgcctaaacgccaattg(SEQ ID NO.41),RT-pcrB-R:ctgccagtttcttggataccccaaacctg(SEQ ID NO.42);RT-pcrC-F:atgaaaagaatcgttgctctgttaagcacgttgc(SEQ ID NO.43),RT-pcrC-R:cagccttaacgttgggtttcagggactc(SEQ IDNO.44);RT-pcrD-F:gaaacaggcacaggcccatctcgaag(SEQ ID NO.45),RT-pcrD-R:gcaagcgcaaagctttctgcaaactc(SEQ ID NO.46)。
(3)Real Time One Step RT-PCR:使用Takara公司的One Step TB Green®PrimeScript™ PLUS RT-PCR Kit (Perfect Real Time)试剂盒,进行DR-agitata的RT-qPCR,反应体系及条件按照该试剂说明书设置,使用Agilent公司的Mx3005P测量荧光值。RT-qPCR的琼脂糖凝胶电泳结果如图3所示,脱氯基因相较于Dr_1343的相对表达量如图4所示。图3与图4的结果表明,脱氯基因均有稳定的表达,且相较于稳定高表达的Dr_1343有着不低的表达量。
实施例4:DR-agitata降解高氯酸盐的处理体系
(1)将D. radiodurans R1和DR-agitata各置于4管5 mL的TGY培养基中活化,30℃ 200 rpm震荡培养至OD600 1.0左右,并分别做如下四种处理:
(2)①添加5 mg/L ClO4 -(由高氯酸镁提供ClO4 -),50 mg/L乙酸钠(提供过量的电子供体),40 μg/mL氯高铁血红素(亚氯酸盐歧化酶Cld为氯高铁血红素依赖蛋白);②添加10 mg/L ClO4 -,100 mg/L乙酸钠,40 μg/mL氯高铁血红素;③添加50 mg/L ClO4 -,500 mg/L乙酸钠,40 μg/mL氯高铁血红素;④添加100 mg/L ClO4 -,1000 mg/L乙酸钠,40 μg/mL氯高铁血红素。
(3)将各组试管置于Thermo Fisher公司的Oxoid™ AnaeroJar™ 2.5 L厌氧罐内,并加入日本三菱瓦斯化学株式会社的厌氧产气袋AnaeroPackTM-Anaero,密封后置于30℃ 200 rpm震荡培养3 d左右后取出,于有氧条件下30 ℃ 200 rpm震荡培养至饱和。
实施例5:ClO4 -含量的测定
(1)ClO4 -标准曲线的绘制:分别配置10 mL含0.25 mg/L,0.5 mg/L,0.75 mg/L,1.0mg/L ClO4 -的标准溶液,并分别将10 mL标准溶液、0.5 mL 50mM硫酸、1 mL亚甲基蓝、10 mL二氯甲烷加入分液漏斗中,剧烈摇晃30 s后萃取出下层溶液(含ClO4 -与亚甲基蓝反应产生的络合物),再加入0.5 g无水硫酸钠剧烈摇晃,待溶液澄清后,使用Eppendorf公司的BioSpectrometer basic分光光度计于波长655nm处测量吸光值,最后绘制出ClO4 -的标准曲线,如图5所示。
(2)将处理后的菌液取出于12000 rpm离心1 min,吸取上清后通过C18离子色谱预处理柱去除上清中的有机物,分别经过适当的比例稀释后得到8管10 mL的预处理溶液。
(3)效仿(1)中的方法,分别测量出8管预处理溶液在波长655nm处的吸光值,经过计算得出各个处理组的菌液中剩余的ClO4 -含量。如图6所示,D. radiodurans R1四组的结果分别为:4.7 mg/L,9.7 mg/L,46.3 mg/L,96.9 mg/L;DR-agitata四组的结果分别为:4.1mg/L,8.8 mg/L,42.8 mg/L,88.6 mg/L。比较结果可以看出,DR-agitata组减少的ClO4 -含量均大于D. radiodurans R1组,可见DR-agitata对ClO4 -具有一定程度的降解能力。
实施例6:DR-agitata的抗UV辐照表型
将D. radiodurans R1和DR-agitata置于5 mL的TGY培养基中活化,30 ℃ 200rpm震荡培养至OD600 1.0左右,各吸取1 mL菌液于12000 rpm离心1 min后弃上清,用1 mL 1×PBS溶液重悬菌体,于12000 rpm离心1 min后弃上清,再加入100 μL 1×PBS溶液重悬菌体,并用1×PBS溶液依次将菌液稀释101、102、103、104、105、106倍,吸取5 μL菌液点于TGY固体培养基上,依次经过0、100、200、400、800 J/m2的UV辐照处理后于30 ℃倒置培养2 d左右,表型结果如图7所示。结果表明,DR-agitata对UV辐照有着极强的抗性,即便是经过800J/m2的强UV辐照后仍有很高的存活率。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明的保护范围应以所附权利要求为准。
序列表
<110> 浙江大学
<120> 一种降解高氯酸盐的抗辐射细菌的制备及其应用
<141> 2022-06-28
<160> 46
<170> SIPOSequenceListing 1.0
<210> 1
<211> 834
<212> DNA
<213> 活跃脱氯单胞菌(Dechloromonas agitata CKB)
<400> 1
atgcgcaagt ctacaggatt gctgctaacg ttcatggcac tactgtcggt gggcagttcg 60
caggcgcaac aagccaacat ggatgcgaag ccgccaatgg ccatgcccga catgaccaaa 120
attcttaccg cacccggggt tttcgggaat ttctccacct acaaggtgcg tcctgactac 180
tacaagctgt ccatggccga acgcaagggg gcggctgccg aggtggtcgc agttgttgag 240
