CN114717145B - 一株黄瓜金黄杆菌及其在降解海藻渣中的应用 - Google Patents
一株黄瓜金黄杆菌及其在降解海藻渣中的应用 Download PDFInfo
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- C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
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Abstract
本发明提供一株黄瓜金黄杆菌及其相应产品和应用。所述黄瓜金黄杆菌3T34的分类命名为黄瓜金黄杆菌(Chryseobacterium cucumeris),该菌株于2020年5月20日保藏在中国微生物菌种保藏管理委员会普通微生物中心,其保藏编号为CGMCC No.19790。该金黄杆菌不仅能高效降解海藻渣,而且含有能够降解乙烯合成前体的ACC脱氨酶。该菌株不仅能为植物提供转化降解海藻渣提供植物生长所需营养元素,并且在逆境条件下,能够增强植物的耐受性和促进植物的生长。
Description
发明领域:
本发明属于微生物和微生物肥料领域,具体涉及一株黄瓜金黄杆菌及其在降解海藻渣和作为植物根际促生菌在微生物肥料中的应用。
背景技术:
我国海洋海藻资源十分丰富,有经济价值的海藻有褐藻、红藻、绿藻和少量蓝藻等大型种类。迄今为止,海藻已在食品、海洋药物、动物饲料、有机肥料、化妆品及生物能源等领域和范围体现出巨大的开发潜力和经济价值。在海藻的加工利用过程中,由于现有工艺条件的限制,海藻中养分不能被完全提取出来,剩余的部分残留在废弃的海藻渣中。因此,这些海藻渣富含丰富的营养,除含有部分水分外,还有海藻酸、甘露醇、甜菜碱、壳聚糖、多酚等,另外还包括海藻纤维、果胶、蛋白质、木质素等物质。因此,如何将海藻渣进一步加工利用,最大程度地发挥其价值,成为人们日益关心的问题。本文以海藻酸提取后的废渣为原料,采用微生物降解解的方法提取剩余营养物质和生产微生物菌剂,从而为海藻渣的废物利用和植物生物刺激素在农业中的应用开辟了一条新的途径。
海藻裂解酶来源广泛,种类多样,已接近100种海藻酸盐裂解酶从海洋和陆地细菌、海洋软体动物和藻类等不同物种中被分离、鉴定、克隆和纯化。海藻酸裂解酶按照产酶位置可分为胞内酶和胞外酶,按照酶切方式可分为内切酶和外切酶。海洋细菌是海藻酸裂解酶最主要的来源,如海洋弧菌(Vibriosp.)、假交替单胞菌属(Pseudoalteromonas sp.)等从腐烂的海带中分离的细菌。这些细菌限制了在农业中的应用发展。因此,从农田土壤中筛选高效降解褐藻胶的新菌种,寻找适合海藻提取物生产的褐藻胶裂解酶,是开发褐藻资源,探索其高值化利用新途径的必然要求。
发明内容
本研究从山东青岛即墨农场的土壤中筛选得到一株高产海藻酸裂解酶的黄瓜金黄杆菌3T34,该菌株产酶发酵时间短,转化利用海藻渣能力强。通过基因组测序、转录组测序发现,该菌在高效降解海藻的同时,还能够产生ACC脱氨酶提高植物逆境抗性。
一方面,本申请提供了一株黄瓜金黄杆菌3T34,其特征在于:所述黄瓜金黄杆菌3T34的分类命名为黄瓜金黄杆菌(Chryseobacteriumcucumeris),该菌株于2020年5月9日保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为:北京市朝阳区北辰西路1号院3号,其保藏编号为CGMCC No.19790。
另一方面,本申请提供了上述瓜金黄杆菌3T34在降解海藻渣中的应用。
另一方面,本申请提供了上述瓜金黄杆菌3T34在制备微生物肥料中的应用。
另一方面,本申请提供了一种微生物菌剂,其包含上述瓜金黄杆菌。
另一方面,本申请提供了一种微生物肥料,其包含上述瓜金黄杆菌。
进一步地,所述微生物肥料由上述瓜金黄杆菌发酵海藻渣制备而成。
进一步地,所述发酵中干重海藻渣的加入量为培养基的2.5%w/v。
进一步地,所述发酵所用培养基中菌液加入量为培养基的1%v/v。
进一步地,所述发酵所用的培养基还包含磷酸二氢钾、七水硫酸镁、磷酸氢二钾、无水氯化钙、乙二胺四乙酸二钠。
进一步地,所述发酵过程中180rpm震荡通气。
本申请中的海藻渣可选各种海藻,包括但不限于红藻、绿藻、褐藻;海藻渣包括但不限于各种加工目的和加工方式生产中的产生的海藻渣。
除了上述成分外,本申请的微生物肥料中还可以包含其他微生物、营养成分。
附图说明
图1为筛选菌株及其在碘液染色中产生的透明圈照片。
