CN106967639A - 一种暗灰链霉菌及其在降解烟碱类杀虫剂中的应用 - Google Patents
一种暗灰链霉菌及其在降解烟碱类杀虫剂中的应用 Download PDFInfo
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Abstract
本发明公开了一种暗灰链霉菌,经鉴定为暗灰链霉菌Streptomyces canus,菌株名为gl2‑18,保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为:CGMCC No.13662,保藏日期为2017年02月15日。相对于现有技术,发明的新菌株暗灰链霉菌CGMCC 13662,无论其静息细胞还是其生长细胞,均可有效降解烟碱类杀虫剂,特别是啶虫脒和噻虫啉,可用作农药降解菌剂,具有很好的应用前景。
Description
技术领域
本发明属于微生物生物技术领域,具体涉及一种暗灰链霉菌及其在降解烟碱类杀虫剂中的应用。
背景技术
烟碱类杀虫剂,是一种作用于昆虫神经***突触部位烟碱乙酰胆碱受体的杀虫剂。啶虫脒是一种硝基亚甲基类杂环类杀虫剂,作用于昆虫神经***突触部位的烟碱乙酰胆碱受体。啶虫脒对刺吸口器害虫高效广谱,广泛用于水稻、蔬菜、果树和茶叶的蚜虫、飞虱、蓟马和部分鳞翅目害虫等的防治。然而长期大量使用啶虫脒已对食品安全和生态环境产生了严重影响,如对传粉昆虫蜜蜂和鸟类毒性高而引起的生态链问题;在谷物、蔬菜和茶叶中的残留而引起的食品安全问题;污染土壤和地表水而引起环境安全问题。
微生物降解是农药在土壤和水环境中降解的主要途径之一,也是消除农药环境污染的最经济有效的方法之一。近几年来,一些微生物如草木樨剑菌Ensifer melilotiCGMCC7333,贪噬菌Variovorax boronicumulans CGMCC 4969,噬染料菌Pigmentiphaga sp.AAP-1和D-2、粘质红酵母Rhodotorulamucilaginosa IM-2、嗜麦芽寡养单胞菌Stenotrophomonas sp.THZ-XP和Stenotrophomonas maltophilia CGMCC 1.1788以及红球菌Rhodococcus sp.BCH2等被报道可降解啶虫脒或噻虫啉,然而目前尚未有放线菌降解啶虫脒和噻虫啉的研究和专利报道。
发明内容
发明目的:为了克服现有技术的不足,本发明提供了一种暗灰链霉菌及其在降解烟碱类杀虫剂中的应用。
技术方案:本发明提供了一种暗灰链霉菌,经鉴定为暗灰链霉菌(Streptomycescanus),菌株名为gl2-18,保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为:CGMCC No.13662,保藏日期为2017年02月15日。
所述暗灰链霉菌的培养方法为:将所述暗灰链霉菌CGMCC 13662接种于高氏一号固体培养基上,于温度为30±1℃的培养箱中培养。
本发明还提供了所述的暗灰链霉菌在降解烟碱类杀虫剂中的应用。
优选,所述烟碱类杀虫剂为啶虫脒、噻虫啉或者吡虫啉。
优选,应用方法为:将所述暗灰链霉菌CGMCC 13662的孢子接种于含烟碱类杀虫剂的营养培养液中振荡培养,暗灰链霉菌CGMCC 13662在生长过程中降解烟碱类杀虫剂。
优选,应用方法为:将所述暗灰链霉菌CGMCC 13662的孢子接种于营养培养液中,振荡培养后离心收集和洗涤菌体;将菌体悬浮于含烟碱类杀虫剂的磷酸盐缓冲溶液中,振荡培养,暗灰链霉菌CGMCC13662的静息细胞降解烟碱类杀虫剂。
所述营养培养液优选为ISP4液体培养基。
技术效果:相对于现有技术,发明的新菌株暗灰链霉菌CGMCC 13662,无论其静息细胞还是其生长细胞,均可有效降解烟碱类杀虫剂,特别是啶虫脒和噻虫啉,可用作农药降解菌剂,具有很好的应用前景。
附图说明
图1为本发明暗灰链霉菌CGMCC 13662平板菌落形态和显微形态。
图2为本发明暗灰链霉菌CGMCC 13662生长细胞降解啶虫脒的HPLC图。
图3为本发明暗灰链霉菌CGMCC 13662的静息细胞降解啶虫脒的HPLC图。
图4为本发明暗灰链霉菌CGMCC 13662生长细胞降解噻虫啉的HPLC图。
图5为本发明暗灰链霉菌CGMCC 13662的静息细胞降解噻虫啉的HPLC图。
具体实施方法
结合下面的实验例详细阐述本发明暗灰链霉菌CGMCC 13662及其应用。
实施例1:可生物降解啶虫脒的菌株分离筛选、鉴定和生物学特性
1、菌株分离
