CN112813085A - 焦磷酸酶基因的用途 - Google Patents
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Abstract
本发明公开了一种焦磷酸酶基因的用途,焦磷酸酶基因folQ在提高植物乳杆菌叶酸合成中的应用,所述焦磷酸酶基因folQ的核苷酸序列如SEQ ID NO:1所示;本发明将基因folQ与组成型载体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菌株体内实现基因folQ的过表达,获得过表达菌株;采用LC‑MS法测定菌株产叶酸能力,发现与野生型菌株相比,BDQ菌株产叶酸单谷氨酸含量增加,folQ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,具体涉及焦磷酸酶基因folQ在提高植物乳杆菌(Lactobacillus plantarum)YM- 4-3叶酸生物合成中的应用。
背景技术
乳酸菌(Lactic Acid Bacteria)是一类公认安全的发酵葡萄糖或用乳糖产生乳酸的微生物的统称。其不仅存在于无机环境当中,而且在人类、动物肠道等环境中亦很常见,传统上与发酵食品相关,并且与人类文化和福祉密切相连,在历史上,乳酸菌因其对所发酵食品感官、质量和安全方面所具有的积极贡献而广为人知。
叶酸(Folic acid),维生素B9,是一种水溶性B族维生素。是由蝶呤、对氨基苯甲酸(p-aminobenzoic acid, pABA)和一个或多个谷氨酸结合而成,是一种无色无味的黄色或橙黄色的晶体或晶状粉末,不溶于乙醇、乙醚等有机溶剂,溶于氨水、氢氧化钾等碱性溶液,在酸性条件下极不稳定,在光照条件下,尤其是紫外线的照射下易被破坏。生物体普遍需要叶酸,然而不同的生物体获得该营养物的途径各不相同。动物本身不能够合成叶酸,然而能够通过摄入食物而获得。
研究表明乳酸菌其能够合成叶酸,其叶酸合成的途径由蝶呤分支和对氨基苯甲酸(pABA)分支组成;焦磷酸酶(Dihydroneopterin triphosphate diphosphatase,DHNTPase,)位于蝶呤分支上,由folQ基因编码;它将folE基因催化得到的7,8-二氢雄甾酮三磷酸转化为相应的单磷酸,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。
随经济发展而来的是人类健康的需求不断升高,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在叶酸工业生产中的大规模应用,因此将愈加成熟的基因工程技术对相应菌株基因层面的改造结合最优的培养条件能有效提高叶酸合成量并降低成本,并使得乳酸菌来源的叶酸的工业化生产成为可能。
发明内容
针对现有技术存在的不足,本发明提供了一种焦磷酸酶基因的用途,即焦磷酸酶基因folQ在提高植物乳杆菌(Lactobacillus plantarum)叶酸合成中的应用,所述焦磷酸酶基因folQ的核苷酸序列如SEQ ID NO: 1所示;
本发明从植物乳杆菌(Lactobacillus plantarum)YM-4-3中克隆焦磷酸酶基因folQ,将基因folQ与组成型载体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菌株体内实现基因folQ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BDQ的叶酸产量,证明folQ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、folQ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、folQ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,本发明在叶酸生物合成研究及应用领域具有巨大潜力,本发明适用于工业化生产和市场推广应用。
附图说明
图1为本发明过表达菌株菌液PCR验证,其中M:2000 bp Marker,泳道1、2、3:过表达载体folQ质粒,泳道 4:阴性对照;
图2 为本发明中利用LC-MS检测的叶酸单谷氨酸标准曲线;
图3 为本发明野生型及过表达BDQ菌株72h内OD600变化情况示意图;
图4 为本发明野生型及过表达BDQ菌株叶酸单谷氨酸产量示意图。
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:焦磷酸酶合成基因fol Q克隆
根据YM-4-3 基因组序列设计引物,使用高保真Taq酶对folQ基因进行PCR扩增(扩增体系见表1),扩增产物获得后,对其3’端连接poly A尾巴(产物14.5 μL、Taq buffer 2 μL、dNTP 3 μL、Taq酶0.5μL,72℃反应15min),扩增引物如下:
BDQF:TCCCCCGGGAACAGGCTGGTTGGC;
BDQR:AACTGCAGTCCGTTGCTTGTGCT;
表1 PCR扩增体系如下(50μL):
扩增条件:95℃预变性3min;95℃变性15s;55℃退火15s;72℃延伸1min;循环30次,最后72℃延伸10min;将扩增出的 PCR 产物测序比对。由测序结果得出,获得一段588bp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YM-4-3 folQ过表达菌株
1、实施例1扩增获得的目的基因folQ片段和pMG36e质粒经PstⅠ、SmaⅠ两个限制性内切酶进行酶切,酶切体系如下:
