CN115011534B - 一种茎瘤固氮根瘤菌ors571的突变株、构建方法及应用 - Google Patents
一种茎瘤固氮根瘤菌ors571的突变株、构建方法及应用 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
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Abstract
本发明提供了一种茎瘤固氮根瘤菌ORS571的突变株、构建方法及应用,所述突变株为AZC_0343基因缺失的茎瘤固氮根瘤菌;本发明以茎瘤固氮根瘤菌ORS571的基因组DNA为模板,分别以引物0343‑up‑F/R、0343‑down‑F/R扩增AZC_0343基因的上游片段和下游片段;将扩增产物酶切酶连构建重组质粒,然后将重组质粒导入大肠杆菌,并通过三亲接合方法导入茎瘤固氮根瘤菌ORS571;最后筛选得到所述突变株。本发明所述突变株的固氮能力显著,其固氮酶活性比野生株提升了88.56%,且所述突变株可以增加土壤养分、提高肥料有效性,促进玉米生长,具有开发成微生物接种剂或微生物菌肥的良好前景。
Description
【技术领域】
本发明涉及农业微生物领域,具体涉及一种茎瘤固氮根瘤菌ORS571的突变株、构建方法及应用。
【背景技术】
氮是植物生长所需的三大主要营养素之一,作物体内蛋白质、酶、核酸、叶绿素、维生素等有机化合物的合成都离不开氮的参与。氮在大气中含量丰富(占79%左右),但大多数植物无法直接利用氮气。农业生产中施用氮肥增产是缓解耕地资源不足、保证粮食品质的重要措施;然而,随着施氮量的进一步增加,农作物产量却不再增加,并且氮效率大幅度下降。研究表明过量施氮导致玉米叶片早衰,产籽量降低;并且长期过量施用氮肥会造成土壤板结,污染环境并影响土壤微生态。因此,科学使用肥料,开发新型绿色肥料保证农田可持续生产能力,促进农业绿色发展仍然是努力的方向。
微生物肥料因具有调节植物生长、增加作物产量、改善作物品质、减少化肥使用量、改良土壤、保护生态环境等特点受到广泛关注,成为绿色生态农业发展中不可替代的制胜法宝之一。固氮细菌与植物共生固氮的过程中将大气中的氮气转化为更容易被宿主植物利用的氨。茎瘤固氮根瘤菌(Azorhizobium caulinodans ORS571)属于杆菌,是从豆科植物毛萼田菁茎瘤中分离到的固氮细菌,其细胞直径大于2.0μm且长短不一,呈卵圆状或类球状,其宿主特异性很强。ORS571除了可以和宿主植物共生固氮,还能够在微耗氧的条件下自生固氮或作为内生菌在其他植物体内固氮,为宿主植物增加获取氮源的途径。由此可见,茎瘤固氮根瘤菌ORS571应用于农业,具有不可替代的研究意义。
【发明内容】
本发明的目的在于,提供一种固氮效率高、具有显著促生效果的茎瘤固氮根瘤菌ORS571的突变株、构建方法及应用。
为实现上述目的,本发明采用如下技术方案:
一种茎瘤固氮根瘤菌ORS571的突变株,所述突变株为σ54因子调控基因AZC_0343缺失的茎瘤固氮根瘤菌,其出发菌株为茎瘤固氮根瘤菌ORS571。
所述突变株通过以下方法构建得到:
(1)以茎瘤固氮根瘤菌ORS571为模板,分别以引物0343-up-F/R和引物0343-down-F/R扩增茎瘤固氮根瘤菌AZC_0343基因的上游片段AZC_0343-up和下游片段AZC_0343-down;
(2)酶切酶连构建重组质粒pCM351::AZC_0343up-down;
(3)将上述重组质粒导入大肠杆菌DH5α,并通过三亲接合方法和同源重组原理导入茎瘤固氮根瘤菌ORS571;
(4)使用同时含有氨苄青霉素和庆大霉素抗性的TY固体培养基筛选阳性接合子,并通过PCR验证获得AZC_0343基因敲除的茎瘤固氮根瘤菌ORS571的突变株。
优选地,步骤(1)中所述引物具体为:
0343-up-F:GGTACCGCTCCACGATCATCTGCTG;
0343-up-R:CATATGCCATCGAAATACTTGCCGAC;
