CN101684452A - 大宝山伯克霍尔德菌及其应用 - Google Patents
大宝山伯克霍尔德菌及其应用 Download PDFInfo
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- CN101684452A CN101684452A CN200910040518A CN200910040518A CN101684452A CN 101684452 A CN101684452 A CN 101684452A CN 200910040518 A CN200910040518 A CN 200910040518A CN 200910040518 A CN200910040518 A CN 200910040518A CN 101684452 A CN101684452 A CN 101684452A
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- China
- Prior art keywords
- burkholderia
- dabaoshanensis
- heavy metal
- bacterium colony
- acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种大宝山伯克霍尔德菌(Burkholderia dabaoshanensis GIMN1.004),该菌已在中国典型培养物保藏中心保藏,保藏日期为2009年5月10日,保藏编号为CCTCCNo.M209109。本发明大宝山伯克霍尔德菌具有耐重金属镉的能力,其耐重金属能力强,在对重金属污染的土壤修复中起着重要作用。
Description
技术领域
本发明属于微生物技术领域,特别是涉及一种能耐重金属镉的新的细菌大宝山伯克霍尔德菌及其应用。
背景技术
在珠江三角洲地区,重金属污染是土壤污染的重要构成,严重地威胁着农产品的安全性和人体的健康。土壤的重金属污染具有隐蔽性或潜伏性、不可逆性和长期性,造成的后果很严重,影响对象广泛,影响区域广、危害人口多。研发经济高效的污染土壤修复技术是改善我国环境质量的迫切要求,也是世界科技的研究热点。
重金属污染土壤的修复包括两种策略:一是改变重金属在土壤中的存在形态使其固定从而降低其在环境中的迁移性和生物可利用性(stabilization),二是从土壤中去除重金属(extraction)。微生物在重金属污染土壤的修复中具有重要的意义,筛选能够耐受重金属的微生物菌种资源成为目前重金属污染修复的热点之一。
发明内容
本发明的目的是提供一种能够耐受重金属镉的新的菌种——大宝山伯克霍尔德菌。
该菌命名为大宝山伯克霍尔德菌(Burkholderia dabaoshanensis GIMN1.004),编号为CCTCC No.M209109。
本发明所述的大宝山伯克霍尔德菌是从广东翁源大宝山矿区的重金属污染土壤中分离培养获得。该菌培养条件为:
(1)培养基:MGY
具体配方如下:KCl 0.01%;MgSO4·7H2O 0.025%;(NH4)2SO40.2%;K2HPO40.025%;葡萄糖0.1%;酵母膏0.01%;琼脂2.0%;ddH2O 1000ml;pH4.0;
(2)培养温度:28℃。
本发明大宝山伯克霍尔德菌(Burkholderia dabaoshanensis GIMN1.004)的分子分类地位的确定。
该菌株的16SrDNA序列如SEQ ID NO:1所示。
本发明所述大宝山伯克霍尔德菌具有以下有益效果:
本发明所述大宝山伯克霍尔德菌具有耐受重金属镉的能力,其耐受能力强,可以耐受22mmol/L即2640mg/L镉离子浓度,在对重金属污染的土壤修复中起着重要作用。
本发明所述大宝山伯克霍尔德菌(Burkholderia dabaoshanensis GIMN1.004)已于2009年5月10日保藏于国家知识产权局指定的保藏单位中国典型培养物保藏中心(CCTCC,地址:武汉大学),保藏编号为No.M209109。
附图说明
图1是本发明大宝山伯克霍尔德菌的透射电子显微镜照片(A,3000倍;B:6000倍);
图2是本发明大宝山伯克霍尔德菌及部分相关菌株依据16S rDNA序列构建的***发育树。
具体实施方式
以下通过具体实施例来详细说明本发明。
实施例1:本发明大宝山伯克霍尔德菌的分离
