CN111154675B - 酸快生芽孢杆菌syy15及其应用 - Google Patents
酸快生芽孢杆菌syy15及其应用 Download PDFInfo
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- CN111154675B CN111154675B CN202010024051.3A CN202010024051A CN111154675B CN 111154675 B CN111154675 B CN 111154675B CN 202010024051 A CN202010024051 A CN 202010024051A CN 111154675 B CN111154675 B CN 111154675B
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Abstract
本发明公开了酸快生芽孢杆菌SYY15及其应用。菌株SYY15保藏号为CCTCC NO:M 2019840,该菌不仅能合成ACC脱氨酶,增强作物的抗逆性,还能大量降解难溶性有机磷和无机磷,产生IAA、蛋白酶等多种促生物质。以玉米为例,菌株SYY15对促进植物的抗逆效果明显。与盐胁迫组相比,菌株SYY15处理组的种子发芽系数可以提高34.10%。菌株SYY15应用在农作物种植中不仅能提高农作物产量,还能提高植物体抗病、抗旱等抗逆性,对我国粮食持续增产和保护农田生态环境具有重要意义。
Description
技术领域
本发明涉及农业微生物学,具体地说,涉及一株酸快生芽孢杆菌SYY15及其应用。
背景技术
胁迫被定义为对植物生长有负面影响的因素,任何形式的胁迫都会增加活性氧(ROS)的形成,如H2O2、O2-、和OH-。过量的活性氧易引起氧化应激,通过氧化光合色素、脱脂、蛋白质和核酸来破坏植物。非生物胁迫(大风、极端温度、干旱、盐渍化、洪涝等)在生物质生产和主粮作物的产量上有高达70%的负面影响,威胁到全球的粮食安全。植物经常受到各种环境胁迫,并形成了特定的响应机制,在过去的几十年中,通过各方面的研究,人们已经了解了非生物和生物的抗逆性的作用机理。已有报道,PGPR可以改善叶片水分状况,尤其是在盐碱等非生物的胁迫下。PGPR与抗旱性的相关性也已经被报道了。2016年Habib等报道了PGPR通过活性氧清除酶提高秋葵盐胁迫耐受性和提高水分利用率的研究。也有酸快生芽孢杆菌具有溶磷、解钾的作用,可以促进花生的生长和提高产量(汪强等,2014)。但未有在植物抗逆胁迫促生和盐碱地修复方面的报道,本发明提供了一株酸快生芽孢杆菌SYY15菌株不仅具有促生的功能,还可以提高作物对外界胁迫的耐受性,提高作物抗逆性。
发明内容
本发明的目的是提供一株酸快生芽孢杆菌SYY15及其应用。
为了实现本发明目的,第一方面,本发明提供从河北邢台威县根力多生物科技股份有限公司试验示范基地采集的土壤中分离出的一株酸快生芽孢杆菌SYY15(Bacillusacidiceler SYY15),菌株SYY15不仅能够利用ACC为唯一氮源,还具有较强的溶解难溶性有机磷和无机磷功能,产生IAA(吲哚乙酸)和蛋白酶。该菌现已保藏于中国典型培养物保藏中心,地址:中国武汉,武汉大学,邮编430072,保藏编号CCTCC NO:M 2019840,保藏日期2019年10月21日。
第二方面,本发明提供含有所述酸快生芽孢杆菌SYY15的菌剂。
第三方面,本发明提供由所述酸快生芽孢杆菌SYY15分泌产生的促生物质,如生长素(IAA)等。
第四方面,本发明提供由所述酸快生芽孢杆菌SYY15分泌产生的酶,所述酶包括但不限于ACC脱氨酶、蛋白酶。
第五方面,本发明提供由所述酸快生芽孢杆菌SYY15或其菌剂制备的农用肥料、磷素活化剂或植物促生长剂。
第六方面,本发明提供所述酸快生芽孢杆菌SYY15或其菌剂的以下任一应用:
1)用于促进植物生长发育以及提高作物产量;
2)用于提高植物抗逆性;
3)用于溶磷;
4)用于制备农用肥料;
5)用于制备磷素活化剂;
6)用于制备植物促生长剂。
本发明中所述植物包括但不限于玉米、小麦、大豆、黄瓜。
所述提高植物抗逆性是指提高植物体抗病、抗旱、抗盐碱及重金属胁迫。
第七方面,本发明提供所述酸快生芽孢杆菌SYY15或其菌剂在溶解难溶磷中的应用。
前述的应用,所述难溶磷为难溶性有机磷和无机磷,如植酸钙、卵磷脂、磷酸钙等。
