CN112795527A - 二氢蝶呤醛缩酶基因的用途 - Google Patents
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Abstract
本发明公开一种二氢蝶呤醛缩酶基因folB的新用途,即其在提高植物乳杆菌(Lactobacillus plantarum)叶酸合成中的应用,所述二氢蝶呤醛缩酶基因folB的核苷酸序列如SEQ ID NO:1所示;本发明将folB基因与pMG36e质粒重组构建过表达载体,并导入食源性植物乳杆菌感受态细胞中,进而获得folB基因过表达菌株BDB;采用LC‑MS法测定菌株产叶酸能力,发现与野生型菌株相比,BDB菌株产叶酸量显著增加,folB基因在叶酸合成中起关键作用,本发明在叶酸生物合成研究及应用领域具有巨大潜力。
Description
技术领域
本发明属于微生物基因应用领域,具体涉及二氢蝶呤醛缩酶基因folB在提高植物乳杆菌(Lactobacillus plantarum)叶酸合成中的应用。
背景技术
许多研究人员报道,乳酸菌,如嗜酸乳杆菌、乳酸乳球菌、嗜热链球菌和明串珠菌等具有合成叶酸的能力。不同的乳酸菌生产叶酸的能力显著不同,区间大致为2至214μg/L,应用菌株所生产和消耗叶酸的能力可能是决定叶酸水平的重要因素之一。
叶酸(Folic acid),又称蝶酰谷氨酸(pteroylglutamic acid, PGA),是一种B 族维生素中水溶性化合物的统称。叶酸是人类饮食中必须的微量元素,涉及许多代谢途径,主要在碳转移反应如嘌呤和嘧啶生物合成和氨基酸互变中。研究表明叶酸几乎是参与所有生物代谢的必需微量元素,因为在物质的合成和代谢中起关键作用,所以人类无法离开叶酸而生存。但是人体自身不能够合成叶酸,只能从饮食中摄取,每个成人每日需摄取400μg叶酸才能满足自身生命活动所需,因此,每日补充叶酸是非常重要的。随着叶酸生物合成途径的逐渐明朗,为了提高天然叶酸产量进而满足人类需求,通过基因工程的方法改变植物或微生物中叶酸的代谢量逐渐受到重视。
研究表明乳酸菌能够合成叶酸,其叶酸合成的途径由由蝶呤分支和对氨基苯甲酸(pABA)分支组成。二氢蝶呤醛缩酶(DHNA)释放羟乙醛,产生6-羟甲基-7,8-二氢蝶呤,然后通过羟甲基二氢蝶呤焦磷酸激酶(DHPPK)将其焦磷酸化,用于后续叶酸合成,最终植物乳杆菌合成的叶酸由单个或多个谷氨酸组成。
乳酸菌合成的叶酸因其具有安全性,有效性使其在市场上更具有竞争力。但是通常情况下乳酸菌的叶酸合成量普遍不高,且菌株稳定性较差,提取和纯化成本高等因素制约着乳酸菌叶酸在工业生产中的大规模应用。因此,探究微生物中叶酸合成关键酶基因的功能与地位,有助于了解其产生叶酸的机理,进而提高叶酸产量,为后期开发叶酸发酵食品奠定基础。
发明内容
针对现有技术存在的不足,本发明提供了一种二氢蝶呤醛缩酶基因的用途,即二氢蝶呤醛缩酶基因folB在提高植物乳杆菌(Lactobacillus plantarum)叶酸合成中的用途,所述二氢蝶呤醛缩酶基因folB的核苷酸序列如SEQ ID NO: 1所示,该基因序列长为369bp(碱基)。
本发明从植物乳杆菌(Lactobacillus plantarum)YM-4-3中克隆二氢蝶呤醛缩酶基因folB,将folB基因与乳酸菌穿梭组成型载体pMG36e酶切连接后,获得重组表达载体,将其转入植物乳杆菌YM-4-3中,在YM-4-3菌株体内实现folB基因的过表达,获得过表达菌株,通过实验比较野生型菌株YM-4-3与过表达菌株BDB的叶酸产量,证明folB基因在叶酸合成中起关键作用,本发明在叶酸生物合成的研究及应用领域具有很大潜力。
与现有技术相比本发明具有以下优势:
1、基因来自于食源性植物乳杆菌,具有安全性,可用于后期食品发酵领域;
2、folB基因在叶酸合成中的关键作用,为叶酸合成功能性食品的研发提供一定理论基础。
附图说明
图1为过表达菌株菌液PCR验证结果,其中M为2000bp Marker,泳道 1、2、3为构建成功的过表达载体;
图2为本发明野生型菌株及过表达BDB菌株叶酸总产量结果示意图;
图3为本发明野生型菌株及过表达BDB菌株的单个细胞内叶酸含量示意图。
具体实施方式
下面结合附图和实施例对本发明作进一步详细说明,但本发明保护范围不局限于所述内容,实施例中使用的试剂和方法,如无特殊说明,均采用常规试剂和使用常规方法,下述实施例中的结果如无特别说明,均为三次重复的平均值。