aaatataagg acaaggtcaa ggctgaagcc tacctgacac ggggattcga ggcacagagc 300
gactttttcc tgcgaatcca ctcttacgac atggctgcca cccaggcatt tttggttgac 360
ttccgtgcca cacgctttgg tatgaatgcc gaggtgaccg agaacctggt cggcatgacg 420
aaggacttga actacatcac aaaagataaa tcgccaaatc tcaacgccgg tctgactggc 480
gccacctata gggacgcaac accccgttac gccttcgtga tacccgtgaa gaaaaatgcg 540
gactggtgga atctgaccga cgagcaacgt ctcaaggaaa tggagaccca taccctgccg 600
acgctggcca atctggtcaa tgtaaaacgg aaactttatc actcgaccgg cctcgacgat 660
accgatttca tcacctactt cgaaaccgcc gacctcggtg cattcaacaa tctgatgctg 720
gcgcttgcca aggtccccga gaacaaatac cacgttcgct ggggcagtcc caccgtactg 780
ggcacgattc agtcgttcga cagcgtcgtg aatacgttgt ccatgggacg ctaa 834
<210> 2
<211> 2784
<212> DNA
<213> 活跃脱氯单胞菌(Dechloromonas agitata CKB)
<400> 2
atggcacgac tgagtcgcag agattttttg aaggcatccg ccgccactct gctgggtaat 60
tcacttacct tcaaaacatt agcggccacc atggatttat ccggcgcctt cgaatattcg 120
ggctgggaaa attttcatcg caaccagtgg tcgtgggaca agaaaactcg cggagcgcac 180
ctgatcaact gtactggggc ttgcccgcac ttcgtttata cgaaagatgg tgtggtcatt 240
cgcgaggagc agtccaagga cattccgccg atgccgaata tcccggaatt gaatccccgg 300
gggtgcaaca agggggagtg cgcacaccac tacatgtatg gtccgcatcg cctgaaatat 360
cctttgatcc gggttggcga acgcggggag ggcaagtggc ggcgtgccac gtgggaggag 420
gcactagacc ttatctcaga caagataatc gacacgatca agaatcactc tcccgattgc 480
atcagtgtct actcacctct tccgggcaca gcaccagtat cgttctctgc tggacacagg 540
tttgcgcact atatcggcgc ccatacacat accttctttg actggtacag tgaccatccc 600
actggtcaga cacagacgtg cggtgtccag ggcgactcag cggagtgctc cgactggttc 660
aactcaaaat acataatcct ctggggggca aaccccactc agactcgtat tccagatgcg 720
catttcctgt cagaggcgca gttgaacggc gcgaaggtag tcagcatctc cccggatttc 780
aactcttcta ccatcaaggc ggaccgttgg attcaccctc tgcccggtac tgatggcgcc 840
ctcgcactgg ccatggcgca tgtgatcatc aaggaaaagc tctacgacgc ccacaacctc 900
aaggaacaaa ctgatctacc gtatttgatc agaagggaca ccaagcggtt cttacgtgaa 960
gctgatgtgg ttgccggggg atccaaagat aagttctata tttgggacag caagactggc 1020
aagccggtaa tcacgaaagg ttcctggggc gaccaaccag aacaaaaagc tccgcctgtc 1080
gcatttatgg gaaggaacac ccatacattc ccaaagggct atatcgccct ggagaatctg 1140
gaccccgccc tggaaggcaa atttcaggtc aagttgcagg atggaaatac agttgaagtc 1200
agaccagtat tcgaaattct gaaatcgcgc atcgaggccg acaacaatat cgccaaggca 1260
gcgaaaatca ccggggtgcc ggcaaagacc atcattgaag tggccagaga gtatgcaaca 1320
acgcagccgg cgatgatcat ttgcggtggc ggcactatgc actggtatta cagcgacgtg 1380
ctgttacgtg cgatgcatct gctaaccgca ctcgttggca gcgaaggcaa gaatggcggc 1440
gggatgaatc attacatcgg acagtggaag cctgtcttcc tgccgggtgt ggctgctctc 1500
gccttccccg aaggccctgc gaacgaacgg tcctgccaga cgacgatctg gacttacatc 1560
catgccgaag ttaacgacga gatggcaaat gtcgggatcg ataccgacaa gtatctgatg 1620