图2为根据全基因组测序构建的***树:通过与本地数据库对比,基于31个看家基因(dnaG,frr,infC,nusA,pgk,pyrG,rplA,rplB,rplC,rplD,rplE,rplF,rplK,rplL,rplM,rplN,rplP,rplS,rplT,rpmA,rpoB,rpsB,rpsC,rpsE,rpsI,rpsJ,rpsK,rpsM,rpsS,smpB,tsf)选择在种属水平上最接近的19株菌,通过MEGA 6.0软件选择NJ(Neighbor-Joining)法构建***进化树。
图3为利用16S基因构建的***进化树。
图4为转录组测序韦恩图T34-A为海藻胶无机盐培养基,T34-G为葡萄糖无机盐培养基所培养的菌体。
图5为转录组测序上调和下调的火山图:横坐标为基因在两组样本间表达差异的倍数变化值,即FC值。纵坐标为基因表达量变化差异的统计学检验值,即p值。p值越高则表达差异越显著,横纵坐标的数值都做了对数化处理。图中每个点代表一个特定的基因,红色点表示显著上调的基因,绿色点表示显著下调的基因,灰色点为非显著差异基因(在说明书灰度附图中可能无法完全清晰地展示)。将所有基因映射上去之后,可以获知,在左边的点为表达差异下调的基因,右边的点为表达差异上调的基因,越靠两边和上边的点表达差异越显著。
图6为培养方式和时间对于海藻渣的降解效率的影响图。
图7为金黄杆菌3T34海藻渣发酵液促进西红柿苗健康生长效果图。
具体实施方式
下面结合具体实施例详述本发明,但本发明的保护范围不局限于以下实施例。
实施例1黄瓜金黄杆菌(Chryseobacterium cucumeris)3T34的筛选和鉴定
(1)土壤采集及降解海藻酸钠菌株富集
2019年3月采集山东省青岛市即墨区中国农业科学院烟草研究所即墨试验基地多年植烟土壤。将采集的土壤室温风干过筛,去除大的石块等杂物,取1kg土壤添加1%的海藻酸钠粉末混合后放入花盆,浇足自来水,室温孵育四个星期。
(2)菌株筛选
称取10克孵育土壤,溶于装有90ml无菌水的三角瓶内,充分震荡摇匀得到10-1稀释液。取10-1稀释度的土壤悬浮液到另一瓶装有90ml无菌水的三角瓶,充分震荡摇匀,得到10-2稀释液。以此类推,得到10-3,10-4,10-5,10-6稀释液。吸取100微升后三个稀释梯度的稀释液涂布到筛选培养基上,置30℃培养箱中培养48小时(图1)。培养基配方参照文献Sawant S.S.et al 2015(Sawant S.S.,Salunke B.K.,Kim B.S.A rapid,sensitive,simple plate assay for detection of microbial alginate lyase activity.Enzymeand Microbial Technology(2015)8–13.)待培养基上长出清晰可见的菌落时,事先拍照留作原始菌落大小计算。之后,用灭菌牙签挑取菌落至新的筛选培养基平板上做一备份,30℃培养48小时后放入4℃冰箱。筛选菌种时的培养基配方(0.5g/L蛋白胨,0.3g/L酵母提取物,2g/L海藻酸钠,2g/L硫酸铵,1g/L磷酸二氢钾,0.5g/L七水硫酸镁和5g/L琼脂)。
(3)菌株降解能力确定
采用碘液染色法形成的透明圈,初步确定降解海藻酸钠的菌株。将1ml无菌水滴加到菌落平板上,利用玻璃涂布棒将菌体从平板上剥离掉,革兰氏碘液购自上海生工生物工程公司(产品编号A642190)。滴加碘液大约5ml,放置5-10min观察透明圈大小(典型结果如图1所示)。
(4)菌株的纯化和降解能力确认
根据原始平板上的透明圈大小,将对应备份平板上的克隆挑取到LB平板和筛选平板上。30℃培养48小时后,挑取单克隆接种到5ml液体LB培养基试管或者液体筛选培养基试管内继续培养,查看生长情况后,取5微升点到筛选平板。按照以上条件继续培养24小时,检测透明圈(典型结果如图1所示)。对应的阳性菌株,利用终浓度为20%的甘油保存于-80℃冰箱
(5)海藻渣降解利用试验
海藻渣:青岛明月海藻有限公司提供,含水量80%。无机盐铵盐培养基购自青岛海博生物技术公司(产品编号HB8761)磷酸二氢钾3.0,七水硫酸镁0.1,磷酸氢二钾,无水氯化钙0.01,乙二胺四乙酸二钠0.01,以上称取4.62g添加到一升自来水中。由于海藻渣中氮源和碳源丰富,仅在培养基中加入部分无机盐。按照1%的浓度加入菌液,添加干重2.5%的海藻渣,并在30℃温度下180rpm摇床转速震荡培养一周,40目筛子(孔径0.45mm)过滤称重,取上清液得到海藻液肥。以海藻渣培养前后的干物质减少量作为提取率的指标,研究表明利用菌株能够在一周的培养时间内消化掉84%。