0343-down-F:GGGCCCGTGGTGGTCTTCCGCCATG;
0343-down-R:GAGCTCGAGGAGCGCGTAGATGTCC。
优选地,步骤(2)具体为:将步骤(1)扩增得到的所述上游片段AZC_0343-up连接到pEASY Simple上,分别用Kpn I和Nde I限制性内切酶进行酶切,收集目的片段与经Kpn I和Nde I酶切的pCM351片段连接,连接后转入大肠杆菌筛选阳性重组子,获得pCM351:AZC_0343-up质粒;
将步骤(1)扩增得到的下游片段AZC_0343-down连接到pEASY Simple上,分别用Apa I和Sac I限制性内切酶双酶切AZC_0343-down片段和pCM351:AZC_0343-up质粒,收集并将经Apa I和Sac I双酶切的AZC_0343-down片段和pCM351:AZC_0343-up质粒连接,连接后转入大肠杆菌筛选阳性重组子,获得重组质粒pCM351::AZC_0343up-down。
优选地,步骤(3)中所述的三亲接合方法具体指的是:将含有重组质粒的DH5α供体菌、含有辅助质粒pRK2013的DH5α辅助菌和茎瘤固氮根瘤菌ORS571分别在LB液体培养基(适用于大肠杆菌DH5α)和TY液体培养基(适用于ORS571)中于37℃条件下培养至OD600=0.6;
然后将供体菌、辅助菌和受体菌按3:2:1的体积比混合,4500rpm离心5min,倒掉上清,将剩余的100μL上清重悬菌体悬滴于LB固体培养基,于37℃条件下培养48h进行三亲接合。
优选地,步骤(3)中所述的同源重组原理具体指的是:三亲接合过程中,重组质粒pCM351::AZC_0343up-down导入茎瘤固氮根瘤菌ORS571并与菌株ORS571基因组靠近,两个DNA分子的同源序列自发进行重新组合得重组后的接合子。
优选地,步骤(4)具体为:将步骤(3)得到的重组后的接合子在同时含有氨苄青霉素和庆大霉素的TY固体培养基上培养,筛选可以在同时含有氨苄青霉素和庆大霉素的TY固体培养基上生长的阳性接合子,通过PCR验证获得所述突变株。
本发明的另一目的在于,提供上述突变株在促进玉米生长中的应用。
优选地,所述突变株在促进玉米生长中作为接种剂或微生物菌肥的应用,具体如下:
在TY培养基中37℃条件下过夜培养所述突变株,将其作为接种剂(促生剂)浸泡发芽的玉米种子30min后,将玉米种子转移至灭菌的拌有低氮营养液的蛭石中,于26℃,光照周期12h:12h条件下培养,促进玉米生长。
所述低氮营养液配方是柠檬酸铁0.075g/L,硝酸钙0.03g/L,氯化钾0.075g/L,硫酸镁0.06g/L,磷酸氢钾0.136g/L,硫酸钙0.46g/L,微量元素混合液1mL。
所述微量元素混合液组分为硼酸2.86g/L,硫酸锰1.81g/L,五水硫酸铜0.8g/L,硫酸锌0.22g/L,钼酸0.02g/L,pH=7.0。
本发明的有益效果:
本发明所述突变株具有自生固氮且固氮效率高的优良特性,其固氮效率高达3.79μmol·h-1·g-1,比ORS571提升了88.56%;
本发明所述突变株的趋化能力大于野生株,说明本发明所述菌株更有利于向植物根系运动并定殖;
利用本发明所述突变株处理的玉米种子,生长迅速,说明本发明所述突变株具有显著的促生效果;且所述突变株可以增加土壤养分、提高肥料有效性,具有开发成微生物接种剂或微生物菌肥的良好前景。
【附图说明】
图1是本发明实施例提供的野生株WT(ORS571)和本申请所述突变株的AZC_0343基因验证引物PCR扩增片段的1%琼脂糖凝胶电泳条带图;
图2是本发明实施例提供的野生株WT和突变株AC343在TY液体培养基中的生长曲线;其中,正方形代表野生株WT,圆点代表突变株AC343;
图3是本发明实施例提供的野生株WT和突变株AC343的趋化圈的形成;
图4是本发明实施例提供的野生株WT和突变株AC343的固氮能力;
图5是本发明实施例提供的18天后接菌玉米和不接菌玉米在低氮营养条件下的生长状况对比图。
