CN111269863B - 一种假单胞菌及其应用 - Google Patents
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Abstract
本发明涉及一种假单胞菌,属于微生物技术领域。所述假单胞菌为JDB001株,经质粒消除技术减毒,制作成微生物菌剂应用于水塘的鱼类养殖可大大提高鱼类的成活率。
Description
技术领域
本发明涉及微生物技术领域,具体涉及一种假单胞菌及其应用。
背景技术
假单胞菌,广泛分布于淡水、污水及土壤中,能引起多种水产养殖动物患病,特别是在现代大规模的集中化高密度鱼类养殖的环境下,鱼类极易感染细菌性疾病。鱼类在感染假单胞菌后,引起细菌性败血症,造成鱼类死亡,给鱼类水产养殖业带来巨大的生产损失。现有的鱼类养殖基本都是通过使用嗜水气单胞菌疫苗对鱼塘中的鱼苗进行疾病防治,但疫苗的投放无法做到让所有的鱼苗都能接种生效,仍然存在防治不彻底的现象。
质粒(Plasmid)是附加到细菌细胞中的非细胞染色体或核区DNA原有的能够自主复制的较小的DNA分子。有些质粒含有某种抗药基因;有一些质粒携带的基因则可以赋予细菌细胞额外的生理代谢能力,乃至于在一些细菌中提高它的致病力。
发明内容
为解决现有技术中存在的问题,本发明专利设计了一种非致病性的假单胞菌,所述假单胞菌经质粒消除技术减毒为非致病菌,命名为假单胞菌JDB001株,建议分类命名为:假单胞菌(Pseudomonas sp.),保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为:北京市朝阳区北辰西路1号院3号,保藏时间为:2019年6月17日,保藏号为:CGMCCNo.17947。
假单胞菌JDB001株的质粒消除过程包括以下步骤:
a、质粒消除培养,按60ug/ml加入质粒消除剂(十二烷基硫酸钠)配制营养肉汤培养基,接种假单胞菌,并于摇床41℃环境下培养46小时;
b、无质粒菌落挑选,将上述培养物利用营养琼脂平板培养基划线分离,37℃培养8-12小时,挑取20单个菌落,分别接种于5mL肉汤培养基中,并于摇床上37℃环境下培养8-12小时;
c、提取质粒,利用大质粒提取法检测上述营养肉汤培养的细菌中是否存在质粒;
d、保留菌种,将不含质粒的假单胞菌,接种琼脂斜面培养基,37℃培养8-12小时;
e、培养完毕后,将菌种于4℃冰箱保存。
同时,本发明专利还设计了上述的假单胞菌在鱼塘养殖中的应用。其应用步骤包括,
a、菌种扩增:配制液体肉汤培养基10000ml,分装至数支5ml试管、数个250三角烧瓶、1000ml三角烧瓶,于120℃灭菌30分钟;
取少许无质粒减毒假假单胞菌JDB001株原始菌种,接种于盛有液体肉汤培养基5ml试管,于数显恒温水浴振荡器28℃振摇8-12小时,于4℃冰箱保存,作为试管生产菌种;
取一支试管生产菌种,接种到盛有液体肉汤培养的250ml三角烧瓶中,于数显恒温水浴振荡器28℃振摇8-12小时,作为三角瓶菌种;
将三角瓶菌种按5%的体积比接种到盛有液体肉汤培养的1000ml三角烧瓶中,于数显恒温水浴振荡器28℃振摇8-12小时,获得含有假单胞菌JDB001的微生物菌剂,后于4℃冰箱保存;
以上述方法扩增10L微生物菌剂;
b、将10L微生物菌剂加入到100L含5%红糖的水溶液中,培养4-8小时;
c、按照1L/亩的用量,将上述水溶液加入到鱼塘中,进行养水,养水时间为2-3周,之后放入鱼苗。
相对与现有技术,本发明设计的非致病性假单胞菌的进步之处在于,它替代了传统的通过疫苗对水产养殖的鱼苗进行防治的方法,通过向鱼塘中添加减毒的非致病性的假单胞菌剂,非致病性的假单胞菌释放到养殖水域可竞争性的拮抗致病性假单胞菌,同时,由于水域中添加的是活菌剂,会在水域中不断地繁殖,提高鱼塘中所有鱼苗的有害菌防治,大大减少了鱼苗的得病率,使鱼塘养殖的成活率始终保持在高水平。
附图说明
图1菌种API 20NE的实验结果
假单胞菌JDB001株,建议分类命名为:假单胞菌(Pseudomonas sp.),保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为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,保藏时间为2019年6月17日,保藏号为CGMCC No.17947。
具体实施方式
下面结合附图和具体实施例对本发明做进一步的说明。
实施例1
假单胞菌JDB001株的获得
一、菌种分离
1、按比例配制商品化营养琼脂培养基100ml,120℃灭菌30分钟,制备营养琼脂平板培养基和斜面培养基;
2、刮取少许养殖锦鲤体表和肠道内容物,分别溶解到5ml灭菌生理盐水中;
3、利用营养琼脂培养基划线分离所含细菌,28℃培养12小时,选择边缘光滑、中央凸起、灰白色的圆形菌落,利用营养琼脂培养基平皿中再次划线分离纯化细菌;