37℃酶切4h后,采用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,连接体系如下(10μL):
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: GCGGTTACTTTGGATTTTTG
36eRR: TTCATTCAGTCATCGGCTTTCA ;
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。
5、制备YM-4-3菌株感受态细胞
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12 h,取1mL接种于含有2.5%甘氨酸浓度的50mL MRS 肉汤培养基中,培养至其OD600值达到0.6时停止培养,4℃、4000rpm/min,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℃、8000rpm/min离心5min,再用25mL 冰冷的无菌水洗涤,用25mL 电击缓冲液(0.5mol/L蔗糖,10%甘油),4℃、8000rpm/min,离心10min,重复一次;将菌体重悬于0.8mL 电击缓冲液中,按照每管100 μL 分装于灭菌的离心管中,-80℃保存。
6、表达载体转化 YML4-3 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YML4-3 感受态细胞在冰上解冻后,加入10μL 已构建好且序列正确的过表达载体,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,2s。点击完成后,立即加入890μLMRS 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 悬浮菌液涂布与含50 μg/mL 红霉素的MRS固体 培养基平板,28℃倒置培养。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验证;PCR结果如图1所示;将PCR产物测序验证,发现所含序列与folQ序列一致。
实施例3:叶酸含量及 OD600 值测定
取出活化后的YML4-3菌株和BDQ菌株,分别接种至不含抗生素和含有50μg/mL红霉素的50mL液体MRS培养基中,每隔12 h取样一次,测定样品OD600值,并使用LC-MS测定叶酸含量,具体方法为:
每隔12h得到的菌液使用超声破碎仪进行破碎,条件为:超声5s,停止5s,振幅30%,10min。破碎后,4℃、12000 rpm/min下离心3min,取上清液至液相小瓶中进行LC-Qtrap MS定量分析。
色谱条件:样品通过自动进样器连接双通进入到质谱进行检测;流动相为A (水,含 5mmol/L甲酸铵)和B(甲醇,含5 mmol/L 甲酸铵),等度洗脱(0-3min,30%A)。流速为0.2mL/min,柱温为 35℃,进样量为5µL,样品盘温度为 4℃。
叶酸单谷氨酸标准曲线的绘制:精密称取1mg叶酸单谷氨酸标准品溶于1mL 1%氨水溶液中,配成浓度为1mg/mL的母液,逐级稀释为1μg/mL、100ng/mL、10ng/mL、1ng/mL、0.1ng/mL和0.01 ng/mL的标准品,按照上面的液质检测条件进质谱进行检测。以叶酸单谷氨酸标准品浓度为横坐标,峰面积为纵坐标,绘制工作曲线(图2),为样品中叶酸单谷氨酸的定量检测建立标准曲线。
由图3、4可知,两株菌在0-24h中,每1个OD值中菌株产生的叶酸含量均在增加,且24h时达到顶峰,在这个过程中,BDQ 菌株的产叶酸速率高于YM-4-3,folQ基因提高了菌株产叶酸速率;在24-48h 中,两株菌产叶酸含量均呈现下降趋势,且BDQ菌株下降的较慢。由此得出,folQ过表达菌株在0-48h内提高了叶酸单谷氨酸的产量。
序列表
<110> 昆明理工大学
<120> 焦磷酸酶基因的用途
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 588
<212> DNA
<213> 植物乳杆菌YM-4-3(Lactobacillus plantarum YM-4-3)
<400> 1
atgacgacga cttggttgat tgcatccaac aacgctggta aaagccgcga cttgatcgcg 60
tgtttggctt attatggctt gactgctcgt cagtacttga cagtggcgcc gcggctcgaa 120
tttcccgtgg aaacgacgac gagctatgtc gataacgcgg ttgctaaagc tcgttttggg 180
gcgcaacagc taggggttcc ggttatcgca gatgatagtg gcttagagat ttccgcgtta 240
ccagacttgt taggtgtgac cacggcgcgc gacttagggg ttgcagtcag tggctttgat 300
cgcaatcagg aaattttaac ggccctacgc gatatccctg acaacgagcg gcaagcgttg 360
atgcgtgcta cgttagcggc tgcctggcca gatgggcgga ccttggccgt acaagcttcg 420
atcaccggct acattgcgtc ctatcaattt ggacgctatt ctgggggatt tgaccgaatt 480
ttctggctac cgcgctatgg tcgaactttt gccgaattac cagcaacgtg gcgcatcccg 540
