CN106520802B - 一种提高乳酸菌耐胁迫能力的gad基因及其应用 - Google Patents
一种提高乳酸菌耐胁迫能力的gad基因及其应用 Download PDFInfo
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- CN106520802B CN106520802B CN201611247840.3A CN201611247840A CN106520802B CN 106520802 B CN106520802 B CN 106520802B CN 201611247840 A CN201611247840 A CN 201611247840A CN 106520802 B CN106520802 B CN 106520802B
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Abstract
本发明公开了一种提高乳酸菌耐胁迫能力的GAD基因，其特征在于，所述基因序列如SEQ ID NO.1所示。所述基因可用于提高乳酸菌耐低温胁迫能力和耐酸胁迫能力。具体方法是向乳酸菌中引入含有GAD基因的重组质粒，通过在乳酸菌中过量表达合成γ‑氨基丁酸的谷氨酸脱羧酶CsGAD以提高乳酸菌耐低温胁迫能力和耐酸胁迫能力。从而提高乳酸乳球菌在低温和酸性环境下的存活率，可用于多种保健食品的开发应用，为工业化生产提供良好的保证。
Description
技术领域
本发明属于生物工程技术领域，具体涉及到一种利用基因工程技术内源表达GABA以改良乳酸菌耐胁迫性能的基因及其应用。
背景技术
乳酸菌(Lacticacid bacteria,LAB)是一群形态、代谢性能和生理学特征不完全相同的革兰氏阳性菌的总称。乳酸菌普遍存在于人类和动物的肠道中，被人们一致认同是安全级的微生物。
乳酸菌具有抑制病原微生物在肠上皮细胞表面黏附、定植和侵袭肠上皮细胞的功能，乳酸菌的代谢产物也具有阻止病原微生物入侵生物屏障作用。由于乳酸菌对动植物和人体均具有良好的益生作用，乳酸菌又被称为“益生菌”，在乳制品及相关产品的生产、制作植物发酵型蛋白饮料、蔬菜深加工等领域有广泛的开发运用。
但是在大规模的工业化生产过程中，乳酸菌无法避免的面临各种不利条件影响其生长发育。包括“低温胁迫”和“酸胁迫”。乳酸菌是嗜温微生物，温度过高过低都会影响乳酸菌的生长增殖。但是在工业化的生产、运输过程中为了保证乳酸菌制品的质量，必须对其进行低温贮存，这样不可避免的会对乳酸菌造成一定的伤害。在冷冻过程会导致细胞膜上形成大量冰晶，导致细胞膜损伤，进而导致细胞的生理、代谢功能受到损伤。同时温度下降会导致细胞中的水分含量减少，胞内外的电解质浓度增加，造成“溶质效应”。此外，低温时，DNA糖基键的断裂使碱基产生了质子化作用，DNA产生脱嘌呤和脱嘧啶反应，使得DNA在自我修复过程中的错误率极大提高了，这可能会产生生物突变体，也是细胞丧失生命力的重要因素之一。
与此同时，乳酸菌作为嗜中性微生物，是发酵生产酸性物质的典型菌株，乳酸菌在增殖过程中所产生的乳酸、乙酸等酸性物质发挥着双重作用。一方面，乳酸菌代谢所产生的高浓度酸抑制了其他微生物的生长增殖，增强了食品的货架保存期，并且可以与醛类、酮类等食品中的一些物质发生反应，进而产生了芳香类化合物，赋予了食品独特的风味。但是如果外界的生长环境过于偏酸性时乳酸菌的代谢和生长就会遭到抑制；同时，由于乳酸菌细胞结构的影响，高浓度的酸会使细胞质中大量积累质子，随即会使得细胞内呈酸性即pH值降低，使得跨膜△pH受到影响，消耗乳酸菌内的质子推动力。进一步影响乳酸菌的生物活性和增殖能力。
目前国内外鲜有直接提高乳酸菌抗性的报导。但是却有研究者通过别的研究方法来间接的帮助乳酸菌抵御外界胁迫环境。例如，可与通过调控氨基酸代谢来提高乳酸菌的抵御酸胁迫能力；通过调控细胞膜上脂肪酸的组成进而调控细胞膜和细胞壁的生理功能来提高乳酸菌的抗性；通过引入别的代谢途径来改善乳酸菌的抗性等，但是尚无报道表明能同时提高乳酸菌的抗酸和抗低温能力，因此提供一种提高乳酸菌综合胁迫抗性的方法尤为重要。