借由上述技术方案,本发明至少具有下列优点及有益效果:
本发明提供的酸快生芽孢杆菌SYY15,该菌不仅能合成ACC脱氨酶,增强作物的抗逆性,还能大量降解难溶性有机磷和无机磷,产生生长素(IAA)、蛋白酶等多种促生物质。以玉米(郑单958)为例,菌株SYY15对促进植物的抗逆效果明显。与盐胁迫组相比,菌株SYY15处理组的种子发芽系数可以提高34.10%左右。菌株SYY15应用在农作物种植中不仅能提高农作物产量,还能提高植物体抗病、抗旱等抗逆性,对我国粮食持续增产和保护农田生态环境具有重要意义。
附图说明
图1为本发明实施例2中菌株SYY15在以ACC为唯一氮源培养基中的生长情况。其中,CK:不接菌空白培养基对照。
图2为本发明实施例3中菌株SYY15的溶磷能力测定结果。
图3为本发明实施例4中菌株SYY15产蛋白酶的能力测定结果。
图4为本发明实施例7中菌株SYY15对玉米种子发芽系数的影响。
图5为本发明实施例8中菌株SYY15对玉米促生效果。
图6为本发明实施例1中菌株SYY15的16S rRNA基因的分子进化树。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段,所用原料均为市售商品。
实施例1 菌株SYY15的分离及鉴定
从河北邢台威县根力多生物科技股份有限公司试验示范基地采集土壤,取10g土样加入装有100ml无菌生理盐水的三角瓶中,静止20min后,28℃,200rpm摇床震荡30min,取1ml样本加入9ml无菌生理盐水中,再依次稀释102,103,104倍,分别取以上土壤悬浮液100μl在LB培养基(酵母粉5g/L,蛋白胨10g/L,NaCl 10g/L,琼脂15g/L)平板上均匀涂板,28℃培养箱内培养48h,用无菌牙签挑取细菌单菌落划线纯化后,保存备用。
综合菌株SYY15的生理生化特性、分子生物学检测结果,将其鉴定为酸快生芽孢杆菌(Bacillus acidiceler)。具体鉴定结果如下:
1、分子生物学鉴定
首先对菌株SYY15进行分子生物学的鉴定,使用引物27F:5′-AGAGTTTGATCCTGGTCAGAACGAACGCT-3′和1492R:5′-TACGGCTACCTTGTTACGACTTCACCCC-3′扩增其16S rRNA基因序列,并进行测序,测序结果如SEQ ID NO:1所示。
PCR反应体系为:2×Taq Mix 12.5μl,引物27F、1492R各1μl,DNA模板1μl,ddH2O9.5μl。PCR反应条件为:94℃预变性5min;94℃变性30s,56℃退火30s,72℃延伸1min,30个循环;72℃延伸5min。其中,Taq Mix购自TaKaRa公司。
PCR扩增产物用1%琼脂糖凝胶电泳分析,用OMEGA公司的纯化试剂盒GelExtraction Kit纯化回收后连接TaKaRa公司的T载体pMD18-T。用ABI 3730 DNA测序仪进行测序,基因序列已提交至GenBank,收录号:MN559043。将序列用GEGA5进行分子进化树分析(图6),根据进化树初步将SYY15判定为酸快生芽孢杆菌。
2、生理生化特性
在分子生物学检测的基础上,对菌株进行生理生化鉴定,首先对其进行革兰氏染色,结果菌株SYY15为革兰氏阳性;将菌活化后分别接种到NaCl浓度为3%、5%的LB培养基中,28℃培养48h,结果只有3%NaCl条件下有菌生长;将菌活化后接种于硝酸盐液体培养基中,30℃培养1、3、5天,在白色磁盘小孔中倒入少许培养液,然后在其中分别滴1滴试剂A(对氨基苯磺酸0.5g溶于10%醋酸150mL)和试剂B(α-萘乙酸0.1g,溶于蒸馏水20mL和10%醋酸150mL),结果培养液变为粉红色,表明菌株SYY15硝酸盐还原阳性;将菌株接种于脱脂牛奶平板培养基,适温培养1、3、5天观察,出现透明圈的为阳性,否则为阴性。酪蛋白水解试验显示菌株SYY15为阳性。将菌活化后接入淀粉的固体培养上,28℃培养48h后,用碘液染色,未见水解圈出现,淀粉水解阴性;将菌株接种于明胶培养中,20℃培养7天,培养基出现融化现象表明明胶水解阳性。用枪头挑取单菌落放在载玻片上,加一滴3%H2O2溶液于菌落上,有气泡出现,表明菌株接触酶阳性;将菌株SYY15接种于甲基红培养基(蛋白胨5g,葡萄糖5g,氯化钠5g,蒸馏水1000ml,pH7.0-7.2),30℃培养1-2天。在培养基中加入几滴甲基红试剂,如培养液呈现红色,为甲基红阳性,黄色为阴性。