ctgacacacc ggggacgggc cgccttaaaa ttaatcacaa aactttaa 588
<210> 2
<211> 24
<212> DNA
<213> 人工序列(Artificial)
<400> 2
tcccccggga acaggctggt tggc 24
<210> 3
<211> 23
<212> DNA
<213> 人工序列(Artificial)
<400> 3
aactgcagtc cgttgcttgt gct 23
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial)
<400> 4
gcggttactt tggatttttg 20
<210> 5
<211> 22
<212> DNA
<213> 人工序列(Artificial)
<400> 5
ttcattcagt catcggcttt ca 22
Claims (1)
1.焦磷酸酶基因folQ在提高植物乳杆菌(Lactobacillus plantarum)叶酸合成中的应用,所述焦磷酸酶基因folQ的核苷酸序列如SEQ ID NO: 1所示。
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111235169A (zh) * | 2020-02-03 | 2020-06-05 | 昆明理工大学 | 一种GTP环化水解酶I基因folE及应用 |
CN112795527A (zh) * | 2021-03-05 | 2021-05-14 | 昆明理工大学 | 二氢蝶呤醛缩酶基因的用途 |
CN112852844A (zh) * | 2021-03-05 | 2021-05-28 | 昆明理工大学 | 羟甲基二氢蝶呤焦磷酸激酶基因folK的用途 |
CN112961878A (zh) * | 2021-03-08 | 2021-06-15 | 昆明理工大学 | 一种植物乳杆菌的基因在叶酸生物生成中的应用 |
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Citations (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU1040102A (en) * | 2000-10-20 | 2002-04-29 | Bioteknologisk Inst | Improved fermentation method for production of heterologous gene products in lactic acid bacteria |
US20090221027A1 (en) * | 2005-07-18 | 2009-09-03 | Basf Ag | Use of a bacillus meti gene to improve methionine production in microorganisms |
US20160340698A1 (en) * | 2015-05-18 | 2016-11-24 | Samsung Electronics Co., Ltd. | Genetically engineered yeast cell having increased nadph production, method of increasing nadph level in yeast cell, method of preparing yeast cell, and method of producing lactate using yeast cell |
CN107474124A (zh) * | 2017-08-25 | 2017-12-15 | 中国农业科学院生物技术研究所 | OsAPBP2蛋白在促进植物叶酸合成中的应用 |
KR20180134601A (ko) * | 2017-06-09 | 2018-12-19 | 주식회사 쎌바이오텍 | 임산부를 위한 기능성 유산균 조성물 |
CN109735556A (zh) * | 2019-02-22 | 2019-05-10 | 昆明理工大学 | 引导糖基转移酶基因的用途 |
CN109810991A (zh) * | 2019-03-02 | 2019-05-28 | 昆明理工大学 | 二氢蝶酸合酶基因folP的用途 |
CN110582296A (zh) * | 2016-11-11 | 2019-12-17 | 勃林格殷格翰动物保健有限公司 | 通过减弱细菌叶酸转运来减弱细菌毒力 |
CN111235169A (zh) * | 2020-02-03 | 2020-06-05 | 昆明理工大学 | 一种GTP环化水解酶I基因folE及应用 |
CN112280795A (zh) * | 2020-11-17 | 2021-01-29 | 昆明理工大学 | 糖基转移酶基因的用途 |
CN112795527A (zh) * | 2021-03-05 | 2021-05-14 | 昆明理工大学 | 二氢蝶呤醛缩酶基因的用途 |
CN112852844A (zh) * | 2021-03-05 | 2021-05-28 | 昆明理工大学 | 羟甲基二氢蝶呤焦磷酸激酶基因folK的用途 |
CN112961878A (zh) * | 2021-03-08 | 2021-06-15 | 昆明理工大学 | 一种植物乳杆菌的基因在叶酸生物生成中的应用 |