【具体实施方式】
为了使本发明的目的、技术方案及优点更加清楚明白,本发明用以下具体实施例进行说明,但绝非仅限于此。以下所述为本发明较好的实施例,仅仅用于描述本发明,不能理解为对本发明的限制,应当指出的是在本发明的精神和原则之内所做的任何修改、等同替换和改进,均应包含在本发明的保护范围之内。
实施例用到的菌株和质粒如下:
茎瘤固氮根瘤菌ORS571由Toshihiro Aono馈赠,该菌株已被保藏,保藏信息为德国菌种保藏中心DSMZ,Azorhizobium caulinodans ORS571,DSM No.:5975;文献有Dreyfus,B.,Garcia,J.L.,Gillis,M.(1988).Characterization of Azorhizobiumcaulinodans gen.nov.a stem nodulating nitrogen-fixing bacterium isolated fromSesbania rastrata.Int.J.Syst.Bacteriol.38:89-98。
大肠杆菌DH5α、克隆载体pEASY Simple均购自全式金公司,克隆载体pEASYSimple具有卡那霉素和氨苄青霉素双抗性;
质粒pCM351,具有庆大霉素和四环素抗性;
辅助质粒pRK2013,具有卡那霉素抗性。
本发明实施例用到的培养基如下:
TY培养基(固体培养基额外加琼脂粉15g/L):胰蛋白胨5g/L,酵母粉3g/L,无水氯化钙0.6g/L,pH=7.0,121℃,灭菌20min。
LB培养基:胰蛋白胨10g/L,酵母提取物5g/L,氯化钠10g/L,琼脂粉15g/L。pH=7.0,121℃,灭菌20min。
L3半固体培养基:KH2PO4 1.36mg/L,MgSO4 100mg/L,NaCl 50mg/L,CaCl2 40mg/L,FeCl3 5.4mg/L,Na2MoO4 5mg/L,生物素2mg/L,烟酸4mg/L,泛酸4mg/L,碳源为乳酸钠10mmol/L,用于加氮培养时,加入NH4Cl 0.53mg/L,高压蒸汽锅121℃灭菌20min。
植物低氮营养液:柠檬酸铁0.075g/L,硝酸钙0.03g/L,氯化钾0.075g/L,硫酸镁0.06g/L,磷酸氢钾0.136g/L,硫酸钙0.46g/L,微量元素混合液1mL;微量元素混合液组分为硼酸2.86g/L,硫酸锰1.81g/L,五水硫酸铜0.8g/L,硫酸锌0.22g/L,钼酸0.02g/L,pH=7.0,高压蒸汽锅121℃灭菌20min。
表1实例中使用到的引物
注:划线部分为添加的限制性核酸内切酶识别序列。
实施例1:AZC_0343基因上下游片段的扩增
用天根细菌基因组提取试剂盒提取茎瘤固氮根瘤菌ORS571的基因组(提取方法参照内附说明书),通过Nanodrop 2000测定提取的样品浓度(100-300ng/μL为宜),保存于-20℃备用。以提取的基因组DNA为模板,分别用引物0343-up-F和0343-up-R扩增目的基因的上游片段AZC_0343-up,用引物0343-down-F和0343-down-R扩增目的基因的下游片段AZC_0343-down;对实施例1扩增得到的片段通过1%琼脂糖凝胶电泳确定大小与设计长度无异;所述PCR扩增采用表2所示反应体系,按照表3所示反应条件进行。
表2实施例1所述扩增用PCR反应体系
表3实施例1所述PCR反应条件
实施例2:构建重组质粒pCM351::AZC_0343up-down
(1)利用全式金公司pEASY Simple载体试剂盒,将实施例1经PCR扩增得到的上游片段AZC_0343-up整合到pEASY质粒中(按pEASY Simple试剂盒说明书条件进行反应);