从江苏徐州采集土壤,取2g土壤加入含玻璃珠的18mL无菌水中,振荡2h,静置20min后,取100μL样品稀释至10-3和10-4后,涂布于含有50mg/L啶虫脒的矿物盐培养基固体平板上。矿物盐培养基的组成为(g/L):KH2PO41.36,Na2HPO42.13,MgSO4·7H2O0.5和10mL金属离子液,pH 7.0。金属离子液组成为(g/L):CaCl2·2H2O0.40,H3BO30.30,CuSO4·5H2O0.04,KI0.10,FeSO4·7H2O0.20,MnSO4·7H2O0.40,NaMoO4·2H2O0.20和10.0mL/L浓盐酸。样品于30℃、转速为220rpm的摇床中培养7d,取培养液涂布于LB固体平板上并于30℃培养3d,挑取形态不同的菌落划线至LB平板上,30℃培养3d后再次进行菌种平板划线纯化。LB培养基组成为(g/L):蛋白胨10.0、酵母膏5.0、NaCl 10.0,琼脂20g,pH 7.2。
2、菌株降解啶虫脒能力的测定
取上述纯化的菌株划线于高氏一号固体培养基上,于30℃培养箱中培养至长出孢子(约7d)。用无菌水收集孢子并制备孢子悬液,接种于含100mL ISP4液体培养基的500mL锥形瓶中,30℃,220rpm下振荡培养。ISP4培养基的配方为(g/L):可溶性淀粉10,磷酸氢二钾1,七水硫酸镁1,氯化钠1,硫酸铵2,碳酸钙2,硫酸亚铁0.001,氯化锰0.001,七水硫酸锌0.001,pH7.2。当菌体长成菌丝球时,称取0.3g湿重菌体用磷酸盐缓冲液(pH7.5)洗涤两次后,悬浮于含15mL上述磷酸盐缓冲液(含200mg/L的啶虫脒)50mL的离心管中,最终转移至100mL锥形瓶于30℃,220rpm摇床振荡培养96h。采用安捷伦1200型HPLC仪分析检测啶虫脒含量变化。HPLC条件:流动相为30%乙腈和70%的含0.01%乙酸的去离子水,流速为1ml/min;HPLC柱为安捷伦HC-C18反相柱(4.6×250mm,5μm),柱温为30℃;检测波长为235nm。比较零天样品和96h样品中的啶虫脒的含量变化。
3、GL2-18菌株的类分类学鉴定
对上述啶虫脒的HPLC峰面积有明显减少的菌株进行菌种鉴定,一是采用光学显微镜观察菌落显微形态;二是菌落PCR法扩增16S rRNA基因序列,对扩增的基因序列进行blastn分析。在高氏一号平板上GL2-18菌落表面干燥、小而致密,呈现放线菌的典型特征,在光学显微镜下可观察到盘曲的菌丝(如附图1所示)。菌落PCR法扩增16S rRNA的方法如下:从高氏一号固体培养基上用无菌牙签挑取GL2-18单菌落,加入20μL无菌水,100℃加热10min。轻微离心后,取10μL作为模板。引物为16S rRNA基因扩增通用引物K1和K2。K1引物序列为:5′-AACTGAAGAGTTTGATCC-3′(SEQ ID No:1),K2引物序列为:5′-TAGGTTACCTTGTTGTTACGACTT-3′(SEQ ID No:2)。引物由生工生物工程(上海)有限公司合成。反应体系:Taq缓冲液(10×)2.5μL、MgCl2(25mmol/L)1μL、dNTP(2.5mmol/L)2.5μL、K1(50mmol/L)1μL、K2(50mmol/L)1μL、去离子水11.5μL、rTaq酶(2U/μL)0.5μL,共20μL总反应体积。PCR反应条件为:95℃预变性5min后,95℃变性50s,55℃退火1min,72℃延伸1min,共29个循环,72℃延伸10min。所得序列由生工生物工程(上海)有限公司测序后,得到的部分16S rRNA基因序列(SEQ ID No:3)在美国国家生物技术信息中心的Genbank数据库中进行blast搜索。比对结果显示,GL2-18菌株与暗灰链霉菌Streptomyces canus S02、HUBM82686菌株的16S rRNA基因的相似性达到99%。形态观察和16S rRNA基因序列分析结果表明,GL2-18菌株为暗灰链霉菌。
GL2-18菌株于2017年2月15日保藏于中国微生物菌种保藏管理委员会普通微生物中心(北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所),分类命名为暗灰链霉菌Streptomyces canus,保藏编号为CGMCC 13662。
实施例2:暗灰链霉菌CGMCC 13662生长细胞降解啶虫脒
取保藏的暗灰链霉菌CGMCC 13662划线于高氏一号固体培养基上,于30℃培养箱中培养至长出孢子(约7d)。用无菌水收集孢子并制备孢子悬液,接种于装有100mL ISP4液体培养基(含200mg/L啶虫脒)的500mL锥形瓶中,30℃,220rpm下振荡培养。培养4d后,取样进行HPLC分析,结果表明接种暗灰链霉菌CGMCC 13662的实验组的啶虫脒含量为0.26mmol/L(如附图2中A所示);未接种菌的对照组的啶虫脒含量为0.94mmol/L(如附图2中B所示)。结果表明暗灰链霉菌CGMCC 13662的生长细胞培养4d可降解73%啶虫脒。