实施例2黄瓜金黄杆菌的生物学特征分析如下:
(1)菌株的碳源利用试验
各种单一碳水化合物的利用情况试验采用无机盐铵盐溶液配制后添加0.5%碳源。碳水化合物利用试验所用培养基如下:1.0g/L硝酸铵,1.0g/L磷酸二氢钾,1.0g/L磷酸氢二钾,0.2g/L七水硫酸镁,0.02g/L氯化钙,0.05g/L氯化铁,0.8g/L酵母提取物,5g/L碳源。利用300ml摇瓶发酵100ml液体培养基,30℃200rpm震荡培养48小时测定OD600吸光度。发现该金黄杆菌能够利用蛋白胨、淀粉、酪氨酸、吐温20、吐温80、果胶、羧甲基纤维素、半乳糖醛酸、酒石酸钾钠、柠檬酸钠,不能利用乳糖、岩藻糖、甘露醇、壳聚糖。能利用果糖,麦芽糖和海藻糖产酸,不能利用纤维二塘,甘露糖,木糖。能够进行七叶皂苷降解。
(2)API鉴定试验
操作步骤如下,利用接种针从分离物平板上挑取单个菌落至0.9%灭菌生理盐水中,仔细研磨震荡,使菌体均一,按照API试剂盒使用说明书,将菌悬液充满管部或者杯部,部分小孔用矿物油覆盖以形成厌氧环境,盖上培养盒,孵育18-24小时。B3T34菌落半透明黄色,圆形。能够在10℃-38℃生长,pH 5.0–8.0(最适温度,pH 6.0–8.0)LB,含有1–3%NaClNB(最适盐度1–2%NaCl),氧化酶和过氧化氢酶活性为正,但不能还原亚硝酸盐。API20E试剂盒(梅里埃公司)检测发现,β-半乳糖酶,脲酶,明胶酶和产生吲哚为阳性。然而,均不产生或者利用精氨酸二氢酶、赖氨酸和鸟氨酸脱羧酶、柠檬酸利用、H2S生成、色氨酸脱氨酶、乙偶姻生成和硝酸盐还原均为负值。不能发酵D-葡萄糖、D-甘露醇、肌醇、D-蔗糖、杏仁苷、L-***糖、D-蜜二糖、L-鼠李糖或D-山梨醇产酸。
(3)16S基因鉴定试验
利用细菌通用引物16S-27F(5’-AGAGTTTGATCMTGGCTCAG-3’)和16S-1492R(5’-TACGGYTACCTTGTTACGACTT-3’),对所分离菌株进行菌落PCR扩增。利用灭菌牙签蘸取少许菌落,放到50微升灭菌重蒸水中悬浮,看到菌液少许浑浊为宜。在50微升PCR体系中,先后加入两倍Taq PCRMaster Mix 25μl,无菌水22μl,上下游引物(10μM)各1μl,上述稀释菌液1μl,混合离心后进行PCR扩增。扩增条件为95℃预变性3min,95℃变性30second,55℃退火30sec,72℃延伸1.5min,30个循环,最后72℃延伸10min,送交青岛擎科生物技术公司检测测序。在NCBI(http://www.ncbi.nlm.nih.gov/)数据库上进行BLAST比对分析,再次在NZBioCloud网站(https://www.ezbiocloud.net/)上确认。从NCBI数据库下载近似菌株的16S序列,利用mega 6.0构建***进化树。根据菌落大小,色泽,生长速度等特征,发现从筛选平板上鉴定到的菌株90%以上都是芽孢杆菌属,本专利中申请所用的菌株大约占到了富集菌株的5%,经过16S进一步鉴定为黄瓜金黄杆菌(Chryseobacterium cucumeris B3T34)(图3),保藏编号为:CGMCC No.19790。16SrRNA序列为SEQ ID NO.1。
(4)基因组测序分析碳水化合物利用酶系基因和促生基因
利用LB培养基培养菌体到对数生长期,离心收集菌体送交上海美吉测序公司。经测序分析发现菌株B3T34基因组大小为5,069,125bp,组装成36个Scaffold,GC含量为36.85%,编码4750个蛋白质,含有82个tRNA,5个rRNA。组装情况如下:框架总长Large ScafBases(bp)5,069,125bp,框架总数36个;大框架数目为26个,最长框架Largest Scaf Len(bp)为1,166,174bp;Scaf N50(bp)为723,819bp,Scaf N90(bp)118,797bp.C测序深度为259.88倍碳水化合物降解酶系:糖苷水解酶GH家族59个,糖基转移酶58个,碳水化合物酯酶49个,具有降解木质素活性的单元有9个,碳水化合物结合域2个。分泌***预测仅发现一个I型分泌***,没有发现II,III,IV,V,VI型分泌***,说明没有潜在的植物病原性。