(2)吸取步骤(1)制得的10μL反应物与50μL刚解冻的DH5α大肠杆菌感受态细胞混合,冰浴30min使细胞活化后,42℃热激30s以激发感受态,使整合质粒进入细胞,再冰浴3min使质粒稳定存留;然后将其加入500μL LB液体培养基中,于37℃,200rpm培养1h后,4500rpm离心3min后保留100μL上清液与菌体吹打混匀,涂布到含有IPTG和X-gal以及卡那霉素的LB固体平板中,37℃过夜培养进行蓝白斑筛选,挑取白斑接种至试管中,进行菌液测序,测序工作由北京奥科鼎盛生物公司完成;
(3)取测序正确的细菌过夜培养,同时培养含有pCM351质粒的大肠杆菌;使用GenStar公司的StarPrep快速质粒小提试剂盒分别提取菌体质粒pEASY:AZC_0343-up和pCM351;利用Nanodrop测定质粒的浓度并于-20℃保存备用;
(4)将pEASY:AZC_0343-up重组质粒和pCM351质粒分别使用两种限制性内切酶KpnI和Nde I进行酶切处理(采用表4所示反应体系),将反应体系进行电泳,使用Trans公司的EasyPrue Quick Gel Extraction Kit试剂盒对目的条带进行重组片段的切胶回收;
(5)将酶切处理后的pCM351质粒和AZC_0343-up片段以Thermofisher scientificT4 DNA连接酶16℃过夜连接(采用表5所示体系),将连接后的体系导入到DH5α大肠杆菌感受态细胞中,并稀释涂布到含庆大霉素的LB固体LB+Gen平板上筛选阳性重组子;
(6)用枪头挑取单菌落悬浮到10μL无菌水中,以该菌悬液为模板并利用0343-up-F和0343-up-R为引物进行PCR扩增,验证AZC_0343-up片段是否被插入pCM351质粒,将验证正确的含有pCM351:AZC_0343-up重组质粒的菌株进行菌液测序,测序工作由北京奥科鼎盛生物公司完成;对连接正确的含有pCM351:AZC_0343-up重组质粒的菌株-80℃甘油保存,备用;
(7)进一步将PCR扩增的AZC_0343-down片段连接到pEASY质粒上,方法同上;将pEASY:AZC_0343-down重组质粒和pCM351:AZC_0343-up重组质粒分别用Age I和Sac I限制性内切酶进行双酶切(采用表4所示反应体系),切胶回收双酶切成功的AZC_0343-down片段和pCM351:AZC_0343-up,再用Thermofisher scientific T4 DNA连接酶16℃过夜连接(采用表5所示连接体系),连接后转入大肠杆菌DH5α中,在含有庆大霉素抗性的LB平板中筛选阳性重组子;最后得到验证成功的pCM351::AZC_0343up-down重组质粒,备用。
表4双酶切反应体系
表5连接体系
实施例3:将重组质粒pCM351::AZC_0343up-down导入茎瘤固氮根瘤菌ORS571
(1)利用TY液体培养基于37℃条件下过夜培养受体菌(野生型ORS571菌株),分别利用LB液体培养基于37℃条件下过夜培养供体菌(含pCM351::AZC_0343up-down重组菌株的DH5α)和辅助菌(含pRK2013质粒的DH5α),培养至OD600=0.6;
(2)分别收集步骤(1)培养得到的各菌液1mL,4500rpm离心5min后弃上清,以无抗性TY培养基清洗2次,4500rpm离心5min,最后悬浮于1mL无抗性TY培养基中;
(3)分别取300μL供体菌、200μL辅助菌和100μL受体菌,置于2mL离心管中吹打混匀,4500rpm离心5min,去上清,保留100μL上清液将菌体吹打混匀,得混合菌液;
(4)将步骤(3)得到的100μL混合菌液悬滴在TY平板中心,将平板于超净工作台静置吹干,待菌液被吹干后置于37℃恒温培养箱培养2d;
(5)用无菌枪头将菌斑刮下,悬浮至1mL无菌水中;
(6)将菌液梯度稀释103、104倍,分别取100μL不同稀释倍数的菌悬液涂布到含氨苄青霉素和庆大霉素的TY固体培养基中,37℃培养2d;
(7)将步骤(6)所述TY固体培养基中生长的单菌落分别划线到含氨苄青霉素和庆大霉素两种抗性的TY平板上,37℃培养2d;