4、挑取典型菌落,图片染色、镜检,观察是否符合假单胞菌形态,并将典型菌落送专业细菌鉴定机构进行鉴定。
菌落在NA培养基中形态为呈乳白色有点黄,圆形,表面光滑、边缘透明,显微镜下心态呈G-杆菌。
其生化特征通过法国生物梅里埃鉴定,实验条件如表1所示,其实验结果如图1所示。
菌落的16SrDNA测序如SEG SEQ ID NO.1所示,与假单胞菌菌株的同源性达到99%。
二、菌株的质粒消除
a、质粒消除培养,按60ug/ml质粒消除剂(十二烷基硫酸钠)配制营养肉汤培养基,接种假单胞菌,并于摇床41℃环境下培养46小时;
b、无质粒菌落挑选,将上述培养物利用营养琼脂平板培养基划线分离,37℃培养12小时,挑取20单个菌落,分别接种于5mL肉汤培养基中,并于摇床上37℃环境下培养12小时;
c、提取质粒,利用大质粒提取法检测上述营养肉汤培养的细菌中是否存在质粒;
d、保留菌种,将不含质粒的假单胞菌,接种琼脂斜面培养基,37℃培养12小时。
e、培养完毕后,将菌种于4℃冰箱保存。作为原始菌种。
经质粒消除减毒的菌株即为本发明所需的菌株,命名为假单胞菌JDB001,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为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,保藏时间为2019年6月17日,保藏号为CGMCC No.17947。
三、菌种的保藏
1、用接种环蘸取第一阶段分离纯化的无质粒减毒原始菌种接种至琼脂斜面;
2、于培养箱中28℃环境下培养48~72小时。
3、于4℃冰箱保存,作为生产用原始菌种。
实施例2
假单胞菌JDB010株对鱼苗运输应激和致病菌的保护能力实验
在观赏鱼市场购买900尾1-2寸长锦鲫鱼苗,分为三组(每组三个重复,每个重复组100尾)。第一组为对照组,不作任何处理,饲养在20℃观赏鱼缸中饲养,观察一周,计数死亡鱼苗数量;第二组为攻毒组,接种嗜水气单胞菌(108CFU/L),饲养在20℃观赏鱼缸中饲养,观察一周,计数死亡鱼苗数量;第三组为本专利菌保护组,同时接种JDB010菌株(2*108CFU/L)和嗜水气单胞菌(108CFU/L),饲养在20℃观赏鱼缸中饲养,观察一周,计数死亡鱼苗数量。结果如表2所示:
表2鱼苗存活情况
由表2可以看出,第1组为对照组,由于运输应激,存活率为91.7%;第2组:攻毒组,存活率为64.7%;第3组:益生菌保护组,存活率为97.0%。经统计学分析各组间有显著差异。说明,本专利的假单胞菌JDB010株可减轻运输应激,同时可减轻病原菌嗜水气单胞菌的感染。
实施例3
假单胞菌JDB001株在鱼塘养殖中的应用
a、菌种扩增:配制液体肉汤培养基10000ml,分装至数支5ml试管、数个250三角烧瓶、1000ml三角烧瓶,于120℃灭菌30分钟;
取保藏的假单胞菌JDB001株菌种,接种于盛有液体肉汤培养基的5ml试管,于数显恒温水浴振荡器28℃振摇12小时,于4℃冰箱保存,作为试管生产菌种;
取一支试管生产菌种,接种到盛有液体肉汤培养的250三角烧瓶中,于数显恒温水浴振荡器28℃振摇12小时,作为三角瓶菌种;
将三角瓶菌种按5%的体积比接种到盛有液体肉汤培养的1000ml三角烧瓶中,于数显恒温水浴振荡器28℃振摇12小时,获得含有假单胞菌JDB001的微生物菌剂,后于4℃冰箱保存;
以上述方法扩增10L微生物菌剂。
b、将10L微生物菌剂加入到100L含5%红糖的水溶液中,培养4-8小时;
c、按照1L/亩的用量,将生产的菌液加入到鱼塘中,进行养水,养水时间为2-3周,之后放入鱼苗。
d、一年后起塘。并与传统不添加本专利菌剂的鱼塘养殖进行对比,计算成活率和生产成绩。见表3所示,
表3
从表3可以看出,添加本专利菌剂的鱼塘在鱼苗的成活率与成鱼的平均重量上均要远高于传统鱼塘养殖。
上述内容仅为本发明创造的较佳实施例而已,不能以此限定本发明创造的实施范围,即凡是依本发明创造权利要求及发明创造说明内容所做出的简单的等效变化与修饰,皆仍属于本发明创造涵盖的范围。
序列表
<110> 山东中农普宁药业有限公司 山东倍倍安生物科技有限公司
<120> 一种假单胞菌及其应用
<141> 2020-03-16
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1391
<212> DNA
<213> 假单胞菌(Pseudomonas sp.JDB001株的16rDNA序列)
<400> 1
aagctatcta cttctggtgc aacccactcc catggtgtga cgggcggtgt gtacaaggcc 60
cgggaacgta ttcaccgcaa cattctgatt tgcgattact agcgattccg acttcacgga 120