cacgcgatcg atacgcgcca aatgccgaat taccctcgag acggcagaga tcccaaagta 1680
tttatcgtct atcgcggcaa ctggctaaac caagccaagg gacagaaata tgtcttgcgc 1740
aatctctggc ccaagctgga tttgattgtt gacatcaata tccgcatgga ctcaacggcg 1800
ctgtattcgg atgttgtgct gccttccgct cactggtacg aaaagctcga cctcaacgtc 1860
accgccgagc acacctatat caacatgacg gaaccggcaa tcaagccgat gtgggaatcc 1920
aagaccgatt ggcagatttt cctcgcactc gccaaacgcg ttgaaatgtc agccaagcgc 1980
aagagttttg agaggtttta cgacgaacaa ttcaagtggg cacgcgacct gacgaacctg 2040
tggaaccaga tgaccatgga cggcaagttg gccgaggatg aggctgctgc ccaatacatt 2100
ctggacactg ccccccattc gaaaggcatt acgctccaga tgctgaggga aaaaggtgag 2160
cgcttcaagg cgaattggac ttcgccgatg aaggaaggcg tgccctatac cccgttccag 2220
aattacattg tcgacaagaa gccttggccg acactcacgg ggcgccagca gttctatctg 2280
gaccatgaag tgttcttcga aatgggtgtc gaattgccga cctacaaggc tccggtcgat 2340
gcggacaaat tccccttccg cttcaactcg ccacacagtc gtcattcgat ccactcgacc 2400
ttcaaggata gtgtgctgat gctacggctt cagcgcggcg gtccctgcgt agaaatctcg 2460
ccgatcgatg ccacggcaat tggagtcaag gacaacgact gggtagaaat ctggaatagc 2520
cacggcaagg tgatctgccg ggccaagatc cgtgccggtg agcaacgtgg ccgcgtctcc 2580
atgtggcaca ccccggaact gtacatggat ctcctcgaag gcagcacgca aagcgtatgc 2640
cccgtccgca tcacgccaac gcacttggtg ggcaactacg gacatttggt gtttcgcccg 2700
aactactacg gaccggccgg cagccagcga gacgttcgtg tcgatgtgaa acgctatatc 2760
ggcgcaactc cgatcagtct ctag 2784
<210> 3
<211> 1002
<212> DNA
<213> 活跃脱氯单胞菌(Dechloromonas agitata CKB)
<400> 3
atgtcaaata tgacgaagtc gcctaaacgc caattggcat atgttgccga cctgaataag 60
tgcattggct gccagacatg cactgtcgca tgcaagactc tttggactag tggacctgga 120
caggactaca tgtattggcg gaatgtcgaa accgccccag gtttggggta tccaagaaac 180
tggcagagca agggcggtgg atacaaggat ggagtcctgc agaaagggaa gattccgccg 240
atgatcgatt atggcgttcc cttcgaattc gactacgccg gtcggctttt cgaagggaaa 300
aaggaaagag cgcgaccgag tccgacgcct cggtatgctc ccaattggga tgaggatcag 360
ggagcgggcg aatatcccaa caactccttt ttctatgtgc cgcggatgtg caaccactgc 420
gccaagccgg catgtcttga agcctgcccg aatgaagcga tatacaagcg cgagcaagac 480
ggcctggtgg tgatccatca ggagaagtgc aaaggggctc aggcatgtat ccagtcctgc 540
ccatatgcca aaccatactt taatgctcag gtcaataaag ccaacaagtg catcggttgc 600
ttcccgcgga ttgaaaaagg ggttgcaccg gcatgcgttg ctgagtgtgc gggaagagcc 660
atgcatgtcg gcttcatcga tgaccaagaa agctccgtgt tcaagctggt caagcggttc 720
ggcgtggcac tgcctttgca ccccgagtac ggcaccgaac ccaacgtgtt ctatgtccct 780
cccgttctcg gaccgcgcgt agaaatgccg aatggcgaac ataccgccga cccgaaaatt 840
tcgatgactc agcttgaaca gttgtttggc aagcaggtcc gcgaggtttt gaagacgctg 900
caggccgaac gcgagaaaaa gatcaagaac cagccgtctg aactgatgga catcctgatc 960
ggccgacgca gtgcggacat gatgatttcg cctatgacct ga 1002
<210> 4
<211> 717
<212> DNA