(8)刮取步骤(7)平板中长势明显的菌落并用10μL无菌水吹打混匀,以该菌悬液为模板,以0343-up-F和0343-down-R为引物,以野生型菌落的菌悬液为对照模板分别进行PCR扩增。
如图1所示,野生型扩增出1804bp的片段,接合子扩增出2302bp的片段,片段大小与预期相符,说明该接合子为成功同源双交换的基因敲除突变株;将PCR验证成功的菌液由北京奥科鼎盛生物公司进行基因组测序,确定基因被正确敲除后,将突变菌株命名为AC343与30%甘油1:1混合后于-80℃保种。
实施例4:茎瘤固氮根瘤菌突变株AC343的生长状况与运动能力测定
通过监测菌株在波长600nm处的吸光值得到菌株的生长曲线:将野生型ORS571,缺失突变株AC343接种到TY培养基过夜培养至对数期,用无菌TY培养基调节细菌的起始浓度为OD600=0.01,取30mL菌液置于100mL锥形瓶中,每株菌设置三组平行。将所有锥形瓶放置在37℃,200rpm条件下振荡培养,每隔2h测定菌液OD600值,取三组平均值做记录,将所有OD600值的测量结果绘制成如图2所示的生长曲线;由图2可以看出,两株菌的生长状况基本一致,没有显著差异。
运动能力测定:将野生株WT,突变株AC343分别接种于TY液体培养基并在37℃,200rpm条件下过夜培养,收集菌液将其调整至OD600=1.0,各取5μL垂直接种到L3半固体平板中,37℃恒温静置培养2d,观察细菌趋化圈的大小;从图3可以看出,突变株的趋化能力大于野生株。趋化运动是细菌实现与植物共生的前提,突变株AC343趋化能力增强,说明菌株更有利于向植物根系运动并定殖。
实施案例5:所述突变株AC343的固氮能力
采用乙炔还原法(ARA)测定突变株AC343和野生株WT的固氮酶活性,实验操作流程如下:
(1)将所述菌株活化并过夜培养,使其生长状态保持一致,向5mL密封玻璃瓶中注入3mL的L3半固体培养基,使其凝固,备用;
(2)将各菌株用无菌L3液体培养基清洗并重悬至OD600=1.0后,吸取10μL重悬菌液接种到L3半固体培养基内,用橡胶塞和封口膜密封玻璃瓶口;37℃培养8h后,在无菌条件下将体积分数10%的空气抽出,注入体积分数10%的高纯乙炔气体,继续以37℃恒温培养24h;
(3)用微量进样器从密封玻璃瓶中抽取100μL混合气体,利用气相色谱仪测定气体组分;每个菌株设置三组平行,以无菌注射乙炔气体作为阴性对照,进样100μL标定乙炔峰的位置,以纯乙烯进样100μL进行乙烯峰物质的量的标定;固氮酶活性以单位时间内乙烯的生成量计算。
固氮酶活性计算公式是:ARA=Asa×(Vt/Vs)/(Ast/H/P)
式中,ARA为固氮酶活性,单位为μmol·h-1·g-1;Asa为乙烯峰面积,单位为cm2;Vt为瓶气相体积,单位为mL;Vs为进样量,单位为mL;Ast为1nmol标准乙烯峰面积,单位为cm2;H为反应时间,单位为h;P为蛋白含量,单位为g。
测定的固氮酶能力结果如图4所示,由4图可知,突变株AC343具有良好的固氮能力,其固氮酶活性显著大于野生株WT(ORS571),比野生株WT(ORS571)提升了88.56%。
实施案例6:突变株AC343对玉米的促生实验
(1)挑取大小均匀的玉米种子用无菌水反复冲洗3次,在超净台的玻璃器皿中摊平吹风10min后,先浸泡于99%酒精中1h,再用3%次氯酸钠浸泡30min,随后立即用无菌水冲洗10次以上,然后将种子浸泡在无菌水中30min,浸泡后的种子放置在水琼脂板上,在植物培养箱中催芽3d,挑选生长状况相同的玉米种子备用;
(2)在TY培养基中37℃条件下过夜培养野生株WT和突变株AC343,将菌液浓度均调整至OD600=0.8,分别将其作为促生剂浸泡发芽的玉米种子30min,空白对照组玉米种子用无菌TY液体培养基同样条件下浸泡30min,之后各处理组玉米种子用无菌水冲洗3次,将各玉米种子转移至灭菌的伴有植物低氮营养液的蛭石中,于26℃,光照周期12h:12h条件下培养。
培养18天后,观察玉米的生长状况,其结果如图5所示,由图5可知,利用突变株AC343处理过的玉米种子长势最好,说明所述突变株AC343促生作用显著,且其促生效果大于野生株。
