CN108531423A - 一种深海盐单胞菌及其在诱导厚壳贻贝稚贝附着中的应用 - Google Patents
一种深海盐单胞菌及其在诱导厚壳贻贝稚贝附着中的应用 Download PDFInfo
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- 230000006698 induction Effects 0.000 title claims abstract description 8
- 241000894006 Bacteria Species 0.000 claims abstract description 22
- 241001653918 Halomonas sp. Species 0.000 claims abstract description 5
- 150000003839 salts Chemical class 0.000 claims abstract description 4
- 239000013535 sea water Substances 0.000 claims description 8
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 2
- 244000005700 microbiome Species 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 5
- 235000015170 shellfish Nutrition 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000011521 glass Substances 0.000 description 3
- 235000002639 sodium chloride Nutrition 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 241000237519 Bivalvia Species 0.000 description 2
- 241000237525 Mytilidae Species 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
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- 108020004707 nucleic acids Proteins 0.000 description 2
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- 102000039446 nucleic acids Human genes 0.000 description 2
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- 238000012163 sequencing technique Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 241000143233 Mytilus coruscus Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- DPKHZNPWBDQZCN-UHFFFAOYSA-N acridine orange free base Chemical compound C1=CC(N(C)C)=CC2=NC3=CC(N(C)C)=CC=C3C=C21 DPKHZNPWBDQZCN-UHFFFAOYSA-N 0.000 description 1
- 230000010062 adhesion mechanism Effects 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000001418 larval effect Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000029052 metamorphosis Effects 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
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Abstract
一种深海盐单胞菌,保藏编号为:CCTCC M 2018066,分类命名:盐单细胞菌Halomonas sp.。该细菌形成的微生物被膜对厚壳贻贝稚贝的附着具有相对较高的诱导活性。还涉及一种上述深海盐单胞菌在诱导厚壳贻贝稚贝附着中的应用。
Description
技术领域
本发明涉及一株深海细菌及其在诱导厚壳贻贝稚贝附着方面的应用,具体涉及一株深海盐单胞细菌(Halomonas sp.ROE5-16)的分离、纯化、鉴定和保藏以及其在不同初始细菌密度条件下形成的微生物被膜对厚壳贻贝稚贝附着的影响作用。
背景技术
厚壳贻贝(Mytilus coruscus),隶属于软体动物门(Mollusca),双壳纲(Bivalvia),贻贝目(Mytilodia),贻贝科(Mytilidae),是我国重要的海产贝类养殖品种,分布于黄海、渤海和东海沿岸。厚壳贻贝是营养价值较高的海水经济贝类,具有较高的食用价值、广阔的养殖前景和市场开发潜力。厚壳贻贝作为一种重要的海贝养殖品种,其苗种主要依靠天然苗种和半人工采苗获得,种苗问题制约了厚壳贻贝养殖产业的发展。在厚壳贻贝生活史中,浮游生活阶段的幼虫成熟后,便会附着变态成为稚贝,并最终发育为成贝。在由稚贝向成贝生长发育的过程中,当环境发生变化时,厚壳贻贝稚贝能够自行切断足丝,开始爬行并重新选择适宜的附着基进行再次附着。因此,解决种苗问题、探索利用细菌形成微生物被膜诱导厚壳贻贝附着技术等生物工程技术显得尤为必要。