从广东大宝山重金属污染区采集0-30cm土样,取10g土壤放入装有90mL无菌水的三角瓶中(带玻珠),在180rpm转速下振荡30分钟,静置10分钟后取上层液稀释10倍后取0.1mL均匀涂布在MGY琼脂培养基板上,翻转平板置于28℃培养箱中培养2天,即可获得本发明所述的大宝山伯克霍尔德菌(Burkholderia dabaoshanensis GIMN1.004)。所述大宝山伯克霍尔德菌(Burkholderia dabaoshanensis GIMN1.004)已于2009年5月10日保藏于国家知识产权局指定的保藏单位中国典型培养物保藏中心(CCTCC,地址:武汉大学),保藏编号为No.M209109
MGY琼脂培养基:
KCl 0.01%;MgSO4·7H2O 0.025%;(NH4)2SO4 0.2%;K2HPO4 0.025%;葡萄糖0.1%;酵母膏0.01%;琼脂2.0%;ddH2O 1000ml;pH4.0;
本发明的大宝山伯克霍尔德菌(Burkholderiadabaoshanensis GIMN1.004)具有下述性质:
1.形态特征
大宝山伯克霍尔德菌在MGY琼脂培养基平板上能产生紫色颗粒;在加有重金属的MGY培养基中的菌落先呈淡粉色后逐渐转为乳白色,菌落圆形,表面皱缩。在NA培养基中菌落呈蓝灰色;菌株GIMN1.004革兰氏染色为阴性,菌体杆状;通过投射电镜,菌体内可观察到颗粒状物质。如图1所示。
2.生理生化特征
通过Biolog试验,本发明的大宝山伯克霍尔德氏菌能利用一下碳源:
Adonitol Amygdalin Arbutin D-Cellobiose a-Cydodextrin B-Cyclodextrin i-ErythritolD-Fructose L-Fucose D-Galacturonic Acid Gertibiose Glucose-1-PhosphateGlucose-6-Phosphate a-D-Lactose Lactulose Maltose Maltotriose D-MetezitoseD-Melibiose 3-Methyl-D-Glucose B-Methyl-D-Galactoside B-Methyl-D-GlucosideL-Rhamnose Stachyose Sucrose Turanose Formic Acid a-Hydroxybutyric Acid ItaconicAcid a-Ketobutyric Acid a-Ketovaleric Acid D,L-Lactic Acid L-Lactic Acid D-LacticAcid Methyl Ester D-Malic Acid Propionic Acid Pyruvic Acid Methyl Ester D-SaccharicAcid Succinamic Acid Succinic Acid Succinic Acid Mono-Methyl Ester Urocanic AcidL-Alaninamide L-Alanyl-L Threonine L-Asparagine L-Glutamic Acid Glycyl-L-AsparticAcid Glycyl-L-Glutamine Glycyl-L-Proline 2/-Deoxy Adenosine Thymidine UridineUridine-5/-Monophosphate m-Inositol a-Methyl-D-Galactoside a-Methyl-D-GlucosideD-Raffinose Salicin Acetic Acid Fumaric Acid L-Alanyl-L-Glutamine L-MethionineN-Acetyl-D-Glucosamine N-Acetyl-B-D-Mannosamine D-Gluconic Acid D-GlucosaminicAcid Pyruvic Acid L-Alanyl-L-Histidine Inosine a-D-Lactose Palatinose L-AlanineL-Serine。
3.其他性质
甲基萘醌的主要类型为MK-8,细胞壁脂肪酸种类和含量如表1。
表1 细胞壁脂肪酸种类和含量
实施例2:本发明大宝山伯克霍尔德氏菌16SrDNA的序列测定
1、PCR模板DNA的快速制备
将细菌划线接种在NA培养基的平板上,30℃培养过夜。取单一菌落悬浮于10μl无菌蒸馏水中,于沸水中加热5min,离心,取上清作为PCR模板DNA.