根据全基因组测序构建***树通过与本地数据库对比,基于31个看家基因(dnaG,frr,infC,nusA,pgk,pyrG,rplA,rplB,rplC,rplD,rplE,rplF,rplK,rplL,rplM,rplN,rplP,rplS,rplT,rpmA,rpoB,rpsB,rpsC,rpsE,rpsI,rpsJ,rpsK,rpsM,rpsS,smpB,tsf)选择在种属水平上最接近的19株菌,通过MEGA 6.0软件选择NJ(Neighbor-Joining)法构建***进化树,显示该菌与产吲哚金黄杆菌亲缘关系最近(图2)。最近报道,从4年生西洋参植物根组织中分离到的产吲哚金黄杆菌PgBE177对病原菌丁香假单胞菌具有拮抗活性,并且具有刺激植物生长的潜力(Complete Genome Sequence of the Endophytic BacteriumChryseobacterium indologenes PgBE177,Isolated from Panax quinquefoliusMicrobiol Resour Announc.2018)。
基因组分析发现,3T34菌株还有ACC脱氨酶。ACC是乙烯生物合成的直接前体,经过ACC氧化酶的作用,产生乙烯。乙烯的的生物合成途径:Met(蛋氨酸)-SAM(S-腺苷蛋氨酸)-ACC(1-氨基环丙烷羧酸)植物体内的甲硫氨酸首先在三磷酸腺苷参与下,转变成S-腺苷甲硫氨酸(简称SAM),在ACC合成酶的作用下SAM被转化为1-氨基环丙烷-1-羧酸(简称ACC)。有些植物促生菌能够分泌ACC脱氨酶,将ACC催化形成酮丁酸和铵离子,不但为植株的生长提供营养,而且减轻了逆境下乙烯的积累导致的植物萎蔫死亡。该菌株的基因1575编码ACC脱氨酶,蛋白序列为SEQ ID NO.2。
(5)转录组测序比较分析降解酶系
比较转录组的测序所用培养基配方:无机盐铵盐培养基购自青岛海博生物技术公司(产品编号HB8761)磷酸二氢钾3.0g/L,七水硫酸镁0.1g/L,磷酸氢二钾1.0g/L,无水氯化钙0.01g/L,乙二胺四乙酸二钠0.01g/L,以上称取4.62g,添加硝酸铵2.0g。先将菌体培养在LB培养基中生长到OD600大约3.0,离心收集菌体添加到分别还有1%的海藻酸钠或者1%的葡萄糖无机盐铵盐液体培养基(0.8g/L酵母粉)接种后的OD600约为0.5。30℃震荡培养两小时,离心收集提交给上海美吉测序公司。
经过测序分析,菌株B3T34在褐藻胶Alginate和葡萄糖Glucose无机盐培养基中的转录组测序原始数据质控分析中Raw Q20和Raw Q30都分别达到了96%和91%,cleanreads分别达到了2600万条和3245万条,clean bases分别达到了3432Mb和4164Mb。
菌株B3T34在褐藻胶Alginate无机盐培养基中的转录组测序总共得到了TotalReads数目为2600万条,比对到基因组中的有2576万条,占比99.04%,独一比对率为96.52%。在葡萄糖Glucose无机盐培养基中的转录组测序总共得到了Total Reads数目为3246万条,其中有3193万条比对到了该菌株的基因组,比对率为98.38%,独一比对率为94.97%。
通过韦恩图和火山图可以看到上调和下调的基因数量差异(图4和图5)。比对发现该菌中还有大量可以降解多糖交联的酶系得到了高表达,比如α-L-岩藻糖苷酶,α-N-***呋喃糖苷酶,β-半乳糖苷酶,β1,4木聚糖酶等。尤其是海藻糖酶的表达量最高,log2FC达到了3.82(表1)。
表1 菌株3T34中高表达碳水化合物水解酶系
ACC脱氨酶gene1575(SEQ ID NO.3)在海藻胶培养基中也得到了高表达,说明在使用该菌株降解生成的海藻肥,不仅能为植物提供营养,还能提高植物的抗逆性。
实施例3黄瓜金黄杆菌(Chryseobacterium cucumeris)3T34的海藻渣降解效果评价
(1)菌株的活化和增殖
从-80℃冰箱取出甘油保藏菌株3T34,划线分离于LB培养固体基。30℃培养24小时,可见黄色边缘完整的小菌落。利用灭菌牙签挑取一个菌落接种于盛有10ml LB液体培养基的10×200mm试管中,置于30℃摇床180rpm震荡培养24小时,留作接种液。LB培养基购自青岛海博生物技术公司,其成分为:胰蛋白胨10.0g/L,酵母浸粉5.0g/L,氯化钠10.0g/L。每升超纯水中加入25克LB粉末,如果配制固体培养基的话,再加15-20克琼脂。121℃15min高压灭菌备用。
(2)发酵培养基培养