实施例1:二氢蝶呤醛缩酶(DHNA)基因folB的克隆
采用CTAB/酶法提取植物乳杆菌YM-4-3基因组总DNA,采用如下引物对基因folB进行PCR扩增;
BDB-F:ATACCCGGGCATGGGCATGATTCGAATTA;
BDB-R:CCCAAGCTTCTACTTGCCATTCGGCGTCC;
PCR扩增体系如下(50μL):
PCR扩增条件:95℃预变性5min;95℃变性30s;60℃退火15s;72℃延伸30s;循环30次,最后72℃延伸10min;将扩增出的 PCR 产物测序比对。测序结果显示,获得一段369bp长的序列,核苷酸序列如SEQ ID NO: 1所示。
实施例2:以质粒 pMG36e 为骨架构建表达载体
1、实施例1扩增获得的目的基因folB片段和pMG36e 质粒经HindⅢ、XmaⅠ两个限制性内切酶进行酶切,酶切体系如下:
37℃酶切4h后,2%琼脂糖凝胶电泳鉴定,参考胶回收试剂盒说明书进行酶切产物回收,-20℃保存。
2、酶切产物连接
酶切产物纯化后使用T4连接酶连接,16℃连接过夜,连接体系(10μL)如下:
3、连接产物转化大肠杆菌 DH5α 菌株及验证
1) 从-80℃冰箱中拿出制备好的大肠杆菌DH5α感受态,冰上解冻后,加入全部连接反应好的连接产物,轻轻吹打混匀,冰上放置30min;
2) 42℃加热 45s 后,冰上放置2min;
3) 加入890µL SOC培养基,37℃摇床培养60min;
4) 将菌液在8000rpm/min条件下离心1min,取出900µL上清,剩余100μL菌悬液;
5) 将菌悬液涂布于含有500µg/mL红霉素的LB平板上,筛选转化子(28℃倒置培养);
6)挑选单个菌落使用 36eFF 和 36eRR 引物进行菌液PCR验证,挑选阳性克隆;送至测序公司测序;
36eFF:ATTCGGTCCTCGGGATATG
36eRR:TTCATTCAGTCATCGGCTTTCA;
验证结果见图1,图中泳道1、2、3构建成功的过表达载体,对其进行测序验证,测序获得的序列与理论值相同,重组过表达载体构建成功。
4、重组表达载体提取
挑选测序正确的含有重组载体的大肠杆菌在28℃、200rpm/min下过夜培养,使用Genestar Starprep Plasmid Miniprep Kit 试剂盒进行质粒提取,操作步骤参考说明书;琼脂糖凝胶电泳验证质粒是否提取成功。
实施例3:表达载体转化YM-4-3菌株感受态细胞
1、YM-4-3 菌株感受态细胞的制备
YM-4-3菌株解冻后按4‰接种量接种至MRS肉汤培养基中,37℃培养12h,取1mL接种于含有2.5%甘氨酸的50mL MRS肉汤培养基中,培养至其OD 600值达到0.6时停止培养,4℃、4000 rpm/min下离心10min 收集菌液;用25mL 冰冷的无菌水洗涤两次,再次离心,弃上清,将菌体重悬于0.05mol/L冰冷的EDTA溶液中,冰浴5min,再加入25mL冰冷的无菌水,于4℃、8000 rpm/min离心5min,再用25mL冰冷的无菌水洗涤,用25mL电击缓冲液(0.5 mol/L蔗糖,10%甘油),在4℃、8000 rpm/min下离心10min,重复一次;将菌体重悬于0.8mL电击缓冲液中,每个100μL分装于灭菌的离心管中,-80℃保存。
2、表达载体转化 YML 4-3 菌株感受态细胞
取出于无水乙醇中浸泡的电转杯,转移至75%的乙醇中浸泡3-4 h,放在超净工作台中风干乙醇并紫外灭菌。取YM-4-3感受态细胞在冰上解冻后,加入10µL已构建好且序列正确的表达载体,将混合液转移至电转杯凹槽处,盖好盖子,放置电转仪中,2.5 kV处理2s;电击完成后,立即加入890µL MRS 肉汤培养基,轻轻吹打混匀后,转移至干净的离心管中,28℃培养4h,培养好的菌液8000rpm/min离心2min,留100μL悬浮菌液涂布于含50µg/mL红霉素的MRS 固体培养基平板,28℃倒置培养;
(3)PCR 法筛选重组菌株
挑选单个菌落接种至含有50µg/mL红霉素的MRS液体培养基中,37℃静置培养过夜,使用36eFF和36eRR引物进行菌液 PCR,挑选阳性克隆,从而获得过表达folB基因的菌株BDB。
实施例4:叶酸含量及 OD600 值测定
1、菌株培养