实施例3:暗灰链霉菌CGMCC 13662的静息细胞降解啶虫脒
取保藏的暗灰链霉菌CGMCC 13662划线于高氏一号固体培养基上,于30℃培养箱中培养至长出孢子。用无菌水收集孢子并制备孢子悬液,接种于含30mL ISP4液体培养基的100mL锥形瓶中,30℃,220rpm下振荡培养。当菌体长成菌丝球时,称取0.3湿菌体用0.2mol/L磷酸盐缓冲液(pH7.5)洗涤两次后,悬浮于含15mL上述磷酸盐缓冲液(含200mg/L的啶虫脒)50mL的离心管中,然后转移至100mL三角瓶中,于30℃,220rpm摇床振荡培养96h。HPLC分析啶虫脒的降解量,如附图3所示,结果显示啶虫脒减少了0.47mmol/L,啶虫脒的降解率为52.5%。
实施例4:
与实例二基本相同,只是降解时间为6d。结果显示啶虫脒减少0.82mmol/L,啶虫脒的降解率为90.2%。
实施例5:
与实例二基本相同,只是降解时间为8d,结果显示啶虫脒减少0.91mmol/L,啶虫脒的降解率96.7%。
实施例6:
与实例二基本相同,降解10d后应用HPLC检测啶虫脒含量变化,结果显示啶虫脒减少0.93mmol/L,啶虫脒的降解率为98.5%。
实施例7:暗灰链霉菌CGMCC 13662的生长细胞降解噻虫啉
与实例2的条件基本相同,底物为噻虫啉,浓度为100mg/L,降解时间为4d。结果(图4)显示噻虫啉减少0.40mmol/L,噻虫啉的降解率为90.9%。
实施例8:暗灰链霉菌CGMCC 13662的静息细胞降解噻虫啉
与实例3的条件基本相同,底物为噻虫啉,浓度为100mg/L,降解时间为2d。结果(图5)显示噻虫啉减少0.36mmol/L,噻虫啉的降解率为95%。
采用上述实施例的方法,以烟碱类杀虫剂吡虫啉为例,经过检测,结果表明,本发明暗灰链霉菌CGMCC 13662降解吡虫啉的效果与降解啶虫脒效果类似。
本发明并不仅仅限于上述的实施例。
SEQUENCE LISTING
<110> 南京师范大学
<120> 一种暗灰链霉菌及其在降解烟碱类杀虫剂中的应用
<130> 2017.04
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213> 人工序列
<400> 1
aactgaagag tttgatcc 18
<210> 2
<211> 24
<212> DNA
<213> 人工序列
<400> 2
taggttacct tgttgttacg actt 24
<210> 3
<211> 1399
<212> DNA
<213> 暗灰链霉菌(Streptomyces canus)
<400> 3
taacacatgc aagtcgaacg atgaaccact tcggtgggga ttagtggcga acgggtgagt 60
aacacgtggg caatctgccc ttcactctgg gacaagccct ggaaacgggg tctaataccg 120
gataccacta ccgcaggcat ctgtggtggt tgaaagctcc ggcggtgaag gatgagcccg 180
cggcctatca gcttgttggt gaggtaatgg ctcaccaagg cgacgacggg tagccggcct 240
gagagggcga ccggccacac tgggactgag acacggccca gactcctacg ggaggcagca 300
gtggggaata ttgcacaatg ggcgaaagcc tgatgcagcg acgccgcgtg agggatgacg 360
gccttcgggt tgtaaacctc tttcagcagg gaagaagcga aagtgacggt acctgcagaa 420
gaagcgccgg ctaactacgt gccagcagcc gcggtaatac gtagggcgca agcgttgtcc 480
ggaattattg ggcgtaaaga gctcgtaggc ggcttgtcac gtcgggtgtg aaagcccggg 540
gcttaacccc gggtctgcat tcgatacggg ctagctagag tgtggtaggg gagatcggaa 600
ttcctggtgt agcggtgaaa tgcgcagata tcaggaggaa caccggtggc gaaggcggat 660
ctctgggcca ttactgacgc tgaggagcga aagcgtgggg agcgaacagg attagatacc 720
ctggtagtcc acgccgtaaa cggtgggaac taggtgttgg cgacattcca cgtcgtcggt 780