试验结果显示菌株SYY15为M.R.阴性;乙酰甲基甲醇(VP试验)培养基、接种和菌种培养同甲基红试验。做VP试验时,取培养液(约2ml)和等量的40%NaOH相混合,加少量肌酸,充分振荡2-5min后,如培养基出现红色,即为VP阳性。V.P试验结果显示菌株SYY15为阳性。在干净培养皿里放一张滤纸,滴加1%二甲基对苯撑二胺水溶液,仅使滤纸湿润。用白金丝接种环挑取一环菌苔,涂抹在湿润的滤纸上。在10秒内涂抹的菌苔出现红色,表明菌株SYY15氧化酶阳性;将活化后的菌株SYY15接种于西蒙斯氏柠檬酸盐试管斜面培养基,30℃培养3-7天,培养基变蓝者为阳性,否则为阴性。柠檬酸盐利用试验的结果显示菌株SYY15为阳性。菌株SYY15的生理生化检测结果如表1所示。根据进化树和生理生化结果可以将SYY15判定为酸快生芽孢杆菌。
表1 菌株SYY15的生理生化检测结果
实施例2 菌株SYY15产ACC脱氨酶的能力
首先将菌株SYY15在LB培养基上活化。将活化后的菌株接种到DFa培养液(KH2PO44g,Na2HPO4 6g,MgSO4·7H2O 0.2g,葡萄糖2g,葡萄糖酸2g,柠檬酸2g,ACC终浓度为3mM,水1000ml,pH7.2)中,30℃,180r/min条件下摇床培养24h,以不接菌株的培养液做对照,检测培养液在600nm下的吸光值,根据吸光值判断菌株是否能够在以ACC为唯一氮源的培养液中生长。如果能生长,在转接到DFa培养液中连续培养3次。结果表明,菌株SYY15可以在以ACC作为唯一氮源的培养液中生长,OD600可以达到2.35(图1)。
实施例3 菌株SYY15的溶磷能力测定
首先将菌株在LB培养基上活化。将活化后的菌株接种到NY/T 1847-2010中附录A所示溶磷培养基中(葡萄糖10.00g,(NH4)2SO4 0.50g,MnSO4·7H2O 0.3g,NaCl 0.3g,KCl0.30g,FeSO4·4H2O 0.036g,MnSO4·4H2O 0.03g,蒸馏水1000mL,pH 7.0。有机磷源选用植酸钙,添加量2g。固体培养基:按比例添加1.5%的琼脂粉)。检测是否具有溶磷功能,经检测,菌株SYY15具有溶解有机磷的能力,溶解有机磷的溶解圈直径达到1.34cm(图2)。
在确定了菌株SYY15具有溶磷功能之后再定量测定菌株的溶磷能力大小,将菌株在LB液体培养基上活化后,接种到液体的溶磷培养基上,28℃摇培7天,用钼锑抗比色法测定发酵上清液中的有效磷含量,取100μl上清液,放入50ml容量瓶中,用水稀释至约30ml,加入二硝基酚指示剂2滴,滴加4mol/L NaOH直至溶液刚转为黄色,再加入1mol/L H2SO4 1滴,使溶液的黄色刚刚退去。加入5.00ml钼锑抗显色剂,用水定容,充分摇匀。在室温高于15℃处放置30min后,取200μl加至96孔板,用酶标仪,在882nm波长处测量吸光度。
结果显示,菌株SYY15溶解有机磷的能力达到88.60mg/L。在确定了菌株SYY15具有溶解有机磷的能力后,直接定量测定菌株溶解无机磷的能力,将培养基中的植酸钙替换为磷酸钙,经测定,菌株SYY15溶解无机磷的能力可达152.36mg/L。
实施例4 菌株SYY15产蛋白酶的能力测定
首先将菌株SYY15在LB培养基上活化,将活化后的菌株接种到蛋白酶检测用培养基上(胰蛋白胨5.00g,酵母提取物3.00g,葡萄糖1.00g,琼脂15.00g,蒸馏水1000mL,pH7.0,121℃高压灭菌30min。将灭菌的检测培养基冷却至约50℃时,将脱脂牛奶按10%的比例加入培养基混匀后倒入培养皿,待其冷却后备用),28℃培养48h,观察有无溶解圈出现,结果显示菌株SYY15具有很好的溶解蛋白的能力,高产蛋白酶,其溶解圈直径达到2.84cm(图3)。利用菌株SYY15高产蛋白酶的特性,可将提取的蛋白酶应用于食品工业或者洗涤工业等蛋白酶领域,同时也可以用于农业上微生物肥料中对病原菌细胞膜降解以控制病害,以及堆肥中农业废弃物中蛋白质的降解。
实施例5 菌株SYY15产IAA能力的测定