海藻渣:青岛明月海藻有限公司提供,含水量80%。无机盐铵盐培养基购自青岛海博生物技术公司(产品编号HB8761):磷酸二氢钾3.0,七水硫酸镁0.1,磷酸氢二钾1.0,无水氯化钙0.01,乙二胺四乙酸二钠0.01(g/L),以上称取4.62g添加到一升自来水中。由于海藻渣中氮源和碳源丰富,仅在培养基中加入部分无机盐。按照1%的浓度加入菌液,添加干重(按照海藻渣的干重计)2.5%的海藻渣,并在30℃温度下震荡培养一周,40目筛子(孔径0.45mm)过滤称重,取上清液得到海藻液肥。以海藻渣培养前后的干物质减少量作为提取率的指标,海藻渣干物质的添加量设定为0.5%,1%,2.5%,5%。比较静置培养和摇床培养发现(图6,图6中的四个柱从左到右分别表示0.5%,1%,2.5%,5%的添加量)摇床通气培养能够加速海藻渣的降解,考虑到降解率和总产率的关系,利用该菌株在一周的培养时间内消化掉84%作为生产海藻渣的条件。
实施例4黄瓜金黄杆菌(Chryseobacterium cucumeris)3T34的海藻渣发酵液促进西红柿苗健康生长
将6-7叶期的西红柿幼苗移栽到直径10cm的塑料盆中,使用缺乏营养的基质培养:采用即墨试验基地的农田土壤200克添加等体积的无营养成分蛭石(约20克)。移栽两天后做以下两个处理。处理一:培养一周时间的2.5%海藻渣摇瓶发酵液过滤后的上清液约100ml,平均分为三份浇到三盆中;处理二:等量的未发酵海藻渣添加到无机盐溶液(青岛海博生物技术公司产品编号HB8761剔除铵盐成分)中,未发酵海藻渣的终浓度为2.1%,100ml溶液平均分为三份浇到三盆中。空白对照不添加海藻渣,仅添加无机盐溶液,继续培养两周。以上三个处理,每个处理重复三次。放置于25℃的光照培养箱内,设定日照时间为十二个小时。研究结果如图7所示,发现添加该菌发酵液的西红柿幼苗生长旺盛,绿叶多而深绿,而对照和仅添加海藻渣的西红柿幼苗生长不佳,叶面少并且发黄。这说明了该菌株3T34通过改善根际环境,能够促进西红柿在贫瘠的土壤中生长。
SEQUENCE LISTING
<110> 广西中烟工业有限责任公司
<120> 一株黄瓜金黄杆菌及其在降解海藻渣中的应用
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 1423
<212> DNA
<213> 16S rRNA
<400> 1
tcatttcggg ggggctacac atgcagccga gcggtagaga tctttcggga tcttgagagc 60
ggcgtacggg tgcggaacac gtgtgcaacc tgcctttatc tgggggatag cctttcgaaa 120
ggaagattaa taccccataa tatactggat ggcatcattc ggtattgaaa actccggtgg 180
atagagatgg gcacgcgcaa gattagatag ttggtgaggt aacggctcac caagtctgcg 240
atctttaggg ggcctgagag ggtgatcccc cacactggta ctgagacacg gaccagactc 300
ctacgggagg cagcagtgag gaatattgga caatgggtga gagcctgatc cagccatccc 360
gcgtgaagga cgacggccct atgggttgta aacttctttt gtatagggat aaacctaccc 420
tcgtgagggt agctgaaggt actatacgaa taagcaccgg ctaactccgt gccagcagcc 480
gcggtaatac ggagggtgca agcgttatcc ggatttattg ggtttaaagg gtccgtaggc 540
ggatctgtaa gtcagtggtg aaatctcaca gcttaactgt gaaactgcca ttgatactgc 600
aggtcttgag tgttgttgaa gtagctggaa taagtagtgt agcggtgaaa tgcatagata 660
ttacttagaa caccaattgc gaaggcaggt tactaagcaa caactgacgc tgatggacga 720
aagcgtgggg agcgaacagg attagatacc ctggtagtcc acgccgtaaa cgatgctaac 780
tcgtttttgg agcgcaagct tcagagacta agcgaaagtg ataagttagc cacctgggga 840
gtacgaacgc aagtttgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggatta 900