<213> 活跃脱氯单胞菌(Dechloromonas agitata CKB)
<400> 4
atgaaaagaa tcgttgctct gttaagcacg ttgctcgtta ccgcgatgcc gagcatgggc 60
gtagcccaac agaccgaata tttaggcttt cgcgcatgca caaagtgtca cgatgcccag 120
ggtgatacct ggcggacatc ggcacatgcc aaagcctttg agtccctgaa acccaacgtt 180
aaggctgata cgaaaagaaa agccaaattg gatccggcca aggactacac gcaagatcaa 240
gactgcattg gctgccatac caccggctac ggactgcctg ggggatttgc catgaatgct 300
tcgccggatg acatgaaact ggttgttggc gttacatgcg agtcatgcca tggagcagga 360
ggaaagtatc gcaacctgca tggagaggcg agtgatcggc tcaagagcca aggggccatg 420
accgaaagaa agctgttggt ggatgcaggt caaaacttcg acatggaaaa agcttgtgta 480
cgttgccacc tgaattacga gggaggaacg acaactcacc atcaaccgaa accaccctac 540
accccattta cgccgaatgt cgatgcgaaa tatcgctttg actacatgaa atcagtgatg 600
gcttcggggc agaaaaaccc gattcatacg catttcaaac tccggggcgt atttaaaggc 660
ggcccgatac ccgatgtaag gacaaagctt caggaagacg cgcccgaacc ggagtaa 717
<210> 5
<211> 675
<212> DNA
<213> 活跃脱氯单胞菌(Dechloromonas agitata CKB)
<400> 5
atgaatagta ttaccgacga tcgactcgtc ttggcggccg ataaagcgtt atgccgaagc 60
cacatttatt cattgttggc cagtggattt ggatatcccg acgaatctgg ttatcaggga 120
ttttcggacg gcaagtttat tgatgaaatc cgtcaggaac tcggggtatg ttggggtgaa 180
ctcttggagc attttaatga ggtacttgct cctgggttga aacttgcctg ctcgcgcgaa 240
gtattcgaat cacagttctt gagtgcattc gagaccaata tgccatcacc tagcgcctcc 300
ttgaatgaag gtgttcacat atttaaatcg gaccgaccaa atttattgct cgaactgaag 360
ggtttttaca gcaacttcgg cttgcaggtc gatagcaaag gcaatgagct tgaagacact 420
ctgacagcag aacttgattt catgcagttt cttgcgttga aacaggcaca ggcccatctc 480
gaaggcatgt cggctgatgc ctacaagttg gcgcagaagg attttctgga gcggcaccta 540
tgtgcttggt tgccattggt gcggagggaa atcgccgaaa aggtgactac cccattcttc 600
gtcactcttg ctgagtttgc agaaagcttt gcgcttgcta atttaagaga attgaaagaa 660
gagctaggag aataa 675
<210> 6
<211> 259
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ccgcccccag atcctgaccg tgcccaagat tgatgtaacc ggggcggccc agggcagcaa 60
gtcccaatcc cagacgggga gcgtcagcag gtagcagggt gttgcgcata agccagtatg 120
gcctgcctac ctggctgtgc cacgcgcgat cacgtctaga tttctgccgg ccacgtcaga 180
attccgacag gaggtgtgtg aataattagg tcacttaaag aaacccttca tgttctagtg 240
acgggtccag ggagagtgt 259
<210> 7
<211> 235
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
agaccatcgg tgtggacggc gggctgtacc cgcactgagc acgggtggct gaacataggt 60
gctgggcaca gatacctgcc tgggaagcag ctggtacgac gtctccgtca ggcatctgca 120
caccccgcag gcacagcacc ggcacggtct cgaattgaac cctgtccact ctggcgccgc 180
ttcggcgcgc cacacgtgta gactggcgca gacttcaatg caggaggtac caagc 235
<210> 8
<211> 335
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ctaaggaaag cctccacatg ccgcatggcg gcggaatgag gtcatgctac ctgcccactc 60
gcaacaggct cgttcggcgg taagacaaat gcagttacgc tacgtcgtgc catcagctac 120