将储存于-80℃的BDB、YM-4-3菌株按体积分数4‰的量接种于新鲜的MRS肉汤培养基中,37℃静置培养18h进行活化后,再次按体积分数4‰的量接种于新鲜的FACM液体培养基中37℃培养18h;接种于FACM液体培养基中的菌液在FACM培养基中连续传代3 次后进行菌落计数,之后按107CFU/mL的浓度接种于30mL FACM 液体培养基中,37℃静置培养,每隔20h取一次样为此时间点下叶酸的产量;在BDB菌株培养转接过程中,培养基均含5μg/mL的红霉素以防质粒丢失;
2、将上述培养的菌液避光超声破碎处理20min,12000 rpm/min离心10 min,取1mL上清直接用于LC-MS分析叶酸含量;
a、色谱条件:色谱柱,Waters ACQUITY UPLC BEH Amide 柱 (2.1 mm×100mm,1.7µm);流动相为甲醇(含5mmol/L甲酸铵)和水(含5mmol/L甲酸铵),梯度洗脱:0-5min,98%-95%甲醇;5-10min,95%-55%甲醇;10-12min,55%甲醇;12-14min,55%-98%甲醇;14-20min,98%甲醇;流速为0.2mL/min,柱温为 35℃,进样量为5µL;
b、质谱条件:4500QTrap 质谱参数设置如下:气帘气 (CUR) 25,碰撞气(CAD) 中等,离子源气 1 (GS1) 45,离子源气 2 (GS2) 50,电喷雾电压 5500V,加热器温度350℃,选用正离子模式进行检测,离子源为 ESI 电离源;
由图2、3可知,过表达菌株BDB产叶酸的能力显著高于野生型菌株;证明folB基因在植物乳杆菌叶酸合成中发挥关键作用;因此,后续可通过过量表达二氢蝶呤醛缩酶的方式提高叶酸产量,以达到扩大生产的目的,为植物乳杆菌YM-4-3菌株及其代谢产物在工业上的应用提供理论基础。
序列表
<110> 昆明理工大学
<120> 二氢蝶呤醛缩酶基因的用途
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 369
<212> DNA
<213> 植物乳杆菌YM-4-3(Lactobacillus plantarum YM-4-3)
<400> 1
atgggcatga ttcgaattaa taatttacgc tttcacacgt ttaacggggt acttccggaa 60
gaacggcgta atggtcaaca actagggcta gatattgcca ttaaatatcc tatcgaaacc 120
aaggttcaac acgatgacgt tcacgagacc atcaattacg cggcggtccg taacgtggtc 180
gatgaatttg taacgaccca ttcatacaag ttgattgaat cgctagctaa ccacttattg 240
cagacgttat tgacaagttt tcccgcggcg gatgcaatca atattaaaat tcgtaaatat 300
agcgtaccaa tgcctggaat ctttgatgat gtggaaattg aggtggaggg gacgccgaat 360
ggcaagtag 369
<210> 2
<211> 29
<212> DNA
<213> 人工序列(Artificial)
<400> 2
atacccgggc atgggcatga ttcgaatta 29
<210> 3
<211> 29
<212> DNA
<213> 人工序列(Artificial)
<400> 3
cccaagcttc tacttgccat tcggcgtcc 29
<210> 4
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<212> DNA
<213> 人工序列(Artificial)
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attcggtcct cgggatatg 19
<210> 5
<211> 22
<212> DNA
<213> 人工序列(Artificial)
<400> 5
ttcattcagt catcggcttt ca 22
Claims (1)
1.二氢蝶呤醛缩酶基因folB在提高植物乳杆菌(Lactobacillus plantarum)叶酸合成中的应用,所述二氢蝶呤醛缩酶基因folB的核苷酸序列如SEQ ID NO: 1所示。
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