CN113215131A (zh) * | 2021-06-04 | 2021-08-06 | 天津大学佐治亚理工深圳学院 | 磷酸水解酶及其应用 |
-
2021
- 2021-03-05 CN CN202110246865.6A patent/CN112813085B/zh active Active
Patent Citations (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU1040102A (en) * | 2000-10-20 | 2002-04-29 | Bioteknologisk Inst | Improved fermentation method for production of heterologous gene products in lactic acid bacteria |
US20090221027A1 (en) * | 2005-07-18 | 2009-09-03 | Basf Ag | Use of a bacillus meti gene to improve methionine production in microorganisms |
US20160340698A1 (en) * | 2015-05-18 | 2016-11-24 | Samsung Electronics Co., Ltd. | Genetically engineered yeast cell having increased nadph production, method of increasing nadph level in yeast cell, method of preparing yeast cell, and method of producing lactate using yeast cell |
CN110582296A (zh) * | 2016-11-11 | 2019-12-17 | 勃林格殷格翰动物保健有限公司 | 通过减弱细菌叶酸转运来减弱细菌毒力 |
KR20180134601A (ko) * | 2017-06-09 | 2018-12-19 | 주식회사 쎌바이오텍 | 임산부를 위한 기능성 유산균 조성물 |
CN107474124A (zh) * | 2017-08-25 | 2017-12-15 | 中国农业科学院生物技术研究所 | OsAPBP2蛋白在促进植物叶酸合成中的应用 |
CN109735556A (zh) * | 2019-02-22 | 2019-05-10 | 昆明理工大学 | 引导糖基转移酶基因的用途 |
CN109810991A (zh) * | 2019-03-02 | 2019-05-28 | 昆明理工大学 | 二氢蝶酸合酶基因folP的用途 |
CN111235169A (zh) * | 2020-02-03 | 2020-06-05 | 昆明理工大学 | 一种GTP环化水解酶I基因folE及应用 |
CN112280795A (zh) * | 2020-11-17 | 2021-01-29 | 昆明理工大学 | 糖基转移酶基因的用途 |
CN112795527A (zh) * | 2021-03-05 | 2021-05-14 | 昆明理工大学 | 二氢蝶呤醛缩酶基因的用途 |
CN112852844A (zh) * | 2021-03-05 | 2021-05-28 | 昆明理工大学 | 羟甲基二氢蝶呤焦磷酸激酶基因folK的用途 |
CN112961878A (zh) * | 2021-03-08 | 2021-06-15 | 昆明理工大学 | 一种植物乳杆菌的基因在叶酸生物生成中的应用 |
CN113215131A (zh) * | 2021-06-04 | 2021-08-06 | 天津大学佐治亚理工深圳学院 | 磷酸水解酶及其应用 |
Non-Patent Citations (11)
Title |
---|
CHEN-JIAN LIU等: "Transcriptomic analysis of de novo folate biosynthetic genes in Lactobacillus plantarum strain 4_3 in fermented soybean", 《FOOD AND FUNCTION》 * |
JEONG,D.-Y.: "Lactiplantibacillus plantarum strain SRCM100442 chromosome, complete genome", 《GENBANK DATABASE》 * |
任贝贝;李晓然;罗义勇;杨恩;柳陈坚;: "植物乳杆菌近亲种中叶酸生物合成途径的基因多态性分析" * |
吴边;柳陈坚;李强坤;李晓然;: "pH对产叶酸植物乳杆菌叶酸产量及相关基因表达的影响" * |
吴边等: "pH对产叶酸植物乳杆菌叶酸产量及相关基因表达的影响", 《安徽农业大学学报》 * |
张海燕;柳陈坚;何树芬;李晓然;: "高产叶酸植物乳杆菌的筛选及应用研究" * |
李强坤: ""植物乳杆菌近亲种叶酸生物合成差异分析及发酵条件优化"", 《中国优秀博硕士学位论文全文数据库(硕士)工程科技I辑》 * |
李强坤;柳陈坚;罗义勇;杨恩;李晓然;: "发酵条件对植物乳杆菌叶酸合成的影响" * |
李强坤等: "发酵条件对植物乳杆菌叶酸合成的影响", 《食品科学》 * |
阚静;李莉;许激扬;: "叶酸的生物合成及其代谢工程研究进展" * |
阚静等: "叶酸的生物合成及其代谢工程研究进展", 《中国生化药物杂志》 * |
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