gtcgagttgc agactccgat ccggactacg acgcgctttt tgggattcgc tcactatcgc 180
tagcttgcag ccctctgtac gcgccattgt agcacgtgtg tagccctggc cgtaagggcc 240
atgatgactt gacgtcatcc ccaccttcct ccggtttatc accggcagtc tcccttgagt 300
tcccaccatt acgtgctggc aacaaaggac aggggttgcg ctcgttgcgg gacttaaccc 360
aacatctcac gacacgagct gacgacagcc atgcagcacc tgtgttctga ttcccgaagg 420
cactcccgta tctctacagg attccagaca tgtcaaggcc aggtaaggtt cttcgcgttg 480
catcgaatta aaccacatgc tccaccgctt gtgcgggccc ccgtcaattc atttgagttt 540
taaccttgcg gccgtactcc ccaggcggtc gatttaacgc gttagctccg gaagccacgt 600
ctcaaggaca cagcctccaa atcgacatcg tttacggcgt ggactaccag ggtatctaat 660
cctgtttgct ccccacgctt tcgcacctga gcgtcagtct ttgtccaggg ggccgccttc 720
gccaccggta ttcctccaga tctctacgca tttcaccgct acacctggaa ttctaccccc 780
ctctacaaga ctctagctgg acagttttaa atgcaattcc caggttgagc ccggggcttt 840
cacatctaac ttatccaacc gcctgcgtgc gctttacgcc cagtaattcc gattaacgct 900
tgcaccctcc gtattaccgc ggctgctggc acggagttag ccggtgcttc ttctgcgagt 960
aacgtcacag ctggcagtta ttagctacca acctttcctc ctcgctgaaa gtgctttaca 1020
acccgaaggc cttcttcaca cacgcggcat ggctgcatca gggtttcccc cattgtgcaa 1080
tattccccac tgctgcctcc cgtaggagtc tggaccgtgt ctcagttcca gtgtggctga 1140
tcatcctctc agaccagcta gggatcgtcg ccttggtgag ccattacctc accaactagc 1200
taatcccacc tgggttcatc caatcgcgca aggcccgaag gtcccctgct ttcccccgta 1260
gggcgtatgc ggtattagct accgtttcca gtagttatcc ccctcgactg ggcagatccc 1320
caggcattac tcacccgtcc gccgctcgcc ggcaaaagta gcaagctact ttcccgctgc 1380
cgctcgactg c 1391
Claims (1)
1.一种假单胞菌(Pseudomonas sp.),其特征在于,所述假单胞菌经质粒消除技术减毒为非致病菌,命名为JDB001株,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏时间为2019年6月17日,保藏号为CGMCC No.17947。
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CN1460712A (zh) * | 2003-06-20 | 2003-12-10 | 符昌锋 | 一种假单胞菌及其制备方法、应用 |
CN105255794B (zh) * | 2015-11-23 | 2019-04-09 | 中国科学院烟台海岸带研究所 | 一种假单胞菌及其应用 |
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CN1460712A (zh) * | 2003-06-20 | 2003-12-10 | 符昌锋 | 一种假单胞菌及其制备方法、应用 |
CN105255794B (zh) * | 2015-11-23 | 2019-04-09 | 中国科学院烟台海岸带研究所 | 一种假单胞菌及其应用 |
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