发明内容
本发明涉及的深海盐单胞菌(Halomonas sp.ROE5-16),分离自太平洋所罗门海,新不列颠海沟(E 151.98°,S 6.04°)水下5000米处的海水。利用ZoBell 2216E固体培养基分离、纯化得到纯种菌落,并通过16S rRNA基因测序与GENEBANK(http://www.ncbi.nlm.nih.gov/) 中核酸数据进行比对后上传至NCBI数据库。最终确定菌名为盐单胞菌,获取序列号为: MG252258,该细菌保藏于中国典型培养物保藏中心(CCTCC),保藏日期为2018年1月29 日,保藏号为CCTCC M 2018066,地址:湖北省武汉市武昌区八一路299号武汉大学校内,邮编:430072。
还涉及一种上述深海盐单胞菌在诱导厚壳贻贝稚贝附着中的应用。
进一步地,将纯种菌株接种到海水LB液体培养基中扩大培养,离心后得到细菌沉淀,用灭菌海水洗涤并制成悬浮液,制得深海盐单胞菌的菌液初始细菌密度为1-500×106cells/ml。
该细菌形成的微生物被膜对厚壳贻贝稚贝的附着具有相对较高的诱导活性。
附图说明
图1为不同初始细菌密度形成的微生物被膜对厚壳贻贝稚贝附着的诱导作用。
具体实施方式
下面通过对最优实施例的描述,对本发明的具体实施方式作进一步详细的说明。
菌株的分离及保藏:
本发明涉及的深海盐单胞菌(Halomonas sp.ROE5-16),分离自太平洋所罗门海,新不列颠海沟(E 151.98°,S 6.04°)水下5000米处的海水。利用ZoBell 2216E固体培养基分离、纯化得到纯种菌落,并通过16S rRNA基因测序与GENEBANK(http://www.ncbi.nlm.nih.gov/) 中核酸数据进行比对后上传至NCBI数据库。最终确定菌名为盐单胞菌,获取序列号为: MG252258,该细菌保藏于中国典型培养物保藏中心(CCTCC),保藏日期为2018年1月29 日,保藏号为CCTCC M 2018066,地址:湖北省武汉市武昌区八一路299号武汉大学校内,邮编:430072。
本发明涉及的深海盐单胞菌对厚壳贻贝稚贝附着的诱导实验。主要步骤是将纯种菌株接种到海水LB液体培养基中扩大培养,离心后得到细菌沉淀,用灭菌海水洗涤并制成悬浮液,重复多次实验。最后将得到的菌液稀释后用吖啶橙染色并在荧光显微镜1000倍镜头下随机选取10个点进行计数,确定菌液总密度为4×109cells/ml。每个培养皿中放入一枚灭菌载玻片,向培养皿中加入灭菌海水及菌液,使细菌初始密度分别为1.0×106cells/ml、1.0×107cells/ml、 1.0×108cells/ml和5×108cells/ml,在18℃、避光条件下培养48h形成微生物被膜。最后将此枚附有微生物被膜的载玻片及10只厚壳贻贝稚贝(壳长1.31±0.02mm;壳高0.89±0.02mm) 放入含有灭菌过滤海水的培养皿中,在12h时记录稚贝的附着率,即载玻片上附着的稚贝个数占该培养皿中稚贝总个数的百分比。
研究发现该细菌形成的微生物被膜对厚壳贻贝稚贝的附着具有相对较高的诱导活性,实验结果如图1所示,在初始密度为1×108cells/ml时,形成的微生物被膜对稚贝附着的诱导率为43.33%,相比与无微生物被膜的对照组提高了16.67%。另外,不同初始密度条件下形成的微生物被膜对稚贝的诱导活性也有所不同,初始密度为1.0×106cells/ml时形成的微生物被膜对稚贝的诱导活性仅为26.66%。由此可得,该株深海盐单胞菌形成的微生物被膜能够诱导厚壳贻贝稚贝的附着,本研究成果对于阐明厚壳贻贝稚贝附着机制以及对厚壳贻贝的人工养殖均具有重要的意义。
上面对本发明进行了示例性描述,显然本发明具体实现并不受上述方式的限制,只要采用了本发明的方法构思和技术方案进行的各种改进,或未经改进直接应用于其它场合的,均在本发明的保护范围之内。
Claims (3)
1.一种深海盐单胞菌,保藏编号为:CCTCC M 2018066,分类命名:盐单细胞菌Halomonas sp.。
2.一种根据权利要求1所述的深海盐单胞菌在诱导厚壳贻贝稚贝附着中的应用。
3.根据权利要求2所述的应用,其特征在于,将纯种菌株接种到海水LB液体培养基中扩大培养,离心后得到细菌沉淀,用灭菌海水洗涤并制成悬浮液,制得深海盐单胞菌的菌液初始细菌密度为1-500×106cells/ml。
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CN112574914A (zh) * | 2020-12-15 | 2021-03-30 | 上海海洋大学 | 深海嗜盐单胞菌及其在诱导厚壳贻贝附着中的应用 |
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CN112159785A (zh) * | 2020-11-03 | 2021-01-01 | 厦门大学 | 麦氏交替单胞菌e6及其应用 |
CN112574914A (zh) * | 2020-12-15 | 2021-03-30 | 上海海洋大学 | 深海嗜盐单胞菌及其在诱导厚壳贻贝附着中的应用 |
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