综上实验表明:所述突变株AC343生长状况良好、固氮能力强、对玉米具有优良的促生效果;高效的固氮能力使其具有增加土壤氮含量,促进植物生长的潜能,因而有望开发成为微生物菌肥。
SEQUENCE LISTING
<110> 山东农业大学、山东蓬勃生物科技有限公司
<120> 一种茎瘤固氮根瘤菌ORS571的突变株、构建方法及应用
<130> 2022-03-09
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213> ORS571
<400> 1
ggtaccgctc cacgatcatc tgctg 25
<210> 2
<211> 26
<212> DNA
<213> ORS571
<400> 2
catatgccat cgaaatactt gccgac 26
<210> 3
<211> 25
<212> DNA
<213> ORS571
<400> 3
gggcccgtgg tggtcttccg ccatg 25
<210> 4
<211> 25
<212> DNA
<213> ORS571
<400> 4
gagctcgagg agcgcgtaga tgtcc 25
Claims (7)
1.一种茎瘤固氮根瘤菌ORS571的突变株,其特征在于,所述突变株为σ54因子调控基因AZC_0343缺失的茎瘤固氮根瘤菌。
2.权利要求1所述突变株的构建方法,其特征在于,包括如下步骤:
(1)以茎瘤固氮根瘤菌ORS571的基因组DNA为模板,分别以0343-up-F/R为引物扩增茎瘤固氮根瘤菌AZC_0343基因的上游片段AZC_0343-up,以0343-down-F/R为引物扩增茎瘤固氮根瘤菌AZC_0343基因的下游片段AZC_0343-down;
(2)酶切酶连构建重组质粒pCM351::AZC_0343up-down;
(3)将上述重组质粒导入大肠杆菌DH5α,并通过三亲接合方法和同源重组原理导入茎瘤固氮根瘤菌ORS571;
(4)使用同时含有氨苄青霉素和庆大霉素抗性的TY固体培养基筛选阳性接合子,并通过PCR验证获得AZC_0343基因敲除的茎瘤固氮根瘤菌ORS571的突变株;
其中,步骤(1)中所述引物具体为:
0343-up-F:GGTACCGCTCCACGATCATCTGCTG;
0343-up-R:CATATGCCATCGAAATACTTGCCGAC;
0343-down-F:GGGCCCGTGGTGGTCTTCCGCCATG;
0343-down-R:GAGCTCGAGGAGCGCGTAGATGTCC。
3.根据权利要求2所述的突变株的构建方法,其特征在于,步骤(2)具体为:将步骤(1)扩增得到的所述上游片段AZC_0343-up连接到pEASY Simple上,分别用Kpn I和Nde I限制性内切酶进行酶切,收集目的片段与经Kpn I和Nde I酶切的pCM351片段连接,连接后转入大肠杆菌筛选阳性重组子,获得pCM351: AZC_0343-up质粒;
将步骤(1)扩增得到的下游片段AZC_0343-down连接到pEASY Simple上,分别用Apa I和Sac I限制性内切酶双酶切AZC_0343-down片段和pCM351: AZC_0343-up质粒,收集并将经Apa I和Sac I双酶切的AZC_0343-down片段和pCM351: AZC_0343-up质粒连接,连接后转入大肠杆菌筛选阳性重组子,获得重组质粒pCM351::AZC_0343up-down。
4.根据权利要求2所述的突变株的构建方法,其特征在于,步骤(3)中所述的三亲接合方法具体指的是:将含有重组质粒的DH5α作为供体菌和含有辅助质粒pRK2013的DH5α作为辅助菌于LB液体培养基中培养,将野生型茎瘤固氮根瘤菌ORS571作为受体菌于TY液体培养基中培养,然后将供体菌、辅助菌和受体菌按3:2:1的体积比混合后于LB固体培养基中,于37 ℃条件下培养48 h进行三亲接合。