γ-氨基丁酸作为一种广泛存在于生物体内的非蛋白质氨基酸，主要由L-谷氨酸经谷氨酸脱羧酶(glutamic acid decarboxylase，GAD)脱羧而成，作为三羧酸循环的重要支路参与氨基酸代谢。同时参与到多种生物体的生长发育和抵御环境胁迫过程中，异源表达高产GABA的GAD基因有助于参与乳酸菌抗胁迫能力的改良。因此，研究GABA在乳酸菌增殖过程中所扮演的角色具有重要的意义。
发明内容
本发明的目的是提供一种提高乳酸乳球菌耐受低温胁迫和酸胁迫能力的GAD基因及其应用，向乳酸菌中引入含有所述GAD基因的质粒，通过在乳酸菌中过量表达合成γ-氨基丁酸的谷氨酸脱羧酶CsGAD以提高乳酸菌耐低温胁迫能力和耐酸胁迫能力，从而提高乳酸乳球菌在低温和酸性环境下的存活率，为工业化生产提供良好的保证。
本发明所请求保护的技术方案如下：
本发明的目的之一请求保护一种提高乳酸菌耐胁迫能力的GAD基因，所述基因序列如SEQ ID NO.1所示。
本发明目的之二是请求保护一种蛋白质，所述蛋白质的氨基酸序列如
SEQ ID NO.2所示.。
本发明的目的之三是请求保护一种构建体，所述构建体包含编码SEQ ID NO.1所示的核苷酸序列。
本发明的目的之所四是请求保护一种构建重组菌的方法，包括以下步骤：a、将所述GAD基因连接到表达载体上获得重组质粒；b将重组质粒转化到宿主菌中获得重组菌。
本发明的目的之所五是请求保护该基因在提高乳酸菌耐胁迫能力中的应用。
所述应用中，乳酸菌耐胁迫能力是指耐低温胁迫能力和耐酸胁迫能力。
本发明的目的之六是请求保护一种生产食品级γ-氨基丁酸的方法，该方法是向乳酸菌中引入含有GAD基因的构建体，通过GAD基因在乳酸菌中过量表达合成γ-氨基丁酸，由于乳酸菌的表达系统本身是食品级的，可以以达到生产富含具有保健效果γ-氨基丁酸的乳酸菌的目的。
与已有技术相比，本发明有益效果表现在：
本发明GAD基因，通过在乳酸菌中过量表达合成γ-氨基丁酸的谷氨酸脱羧酶CsGAD以提高乳酸菌耐低温胁迫能力和耐酸胁迫能力，从而提高乳酸乳球菌在低温和酸性环境下的存活率，同时产生的γ-氨基丁酸可用于多种保健食品的开发应用，为工业化生产提供良好的保证。
附图说明
图1是本发明实施例中正常培养条件下重组菌与对照菌的生长曲线。
图2是本发明实施例中对数期中期重组菌与对照菌在低温冷冻胁迫下生长情况对比。
图3是本发明实施例中稳定期前期重组菌与对照菌在低温冷冻胁迫下生长情况对比。
图4是本发明实施例中稳定期后期重组菌与对照菌在低温冷冻胁迫下生长情况对比。
图5是本发明实施例中重组菌与对照菌在酸胁迫下生长情况对比。
图6是重组菌电泳图谱。
图6中，M：Marker(蛋白分子量标记)；1：空载体诱后；2：重组载体诱前；3：重组载体诱后；4：重组载体诱前上清；5：重组载体诱后上清；6：重组载体诱前沉淀；7：重组载体诱后沉淀。
具体实施方式
以下结合附图通过具体实施方式对本发明技术方案做进一步解释说明。
1.重组表达菌株的构建：
扩增本发明人前期克隆自茶叶叶片中的CsGAD基因序列，该并将其连接到乳酸乳球菌表达载体pNZ8148上，获得重组质粒pNZ8148-CsGAD，再将其电转化入宿主菌L.lactisNZ9000中，得到重组菌株L.lactis NZ9000-CsGAD(乳酸菌L.lactisNZ9000以及表达质粒载体pNZ8048购于湖南长沙赢润生物技术有限公司)。详细步骤如下：
(1)根据已经获得的茶树GAD基因序列，序列如SEQ ID NO.1所示，运用Primer 5.0软件在序列的5’和3’设计一对引物：
CsGAD-up CCG CCATGG ATGGTTCTCTCAAAGATTGC NcoI酶切位点
CsGAD-down CCC AAGCTT CTAGCAAATCACTTGTGT HindIII酶切位点