gccgcagcta acgcattaag ttccccgcct ggggagtacg gccgcaaggc taaaactcaa 840
aggaattgac gggggcccgc acaagcagcg gagcatgtgg cttaattcga cgcaacgcga 900
agaaccttac caaggcttga catacaccgg aaagcatcag agatggtgcc ccccttgtgg 960
tcggtgtaca ggtggtgcat ggctgtcgtc agctcgtgtc gtgagatgtt gggttaagtc 1020
ccgcaacgag cgcaaccctt gtcctgtgtt gccagcatgc ccttcggggt gatggggact 1080
cacaggagac cgccggggtc aactcggagg aaggtgggga cgacgtcaag tcatcatgcc 1140
ccttatgtct tgggctgcac acgtgctaca atggcaggta caatgagctg cgaaaccgtg 1200
aggtagagcg aatctcaaaa agcctgtctc agttcggatt ggggtctgca actcgacccc 1260
atgaagtcgg agttgctagt aatcgcagat cagcattgct gcggtgaata cgttcccggg 1320
ccttgtacac accgcccgtc acgtcacgaa agtcggtaac acccgaagcc ggtggcccaa 1380
ccccttgtgg gagggagct 1399
Claims (7)
1.一种暗灰链霉菌,其特征在于,经鉴定为暗灰链霉菌(Streptomyces canus),菌株名为gl2-18,保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为:CGMCCNo.13662,保藏日期为2017年02月15日。
2.权利要求1所述的暗灰链霉菌的培养方法,其特征在于,将所述暗灰链霉菌CGMCC13662接种于高氏一号固体培养基上,于温度为30±1℃的培养箱中培养。
3.权利要求1所述的暗灰链霉菌在降解烟碱类杀虫剂中的应用。
4.根据权利要求3所述的暗灰链霉菌在降解烟碱类杀虫剂中的应用,其特征在于,所述烟碱类杀虫剂为啶虫脒、噻虫啉或者吡虫啉。
5.根据权利要求3所述的应用,其特征在于,应用方法为:将所述暗灰链霉菌CGMCC13662的孢子接种于含烟碱类杀虫剂的营养培养液中振荡培养,暗灰链霉菌CGMCC13662在生长过程中降解烟碱类杀虫剂。
6.根据权利要求3所述的应用,其特征在于,应用方法为:将所述暗灰链霉菌CGMCC13662的孢子接种于营养培养液中,振荡培养后离心收集和洗涤菌体;将菌体悬浮于含烟碱类杀虫剂的磷酸盐缓冲溶液中,振荡培养,暗灰链霉菌CGMCC 13662的静息细胞降解烟碱类杀虫剂。
7.根据权利要求5或者6所述的应用,其特征在于,所述营养培养液为ISP4液体培养基。
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Cited By (4)
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CN108192843A (zh) * | 2018-01-18 | 2018-06-22 | 湖南农业大学 | 一种固氮菌株及其微生态制剂 |
CN109266582A (zh) * | 2018-10-16 | 2019-01-25 | 环境保护部南京环境科学研究所 | 一种薄层菌及其降解新烟碱类杀虫剂的应用 |
CN109762800A (zh) * | 2018-12-14 | 2019-05-17 | 南京农业大学 | 一种啶虫脒酰胺酶基因aceAB及其编码蛋白质及其应用 |
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CN108192843A (zh) * | 2018-01-18 | 2018-06-22 | 湖南农业大学 | 一种固氮菌株及其微生态制剂 |
CN109266582A (zh) * | 2018-10-16 | 2019-01-25 | 环境保护部南京环境科学研究所 | 一种薄层菌及其降解新烟碱类杀虫剂的应用 |
CN109266582B (zh) * | 2018-10-16 | 2022-01-14 | 南京国环环境研究院有限公司 | 一种薄层菌及其降解新烟碱类杀虫剂的应用 |
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