5.根据权利要求4所述的突变株的构建方法,其特征在于,三亲接合过程中,重组质粒pCM351::AZC_0343up-down导入茎瘤固氮根瘤菌ORS571并与菌株ORS571基因组靠近,两个DNA分子的同源序列自发进行重新组合得重组后的接合子。
6.根据权利要求5所述的突变株的构建方法,其特征在于,步骤(4)具体为:将步骤(3)得到的重组后的接合子在同时含有氨苄青霉素和庆大霉素的TY固体培养基上培养,筛选能够在同时含有氨苄青霉素和庆大霉素的TY固体培养基上生长的阳性接合子,通过PCR验证获得所述突变株。
7.权利要求1所述突变株在促进玉米生长中的应用。
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Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103861094A (zh) * | 2014-03-27 | 2014-06-18 | 山东农业大学 | 一种产气荚膜梭菌α毒素基因工程疫苗及其应用 |
WO2014117639A1 (zh) * | 2013-02-01 | 2014-08-07 | 天津工业生物技术研究所 | 生产萜类化合物的重组微生物及构建方法 |
CN107446873A (zh) * | 2017-08-31 | 2017-12-08 | 中国科学院烟台海岸带研究所 | 促小麦生长的茎瘤固氮根瘤菌及其构建和应用 |
WO2018036479A1 (zh) * | 2016-08-22 | 2018-03-01 | 上海交通大学 | 高产吩嗪-1-甲酰胺的基因工程菌株及其构建方法和用途 |
WO2018192507A1 (zh) * | 2017-04-19 | 2018-10-25 | 山东大学 | 荧光假单胞菌突变菌株的筛选方法及其在生物防治中的应用 |
CN110452864A (zh) * | 2019-08-08 | 2019-11-15 | 华创佳农生物科技(武汉)有限公司 | 一种提高根瘤菌usda110竞争结瘤能力的方法 |
CN111849851A (zh) * | 2020-08-12 | 2020-10-30 | 华南农业大学 | 一种高产绿脓菌素工程菌及其构建方法和应用 |
WO2021077455A1 (zh) * | 2019-10-24 | 2021-04-29 | 天津科技大学 | 一株高产l-缬氨酸的基因工程菌及发酵生产l-缬氨酸方法 |
CN113179882A (zh) * | 2021-04-29 | 2021-07-30 | 长江师范学院 | 一种利用田菁固氮根瘤菌促进茎瘤芥根系结瘤固氮的方法 |
WO2021232536A1 (zh) * | 2020-05-19 | 2021-11-25 | 中国林业科学研究院资源昆虫研究所 | 一种体外大量获得白蜡虫dsRNA的体系构建方法 |
CN113710690A (zh) * | 2019-03-19 | 2021-11-26 | 麻省理工学院 | 与谷类联合的根瘤菌中的固氮的控制 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9309542B2 (en) * | 2012-08-17 | 2016-04-12 | University Of Georgia Research Foundation, Inc. | Recombinant Caldicellulosiruptor bescii and methods of use |
-
2022
- 2022-03-23 CN CN202210293436.9A patent/CN115011534B/zh active Active