利用高保真酶从提取的茶树叶片cDNA中进行PCR扩增，对所得的PCR产物进行纯化回收，回收产物利用普通Taq酶进行加“A”反应(常规操作)后再次回收连接入克隆载体pMD19-T，构建pMD19-CsGAD质粒并转化入大肠杆菌Trans1-T1感受态细胞，氨苄青霉素平板培养过夜后挑取阳性克隆测序。
(2)确定测序结果与原始序列无误后，对提取的高浓度pMD19-CsGAD质粒和pNZ8148质粒(商品化的空表达载体)进行NcoI和HindIII酶的双酶切，回收酶切产物后用T4连接酶进行连接反应，获得pNZ8148-CsGAD表达载体，再次转化入大肠杆菌Trans1-T1感受态细胞，培养过夜后挑菌测序。
(3)测序确定结果无误后，摇菌提取pNZ8148-CsGAD质粒，利用电穿孔仪转入乳酸乳球菌NZ9000感受态细胞，30℃培养并挑取阳性克隆进行菌落PCR和质粒双酶切的验证。电泳检测片段大小正确后表明获得重组乳酸菌L.lactis NZ-CsGAD。
2.PNZ8048-CsGAD的诱导表达与检测
将经测序正确的导入目的质粒的乳酸菌命名为重组菌，将导入NZ9000空载体的乳酸菌命名为对照菌。对获得的重组乳酸菌L.lactis NZ-CsGAD利用nisin(乳酸链球菌素，一种天然生物活性抗菌肽)进行诱导表达，然后利用垂直板电泳对表达蛋白进行检测，通过与对照菌总蛋白量进行对比，确定重组菌中GAD蛋白的表达。
(1)活化重组乳酸菌L.lactis NZ-CsGAD至5ml GM17液体培养基中(含有10ng/mLChl+)，30℃静置培养过夜，随后按照5％接种量放大培养至50ml GM17培养基(含有10ng/mLChl+，2ng/mL nisin)，以导入NZ9000空载体的乳酸菌作为对照菌同时进行处理。诱导培养8h后收集细胞，用无菌生理盐水洗涤2次后加入50mM Tris-HCl缓冲液(pH 7.4)，冰浴下超声破碎5min，离心取上清，加入上样缓冲液后，进行SDS-PAGE分析。
上述GM17肉汤培养基是M17肉汤培养基补充浓度为5g/L的葡萄糖溶液，即为GM17肉汤培养基，M17肉汤培养基为常见商业用乳酸菌培养基，成分为每升水中加入：植物蛋白胨5g，酵母粉5g，聚蛋白胨5g，抗坏血酸0.5g，牛肉膏2.5g，MgSO4·7H2O 0.01g，甘油磷酸二钠19g。
(2)通过电泳图谱检测发现(如图6所示)PNZ8048-CsGAD重组菌相比对照菌而言，上清液中有一明显的表达条带，经与蛋白Marker对比，分子量约为56KD，与目标蛋白GAD的分子量一致，说明CsGAD蛋白成功在乳酸菌L.lactis中被表达。
3.重组菌生长活力检测
对表达成功的重组菌进行生长曲线的测定，以导入NZ9000空载体的乳酸菌作为对照菌。按照上述方法在正常情况下对重组菌和对照菌进行培养，并测定它们的生长曲线，如图1所示，重组菌与对照菌的生长曲线相比发生了明显的变化(对照菌中同样添加nisin)。其中对照菌大概在6h达到了稳定期，而重组菌大约在8h达到稳定期，但是重组菌到达稳定期的最大生物量(OD600)大约是对照菌的1.5倍，说明重组PNZ8048-CsGAD质粒在表达GAD的蛋白情况下大大提高了乳酸菌的生长活力。
4.抗胁迫能力的比较
(1)低温胁迫下耐受力试验
将第3步中的重组菌培养液按照5％的接种量分别接种于新鲜的GM17肉汤液体培养基(含有10ng/mL Chl+，2ng/mL nisin)中。根据前期测定生长曲线，分别培养至对数期中期、稳定期前期和稳定期中后期，取这3个时期的发酵菌液，4℃，6000r/min离心5min分钟后，去上清，无菌生理盐水洗涤2次后，用等量的新鲜的GM17液体培养基重悬后，1mL/管分装，置于4℃和-20℃温度下，每隔一段时间，取出经无菌生理盐水洗涤、离心后，按1:10，1:100，1:1000的稀释度点种于含10ng/mL氯霉素的GM17固体平板，根据所得的数据计算存活个数。
低温胁迫实验结果表明，培养至稳定前期的乳酸菌抗冻能力要好于对数期中期和稳定后期两个培养阶段，其存活菌个数大大高于其他两个阶段(见图2,3,4)，但是无论在哪个培养阶段，无论是出于4℃冷藏还是-20℃冷冻胁迫下，重组菌的存活数目均远高于对照菌几个数量级，说明通过在L.lactis NZ9000中表达CsGAD蛋白的方法可以极为显著地提高乳酸乳球菌的抗冻胁迫能力。