tgtggtttaa tttcgatgat acgcgaggaa ccttaccaag gcttaaatgg gaaatgacag 960
gtttagaaat agacttttct tcggacattt ttcaaggtgc tgcatggttg tcgtcagctc 1020
gtgccgtgag gtgttaggtt aagtcctgca acgagcggca acccctgtca ctagttgcca 1080
tcattaagtt ggggactcta gtgagactgc ctacgcaagt agagaggaag gtggggatga 1140
cgtcaaatca tcacggccct tacgccttgg gccacacacg taatacaatg gccggtacag 1200
agggcagcta cactgcgaag tgatgcaaat ctcgaaagcc ggtctcagtt cggattggag 1260
tctgcaactc gactctatga agctggaatc gctagtaatc gcgcatcagc catggcgcgg 1320
tgaatacgtt cccgggcctt gtacacaccg cccgtcaagc catggaagtc tggggtacct 1380
gaagtcggtg accgtaacag gagctgccta ggtaagcatg tta 1423
<210> 2
<211> 300
<212> PRT
<213> 1-aminocyclopropane-1-carboxylate deaminase
<400> 2
Met Leu Leu Lys Leu Pro Thr Glu Pro Val Tyr Ile Gln Glu Ile Pro
1 5 10 15
Ile Gln Lys Asn Ile Lys Leu Phe Ile Lys Arg Glu Asp Gln Ile His
20 25 30
Pro Leu Ile Ser Gly Asn Lys Tyr Trp Lys Leu Phe His Asn Val Asn
35 40 45
Asn Tyr Leu Glu Lys Asn Gln Gly Asn Pro Tyr Ile Ile Thr Phe Gly
50 55 60
Gly Ala Phe Ser Asn His Ile Ser Ala Val Ser Ala Val Gly Asn Arg
65 70 75 80
Ala Gly Ile Pro Thr Leu Gly Ile Ile Arg Gly Glu Glu Leu Glu His
85 90 95
Lys Trp Arg Asp Asn Pro Thr Leu Leu Phe Ala Lys Arg Asn Gly Met
100 105 110
Asn Leu Lys Phe Val Thr Arg Glu Glu Tyr Arg His Lys Glu Lys Leu
115 120 125
Thr Glu Phe Leu Gln Gln Glu Phe Pro Glu Ala Leu Ile Val Pro Glu
130 135 140
Gly Gly Thr Asn Lys Glu Ala Val Glu Gly Val Lys Met Met Leu Asn
145 150 155 160
Glu Gln Thr Lys Asp Phe Asp Tyr Leu Cys Thr Ala Val Gly Thr Gly
165 170 175
Gly Thr Ile Ala Gly Ile Ser Lys Phe Cys Glu Glu Asn Gln Lys Val
180 185 190
Ile Gly Phe Lys Ala Val Asp Asp Ala Ser Leu Glu Asn Lys Ile Phe
195 200 205
Glu Leu Thr Leu Lys Gln Asn Phe Asn Leu Ile Asp Ser Cys Phe Gly
210 215 220
Gly Tyr Gly Lys Ile Ser Asp Gly Asn Val Arg Phe Ile Asn Asp Phe
225 230 235 240
Lys Glu Arg Tyr Gly Ile Pro Leu Glu Pro Ile Tyr Thr Gly Lys Met
245 250 255
Met Glu Lys Val Phe Glu Met Ile Glu Glu Glu Tyr Phe Pro Glu Asn