首先将菌株SYY15在LB液体培养基上进行活化,将活化后的菌株按照1%的量接种于DF培养基中(蛋白胨5.00g,酵母提取物1.50g,牛肉膏1.50g,NaCl 5.00g,氨酸0.50g,蒸馏水1000mL,pH 7.0,121℃高压蒸汽灭菌30min)。28℃摇培7天,7天后,将发酵液取出,12000rpm离心5min,用Salkowkin比色法测定发酵液中IAA的含量。结果显示菌株SYY15产IAA的量为5.65mg/L。通过HPLC检测,进一步分析确认菌株合成的为IAA,菌株培养7天后,12000rpm离心5min,取上清30mL,用两倍体积乙酸乙酯在恒温振荡器中充分萃取3次,合并萃取液用乙酸乙酯减压蒸馏器蒸馏,然后用5mL甲醇溶解、定容,经过0.22um滤膜过滤。参考文献(连翠飞,蒋继志,李社增,鹿秀云,晁春燕,马平.利用高效液相色谱筛选产植物激素细菌[J].华北农学报,2006(02):66-69;高桂枝,徐爱军,虞梅,苑姗姗.高效液相色谱切变波长法测定艾蒿中内源激素[J].现代农业科技,2007(03):9-11)中的方法进行样品检测。检测仪器:waters2998高效液相色谱,色谱柱:Agilert Zorbax SB-C18 250mm×4.6mm,5um。流动相,甲醇:乙腈:0.6%冰乙酸水溶液(体积比50∶5∶45)。进样量:20μl。流速0.8mL/min。柱温:室温。检测波长:255nm。检测结果为5.80mg/L,略高于比色法检测结果。
实施例6 菌剂制备
1、菌种活化
首先将菌株SYY15在LB培养基上进行活化。将酸快生芽孢杆菌SYY15接种于LB液体培养基中,28℃培养12h。
2、种子液的培养
种子培养基组成为:蛋白胨15g/L,酵母膏8g/L,葡萄糖12g/L,NaCl 10g/L,pH值为7.2。将活化好的菌株SYY15按1.2%的接种量接种于种子液体培养基中,28℃摇床震荡培养,转速为200rpm,培养时间为12h。
3、发酵罐发酵
发酵培养基组成为:豆粕12g/L,木薯淀粉9.5g/L,酵母膏2.8g/L,蛋白胨2.8g/L,蔗糖6g/L,MgSO4·7H2O 0.08g/L,MnSO4·7H2O 0.018/L,聚醚消泡剂1.2g/L,pH值为7.2。将培养好的种子液OD600达到6.0时按5%的接种量接入发酵罐中,28℃,搅拌速度为180rpm,调节通风量保证在溶氧55%以上、罐压0.05MPa。发酵36h菌量可以达到1010cfu/ml,待芽孢量达到95%以上时停止发酵,降温放罐,通过对发酵培养基配方进行优化,得到酸快生芽孢杆菌SYY15的1010cfu/mL菌液,IAA的含量由5.80mg/L提高到12.32mg/L,可以根据需求制成固体或者液体菌剂。
实施例7 菌株SYY15在盐胁迫下提高玉米发芽系数的试验
选取饱满、健康玉米(郑单958)种子,用75%乙醇消毒30s,消毒后,将75%乙醇倒掉,用去离子水冲洗种子3-5次,并浸泡6h。
将处理好的种子放入垫三层滤纸的培养皿中,每个平皿放入20颗种子,每个平皿20ml清水,每个处理三个重复。实验设置4个处理。
处理一:空白清水对照、不添加SYY15菌剂;
处理二:100mM NaCl盐胁迫、不添加菌剂;
处理三:空白清水对照、添加SYY15菌剂;
处理四:100mM NaCl盐胁迫、添加SYY15菌剂;
每个平皿菌剂添加量为稀释到107cfu/mL的菌液1ml,不添加菌剂用清水作对照(CK)。放入28℃培养箱黑暗培养3天,测定发芽系数。
结果表明菌株SYY15具有很好地提高植物抗逆作用(图4),能显著提高种子的发芽率,能提高玉米的发芽系数34.10%(表2)。
表2 盐胁迫下种子发芽系数测定
实施例8 菌株SYY15的植物促生试验
选取饱满、健康玉米(郑单958)种子消毒、清洗、浸泡后放入28℃生化培养箱培养催芽24h左右。将供试菌株从平板挑取单菌落于LB液体培养基中进行活化,将活化后的菌液按1%的接菌量接种于LB培养基中,28℃,150rpm摇培72h并将菌液稀释106cfu/mL。选用圆筒PVC管50cm,底部封口后打孔,管内加沙子,每个管中添加50ml稀释后的菌液。同时用LB培养基做空白对照(CK),生长30天后测定玉米幼苗茎高、茎鲜重、茎干重、根鲜重、根干重。
结果显示菌株SYY15具有很好地促生效果(图5)。可以使玉米苗茎高增加12.91%,茎鲜重增加26.00%,茎干重增加15.91%,根鲜重增加47.63%,根干重增加42.25%(表3)。
表3 菌株SYY15对玉米的促生效果
注:*、**表示显著性差异,*为P<0.05,**为P<0.01。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之做一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 根力多生物科技股份有限公司