2、16SrRNA基因PCR扩增
PCR引物由上海英骏公司合成。
PrimerA:5’-AGAGTTTGATCCTGGCTCAG-3’
PrimerB:5’-AAGGAGGTGATCCACCCCCA-3’
PCR反应体系(总体积50μl):10倍缓冲液5μl,dNTP(2mmol/l)4μl,PrimerA(10pmol/l)1μl,PrimerB(10pmol/l)1μl,Taq酶(2U/l)0.4μl,DNA模板1μl,灭菌ddH2O 37.4μl。
PCR扩增条件:94℃5min;94℃1min,58℃1min,72℃2min,30个循环;72℃7min。
3、序列测定
PCR产物纯化后,送上海英骏公司用3730全自动测序仪测序,测试结果见附图2。其16SrDNA序列如SEQ ID NO:1所示(登录号:FJ210816.1),
测序结果与GenBank数据库中的已知序列进行BLAST比较,确定与本发明的菌株亲缘关系最近的种属。从数据库获得相关种属的16SrDNA序列,构建***发育树。
大宝山伯克霍尔德菌的基因序列与Burkholderia soli sp.nov.(KACC11589)的同源性为99.0%,但是两株菌在菌落形态以及生理生化方面都有较大差异。从***发育树可以看出,大宝山伯克霍尔德菌与KACC11589和Burkholderia sp.A6.2两株菌关系密切,聚成一类构成一个分支,但又独立两株菌。通过DNA-DNA杂交显示,大宝山伯克霍尔德菌与亲缘关系最近的模式菌株KACC11589的DNA-DNA杂交结果为25.0%。
本发明的大宝山伯克霍尔德菌与其同源性最高的Burkholderia soli sp.nov.(KACC11589)菌株微生物学特性的比较见表2。
表2 大宝山伯克霍尔德菌与其同源性最高的Burkholderia soli菌株微生物学特性比较
注:+:阳性;-:阴性;ND:表示没有测定
综合16SrDNA序列分析和DNA-DNA杂交结果(1987年国际***细菌学委员会ICSB规定,DNA同源性小于70%或杂交分子的热解链温度差小于或等于2℃为细菌种的界线,Wayneet al,1987),本发明的伯克霍尔德氏菌是一种新的细菌,命名为大宝山伯克霍尔德菌(Burkholderia Dabaoshanensis GIMN1.004)。
实施例3:本发明所述大宝山伯克霍尔德氏菌耐重金属能力测定
通过测定微生物对重金属的最大耐受浓度(Maximal tolerant concentration,MTC),来确定细菌对某种金属离子的极限耐受浓度。
挑取本发明的大宝山伯克霍尔德氏菌菌株的单菌落,接种至装有50mL液体NA培养基的三角瓶中,在28℃的摇床上培养(180转速)至对数生长期,然后将培养好的菌液用接种针划线接种到一系列包含不同浓度的镉离子的NA固体平板上,翻转平板置于28℃培养箱中培养4d后观察菌落的生长。
如果在某个浓度只能细微地观察到有菌落的生长,并且在略微高于这个浓度的平板上观察不到菌落的生长,这一浓度便是本发明大宝山伯克霍尔德氏菌菌株对这种金属的最大耐受浓度MTC。
测试结果表明,本发明大宝山伯克霍尔德氏菌菌株耐受镉的能力达到22mmol/L即2640mg/L。
序列表
<110>广东省微生物研究所
<120>大宝山伯克霍尔德菌及其应用
<160>1
<170>PatentIn version 3.1
<210>1
<211>1437
<212>DNA
<213>Burkholderia dabaoshanensis
<400>1
acacatgcaa gtcgaacggc agcacggggg caaccctggt ggcgagtggc 50
gaacgggtga gtaatacatc ggaacgtgtc ctggagtggg ggatagcccg 100
gcgaaagccg gattaatacc gcatacgctc tacggaggaa agcgggggat 150
cttcggacct cgcgctcaag gggcggccga tggcggatta gctagttggt 200
agggtaaagg cctaccaagg cgacgatccg tagctggtct gagaggacga 250
ccagccacac tgggactgag acacggccca gactcctacg ggaggcagca 300
gtggggaatt ttggacaatg ggggcaaccc tgatccagca atgccgcgtg 350
tgtgaagaag gccttcgggt tgtaaagcac ttttgtccgg aaagaaatcc 400
tttgggctaa tacctcggag ggatgacggt accggaagaa taagcaccgg 450
ctaactacgt gccagcagcc gcggtaatac gtagggtgcg agcgttaatc 500
ggaattactg ggcgtaaagc gtgcgcaggc ggttcgctaa gaccgatgtg 550
aaatccccgg gcttaacctg ggaactgcat tggtgactgg cgagctagag 600