gcgtcagaga tgaggcaatc agactcatct ctgaaggcca gacttagctg gtatttgcgg 180
gacatccatc ccgtcttcgc acgcccttga gaaagtactg ctttgcacat tagaggccct 240
tggggagtct catgctgcgc tcaacaacga agcaaattca tcgttgagca aacgacttgg 300
aggaaacgaa catgggttgg attatcacta ttctg 335
<210> 9
<211> 380
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
agcccggcat aaacctcacc gccgggacag ttgatgtaca tctggatttc ctgctcgggg 60
ttctgggagt caagcagcag cagctgcgcg acgatggtat tggccatctg ggattcaatg 120
ggagtcccca cgaaaataat ccggtctttc agcagccgcg agtagatgtc atacatccgc 180
tcaccgcgtc cggtctgctc gatcacgtag ggaataacgc tcatgggcgg cattatcgca 240
tgggtcagtg ggaggaaggt gggcaagact gccctcaggt gtagaggaga ttgcggcact 300
tgccgaaaaa ttaatgcggc ccgttttccg cgcccagctc gtctaccctg aaggtccatt 360
tcaatcacag gaggtttgac 380
<210> 10
<211> 260
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
aacgctcagg cctgcaccct tgacctctcg gcgcatcaca ggcgcacacc ggtcaccttg 60
tccggaggca gccacctgcc gcctgttcag gatgagcgct atccgctggt catgggcaag 120
tacgactatt actggctgcg cctgaactga cggcaagccc agattgcttt atgcgggctg 180
ccggcttagg cccggacagg tcttaagatt cggtagggag ccgatttttc agtttctctg 240
ggatggtgca tcatgggatc 260
<210> 11
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gacgggtcca gggagagtgt atgcgcaagt ctacaggatt gc 42
<210> 12
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ctgcaggtcg aatcggatcc ttattctcct agctcttctt tcaattctct taaattagc 59
<210> 13
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
acactctccc tggacccgtc actag 25
<210> 14
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ggatccgatt cgacctgcag gcatg 25
<210> 15
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
aagcttctag aattcgagct cccgggtac 29
<210> 16
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
atgcgcaagt ctacaggatt gctgctaac 29
<210> 17
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
agctcgaatt ctagaagctt ccgcccccag atcctgaccg 40
<210> 18
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
aatcctgtag acttgcgcat acactctccc tggacccgtc actagaac 48
<210> 19
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
ttagcgtccc atggacaacg tattcacg 28
<210> 20
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
atggcacgac tgagtcgcag ag 22
<210> 21
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
cgttgtccat gggacgctaa agaccatcgg tgtggacggc 40
<210> 22
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ctgcgactca gtcgtgccat gcttggtacc tcctgcattg aagtctg 47
<210> 23
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
ctagagactg atcggagttg cgccgatata gc 32
<210> 24
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
atgtcaaata tgacgaagtc gcctaaacgc caattg 36