Patent Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014117639A1 (zh) * | 2013-02-01 | 2014-08-07 | 天津工业生物技术研究所 | 生产萜类化合物的重组微生物及构建方法 |
CN103861094A (zh) * | 2014-03-27 | 2014-06-18 | 山东农业大学 | 一种产气荚膜梭菌α毒素基因工程疫苗及其应用 |
WO2018036479A1 (zh) * | 2016-08-22 | 2018-03-01 | 上海交通大学 | 高产吩嗪-1-甲酰胺的基因工程菌株及其构建方法和用途 |
WO2018192507A1 (zh) * | 2017-04-19 | 2018-10-25 | 山东大学 | 荧光假单胞菌突变菌株的筛选方法及其在生物防治中的应用 |
CN107446873A (zh) * | 2017-08-31 | 2017-12-08 | 中国科学院烟台海岸带研究所 | 促小麦生长的茎瘤固氮根瘤菌及其构建和应用 |
CN113710690A (zh) * | 2019-03-19 | 2021-11-26 | 麻省理工学院 | 与谷类联合的根瘤菌中的固氮的控制 |
CN110452864A (zh) * | 2019-08-08 | 2019-11-15 | 华创佳农生物科技(武汉)有限公司 | 一种提高根瘤菌usda110竞争结瘤能力的方法 |
WO2021077455A1 (zh) * | 2019-10-24 | 2021-04-29 | 天津科技大学 | 一株高产l-缬氨酸的基因工程菌及发酵生产l-缬氨酸方法 |
WO2021232536A1 (zh) * | 2020-05-19 | 2021-11-25 | 中国林业科学研究院资源昆虫研究所 | 一种体外大量获得白蜡虫dsRNA的体系构建方法 |
CN111849851A (zh) * | 2020-08-12 | 2020-10-30 | 华南农业大学 | 一种高产绿脓菌素工程菌及其构建方法和应用 |
CN113179882A (zh) * | 2021-04-29 | 2021-07-30 | 长江师范学院 | 一种利用田菁固氮根瘤菌促进茎瘤芥根系结瘤固氮的方法 |
Non-Patent Citations (6)
Title |
---|
A light-repressed transcript found in Synechococcus PCC 7002 is similar to a chloroplast-specific small subunit ribosomal protein and to a transcription modulator protein associated with sigma 54;Tan X等;J. Biol. Chem;第269卷;第1-10页 * |
Comparative proteomic analysis of Lactobacillus plantarum for the identification of key proteins in bile tolerance;Erwann Hamon等;BMC Microbiology;第63卷(第11期);第1-11页 * |
GenBank: AP009384.1;GenBank;GenBank;第1-2页 * |
茎瘤固氮根瘤菌(Azorhizobium caulinodans ORS571)可溶性趋化受体的功能研究;姜南;中国科学院烟台海岸带研究所;第1-113页 * |
茎瘤固氮根瘤菌ORS571受体TlpA1对琥珀酸的感应机理;隋傅;刘晓琳;解志红;;中国农业科技导报(10);第83-90页 * |
茎瘤固氮根瘤菌ORS571鞭毛马达基因fliN与fliM的功能分析;沈日敏;刘卫;孙雨;李润植;解志红;;微生物学报(05);第138-148页 * |
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