260 265 270
Ser Lys Ile Leu Cys Phe His Thr Gly Gly Leu Gln Gly Ile Glu Gly
275 280 285
Ala Asn Leu Leu Leu Glu Lys Gln Asn Arg Asn Leu
290 295 300
<210> 3
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<213> 1-aminocyclopropane-1-carboxylate deaminase
<400> 3
atgctattaa aacttcccac agaacctgtt tacatccagg aaattcccat tcagaaaaat 60
attaaacttt tcattaaaag ggaagatcag attcatccgt tgatttcagg aaataaatac 120
tggaagcttt ttcacaatgt caataactat ctcgaaaaaa atcagggaaa cccttatatc 180
attacttttg gaggtgcttt ttccaatcac atttctgcgg tttctgccgt aggaaatagg 240
gcgggtattc caacactggg aatcatcaga ggagaagaac tggagcataa atggcgcgat 300
aacccaacct tactttttgc gaaaagaaat gggatgaacc tgaaatttgt cacccgcgaa 360
gaataccgcc acaaagaaaa actgacagaa ttccttcagc aggagttccc tgaagctttg 420
atagttcctg aaggaggaac caataaagag gctgtagaag gagtgaaaat gatgctcaac 480
gaacaaacaa aagattttga ctatctttgc accgcagttg gaaccggtgg taccattgca 540
ggaatttcaa aattttgtga agagaatcag aaagttatag gatttaaagc cgttgacgat 600
gcttcacttg aaaataaaat ttttgaatta actttgaaac agaattttaa tctaatagat 660
tcatgttttg gaggttatgg taaaataagt gatggaaacg tccgttttat caatgatttc 720
aaggaaagat acggtattcc tctggaaccg atttatacag ggaaaatgat ggagaaggtt 780
tttgaaatga tagaagaaga atattttcct gaaaacagta agattttgtg ctttcacact 840
ggtggtttac agggaattga aggagcgaat ctgctgttgg aaaaacagaa tagaaattta 900
attatataa 909
<210> 4
<211> 20
<212> DNA
<213> 16S-27F
<400> 4
agagtttgat cmtggctcag 20
<210> 5
<211> 22
<212> DNA
<213> 16S-1492R
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tacggytacc ttgttacgac tt 22
Claims (6)
1.一株黄瓜金黄杆菌(Chryseobacterium cucumeris)3T34,其特征在于:所述黄瓜金黄杆菌3T34保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCCNo. 19790。
2.根据权利要求1所述的黄瓜金黄杆菌3T34在降解海藻渣中的应用。
3.根据权利要求1所述的黄瓜金黄杆菌3T34在制备微生物肥料中的应用。
4.一种微生物菌剂,其特征在于,其包含根据权利要求1所述的黄瓜金黄杆菌3T34。
5.一种微生物肥料,其特征在于,其包含根据权利要求1所述的黄瓜金黄杆菌3T34。
6.根据权利要求5所述的微生物肥料,其中所述微生物肥料由根据权利要求1所述的瓜金黄杆菌发酵海藻渣制备而成。
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