<210> 25
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
caactccgat cagtctctag ctaaggaaag cctccacatg cc 42
<210> 26
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
gacttcgtca tatttgacat cagaatagtg ataatccaac ccatgttcg 49
<210> 27
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
tcaggtcata ggcgaaatca tcatgtccgc 30
<210> 28
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
atgaaaagaa tcgttgctct gttaagcacg ttgc 34
<210> 29
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
tgatttcgcc tatgacctga agcccggcat aaacctcacc g 41
<210> 30
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
agagcaacga ttcttttcat gtcaaacctc ctgtgattga aatggacctt cagg 54
<210> 31
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
ttactccggt tcgggcgcgt cttc 24
<210> 32
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
atgaatagta ttaccgacga tcgactcgtc ttggc 35
<210> 33
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
acgcgcccga accggagtaa aacgctcagg cctgcaccct tg 42
<210> 34
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
tcgtcggtaa tactattcat gatcccatga tgcaccatcc cagagaaac 49
<210> 35
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
ccaccggcat cttcaccagc c 21
<210> 36
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
ggtcatgatg gccttctcga tgccg 25
<210> 37
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
cacctacttc gaaaccgccg acctc 25
<210> 38
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
ttagcgtccc atggacaacg tattcacg 28
<210> 39
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
atggcacgac tgagtcgcag ag 22
<210> 40
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
cagttgatca ggtgcgctcc gc 22
<210> 41
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
atgtcaaata tgacgaagtc gcctaaacgc caattg 36
<210> 42
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
ctgccagttt cttggatacc ccaaacctg 29
<210> 43
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
atgaaaagaa tcgttgctct gttaagcacg ttgc 34
<210> 44
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
cagccttaac gttgggtttc agggactc 28
<210> 45
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
gaaacaggca caggcccatc tcgaag 26
<210> 46
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
gcaagcgcaa agctttctgc aaactc 26
Claims (7)
1.一种降解高氯酸盐的抗辐射细菌的制备方法,其特征在于,
1)提取Dechloromonas agitata CKB的基因组,D. agitata CKB购自德国菌种库DSMZ,编号:DSM 13637;
2)以D. agitata CKB的基因组DNA为模板,参考pRADK载体的序列,设计出一对用于同源重组的引物,并利用该引物对扩增得到含cld、pcrA、pcrB、pcrC和pcrD的目标脱氯基因;
3)对pRADK载体进行线性化;
4)将目标脱氯基因重组至线性化后的pRADK载体上;
5)将如SEQ ID NO.6所示的启动子P1***至基因cld前;将如SEQ ID NO.7所示的启动子P2***至基因cld与pcrA之间;将如SEQ ID NO.8所示的启动子P3***至基因pcrA与pcrB之间;将如SEQ ID NO.9所示的启动子P4***至基因pcrB与pcrC之间;将启动子如SEQ IDNO.10所示的P5***至基因pcrC与pcrD之间;