tgtggcagag gggggtagaa ttccacgtgt agcagtgaaa tgcgtagaga 650
tgtggaggaa taccgatggc gaaggcagcc ccctgggcta acactgacgc 700
tcatgcacga aagcgtgggg agcaaacagg attagatacc ctggtagtcc 750
acgccctaaa cgatgtcaac tagttgttgg ggattcattt ccttagtaac 800
gtagctaacg cgtgaagttg accgcctggg gagtacggtc gcaagattaa 850
aactcaaagg aattgacggg gacccgcaca agcggtggat gatgtggatt 900
aattcgatgc aacgcgaaaa accttaccta cccttgacat ggacggaatc 950
cctgctgaga ggtgagagtg ctcgaaagag aaccgtcgca caggtgctgc 1000
atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1050
agcgcaaccc ttgtctctag ttgctacgaa agggcactct agagagactg 1100
ccggtgacaa accggaggaa ggtggggatg acgtcaagtc ctcatggccc 1150
ttatgggtag ggcttcacac gtcatacaat ggtcggaaca gagggttgcc 1200
aacccgcgag ggggagctaa tcccagaaaa ccgatcgtag tccggattgc 1250
actctgcaac tcgagtgcat gaagctggaa tcgctagtaa tcgcggatca 1300
gcatgccgcg gtgaatacgt tcccgggtct tgtacacacc gcccgtcaca 1350
ccatgggagt gggttttacc agaagtggct agtctaaccg caaggaggac 1400
ggtcaccacg gtaggattca tgactggggt gaagtcg 1437
Claims (4)
1.大宝山伯克霍尔德菌(Burkholderia dabaoshanensis GIMN1.004),其特征在于:其保藏编号为CCTCC No.M209109。
2.根据权利要求1所述的大宝山伯克霍尔德菌(Burkholderia dabaoshanensis GIMN1.004),其特征在于:该大宝山伯克霍尔德菌(Burkholderia dabaoshanensis GIMN1.004)的16SrDNA序列如SEQ ID NO:1所示。
3.根据权利要求1或2所述的大宝山伯克霍尔德菌(Burkholderia dabaoshanensis GIMN1.004),其特征在于:具有以下的微生物学特征:
a)形态特征:
所述大宝山伯克霍尔德菌在MGY琼脂培养基平板上能产生紫色颗粒;在加有重金属的MGY培养基中的菌落先呈淡粉色后逐渐转为乳白色,菌落圆形,表面皱缩;在NA培养基中菌落呈蓝灰色;菌株GIMN1.004革兰氏染色为阴性,菌体杆状;通过投射电镜,菌体内可观察到颗粒状物质;
b)生理生化特征:
c)其他特征:
所述大宝山伯克霍尔德菌甲基萘醌主要类型为MK-8。
4.权利要求1-3任一项所述的大宝山伯克霍尔德菌(Burkholderia dabaoshanensisGIMN1.004)在耐重金属镉中的应用。
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CN102312229A (zh) * | 2011-09-26 | 2012-01-11 | 武汉材料保护研究所 | 以化学沉积镍磷合金层封闭钢铁表面陶瓷喷涂层孔隙的方法 |
CN102936574A (zh) * | 2012-11-20 | 2013-02-20 | 南京农业大学 | 一种耐重金属的根瘤菌及其促进尾矿区植物修复的方法 |
CN102936574B (zh) * | 2012-11-20 | 2013-11-06 | 南京农业大学 | 一种耐重金属的根瘤菌及其促进尾矿区植物修复的方法 |
CN111676160A (zh) * | 2020-06-16 | 2020-09-18 | 广西中医药大学 | 牛大力内生菌rh5在促进牛大力生长中的应用 |
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CN111690564B (zh) * | 2020-06-16 | 2022-08-30 | 广西中医药大学 | 牛大力耐镉内生菌rh5及其应用 |
CN111676160B (zh) * | 2020-06-16 | 2022-09-23 | 广西中医药大学 | 牛大力内生菌rh5在促进牛大力生长中的应用 |
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