<120> 酸快生芽孢杆菌SYY15及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 850
<212> DNA
<213> 酸快生芽孢杆菌(Bacillus acidiceler)
<400> 1
tgcagtcgag cgaactgatt agaagcttgc ttctatgacg ttagcggcgg acgggtgagt 60
aacacgtggg caacctgcct gtaagactgg gataacttcg ggaaaccgaa gctaataccg 120
gataggatct tctccttcat gggagatgat tgaaagatgg tttcggctat cacttacaga 180
tgggcccgcg gtgcattagc tagttggtga ggtaacggct caccaaggca acgatgcata 240
gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg 300
aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag 360
tgatgaaggc tttcgggtcg taaaactctg ttgttaggga agaacaagta cgagagtaac 420
tgctcgtacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480
ggtaatacgt aggtggcaag cgttatccgg aattattggg cgtaaagcgc gcgcaggcgg 540
tttcttaagt ctgatgtgaa agcccacggc tcaaccgtgg agggtcattg gaaactgggg 600
aacttgagtg cagaagagaa aagcggaatt ccacgtgtag cggtgaaatg cgtagagatg 660
tggaggaaca ccagtggcga aggcggcttt ttggtctgta actgacgctg aggcgcgaaa 720
gcgtggggag caaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780
agtgttagag ggtttccgcc ctttagtgct gcagctaacg cattaagcac tccgcctggg 840
gagtacggtc 850
Claims (8)
1.酸快生芽孢杆菌(Bacillus acidiceler)SYY15,保藏编号为CCTCC NO:M 2019840。
2.含有权利要求1所述酸快生芽孢杆菌SYY15的菌剂。
3.由权利要求1所述酸快生芽孢杆菌SYY15或权利要求2所述菌剂制备的农用肥料、磷素活化剂或植物促生长剂。
4.权利要求1所述酸快生芽孢杆菌SYY15或权利要求2所述菌剂的以下任一应用:
1)用于促进植物生长发育以及提高作物产量;
2)用于提高植物抗逆性;
3)用于除磷;
4)用于制备农用肥料;
5)用于制备磷素活化剂;
6)用于制备植物促生长剂。
5.根据权利要求4所述的应用,其特征在于,所述植物包括玉米、小麦、大豆、黄瓜。
6.根据权利要求4或5所述的应用,其特征在于,所述抗逆性包括抗病、抗旱、抗盐碱及重金属胁迫。
7.权利要求1所述酸快生芽孢杆菌SYY15或权利要求2所述菌剂在溶解难溶磷中的应用。
8.根据权利要求7所述的应用,其特征在于,所述难溶磷为难溶性有机磷。
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Denomination of invention: Acid fast growing Bacillus SYY15 and its application Granted publication date: 20220401 Pledgee: China CITIC Bank Co.,Ltd. Shijiazhuang Branch Pledgor: GENLIDUO BIO-TECH Corp.,Ltd. Registration number: Y2024980001438 |