6)获得质粒pRADP1-P2-P3-P4-P5-agitata,并转入购自美国模式菌种保藏中心,编号为ATCC 13939的耐辐射奇球菌Deinococcus radiodurans R1的感受态,得到一种降解高氯酸盐的抗辐射细菌称为Deinococcus radiodurans-agitata。
2. 根据权利要求1所述的制备方法得到一种降解高氯酸盐的抗辐射细菌,其特征在于,耐辐射奇球菌D. radiodurans R1的感受态通过吸收***了脱氯基因和各个启动子的质粒pRADP1-P2-P3-P4-P5-agitata,从而同时具有降解高氯酸盐和抗辐射能力。
3. 根据权利要求2所述的抗辐射细菌的应用,其特征在于,用于高氯酸盐降解的体系为:5 mg/L~100 mg/L ClO4 -,50 mg/L~1000 mg/L乙酸钠,40 μg/mL氯高铁血红素。
4. 根据权利要求3所述的应用,其特征在于,高氯酸盐降解体系的温度范围为16 ℃~42 ℃。
5. 根据权利要求3所述的应用,其特征在于,高氯酸盐降解体系的最适反应温度为30℃。
6.根据权利要求2所述的抗辐射细菌的应用,其特征在于,用于降解高辐射区的高浓度高氯酸盐。
7.根据权利要求2所述的抗辐射细菌的应用,其特征在于,用于降解高氯酸盐,从而改良火星表面土壤。
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Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2005058223A (ja) * | 2003-07-31 | 2005-03-10 | Institute Of Physical & Chemical Research | 高度好熱菌由来の新規な亜塩素酸ジスムターゼ及びその立体構造の使用 |
CN101671679A (zh) * | 2009-04-30 | 2010-03-17 | 浙江大学 | 一种耐辐射球菌抗逆相关基因及其应用 |
CN101768598A (zh) * | 2010-01-22 | 2010-07-07 | 浙江大学 | 一个定向突变及遗传改造的重组质粒及其应用 |
CN103703019A (zh) * | 2011-06-03 | 2014-04-02 | 加利福尼亚大学董事会 | 作为生物源硫化氢产生控制的氯氧阴离子的微生物代谢 |
CN103937814A (zh) * | 2014-04-16 | 2014-07-23 | 苏州大学 | 一种DNA分子及毕赤酵母重组质粒和高效表达耐辐射球菌PprI蛋白的毕赤酵母重组菌 |
KR20180130046A (ko) * | 2017-05-25 | 2018-12-06 | 한국원자력연구원 | 내산성을 갖는 재조합 미생물 제조방법 및 상기 방법으로 제조된 미생물 |
CN109439636A (zh) * | 2018-10-31 | 2019-03-08 | 浙江大学 | 一种耐辐射奇球菌dna甲基转移酶 |
CN113106048A (zh) * | 2021-04-13 | 2021-07-13 | 南华大学 | 耐辐射基因工程菌Deino-dsrA及其构建方法与应用 |
-
2022
- 2022-06-30 CN CN202210754642.5A patent/CN114990044B/zh active Active
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2005058223A (ja) * | 2003-07-31 | 2005-03-10 | Institute Of Physical & Chemical Research | 高度好熱菌由来の新規な亜塩素酸ジスムターゼ及びその立体構造の使用 |
CN101671679A (zh) * | 2009-04-30 | 2010-03-17 | 浙江大学 | 一种耐辐射球菌抗逆相关基因及其应用 |
CN101768598A (zh) * | 2010-01-22 | 2010-07-07 | 浙江大学 | 一个定向突变及遗传改造的重组质粒及其应用 |
CN103703019A (zh) * | 2011-06-03 | 2014-04-02 | 加利福尼亚大学董事会 | 作为生物源硫化氢产生控制的氯氧阴离子的微生物代谢 |
CN103937814A (zh) * | 2014-04-16 | 2014-07-23 | 苏州大学 | 一种DNA分子及毕赤酵母重组质粒和高效表达耐辐射球菌PprI蛋白的毕赤酵母重组菌 |
KR20180130046A (ko) * | 2017-05-25 | 2018-12-06 | 한국원자력연구원 | 내산성을 갖는 재조합 미생물 제조방법 및 상기 방법으로 제조된 미생물 |
CN109439636A (zh) * | 2018-10-31 | 2019-03-08 | 浙江大学 | 一种耐辐射奇球菌dna甲基转移酶 |
CN113106048A (zh) * | 2021-04-13 | 2021-07-13 | 南华大学 | 耐辐射基因工程菌Deino-dsrA及其构建方法与应用 |
Non-Patent Citations (2)
Title |
---|
Identification, Characterization, and Classification of Genes Encoding Perchlorate Reductase;Kelly S. Bender等;J Bacteriol.;第187卷(第15期);第5092页左栏第1段,图1,第5091页右栏第3段 * |
航天器AIT厂房环境中嗜极微生物的筛选与鉴定;张文德等;应用与环境生物学报;第26卷(第4期);第772页左栏第1段,第771页左栏第1段 * |
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