(2)酸胁迫条件下耐受性试验
将活化后的种子培养液按照5％的接种量分别接种于新鲜的GM17肉汤液体培养基(10ng/mL氯霉素，2ng/mL nisin)中，30℃诱导培养至OD 2.0，将诱导后的培养液以4％的接种量转接至新鲜的GM17(10ng/mLChl+，2ng/mL nisin，pH 5.0，乳酸调节)培养基中。分别测定重组菌和对照菌在胁迫条件下的生长性能。
结果如图5所示。经生长性能试验分析，重组乳酸菌尽管在酸性生长条件下生物量下降，达到稳定期的时间延长，但是相对于对照菌的生物量仍然提高了约1.3倍，说明在L.lactis NZ9000中表达了CsGAD蛋白后，菌株耐酸性明显增强。
依据胁迫实验分析，可知在L.lactis NZ9000中表达了CsGAD蛋白后，菌株抵御冷冻胁迫和酸胁迫的能力显著提高。说明通过在L.lactis NZ9000中表达CsGAD蛋白的方法可以提高乳酸乳球菌胁迫抗性。
本发明通过在乳酸菌中诱导表达产GABA的GAD基因，实现了提高乳酸乳球菌耐低温胁迫和酸胁迫两种能力的目的。
SEQUENCE LISTING
<110> 安徽农业大学
<120> 一种增强乳酸菌耐胁迫能力的GAD基因及其应用
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atggttctgt caaagacaac atcgggggcg agcgatgtat ccgtccactc aaccttcgct 60
tctcgatacg tccgaacacc gcttcctagg ttcaaaatgc cggaaaattc aataccaaag 120
gaagcggcat cgcagataat aaatgacgag ttgatgctgg atgggaatcc aaggctgaat 180
ttggcgtcgt tcgtgacaac gtggacggag ccagagtgcg ataagctcat catggcttcc 240
attaacaaga actatgtcga tatggatgag taccctgtca ccaccgaact ccagaaccgg 300
tgtgtaaaca tgatagcaga tttatttcat gcaccactgg gagacggaga ggctgcagta 360
ggagtgggaa cagttggatc atcggaggca ataatgctgg ctggtttagc attcaagaga 420
aagtggcaga acaagatgaa agctcttggc aaaccctatg ataatcccaa cattgtcacc 480
ggtgccaatg ttcaggtatg ttgggagaaa tttgcaaggt attttgaagt ggagttgaag 540
gaggtgaagc tgagggaagg gtactatgtg atggacccaa ctaaggctgt ggagatggtt 600
gatgagaaca ctatctgtgt tgctgctatt ttgggttcca cactaaatgg agaattcgag 660
gacgttaagc tcttgaatga cctcttgaca gagaagaaca aacaaacagg atgggataca 720
ccaatacatg tagatgcagc aagtggtgga ttcatagcac cattcttgta cccggagctg 780
gagtgggatt tcaggctgga actggtgaag agtataaatg ttagcggaca caagtatgga 840
cttgtgtatg caggtattgg ttggtgcatt tggaggagca aagagaactt gcctgatgaa 900
ctcatctttc atatcaacta tcttggagct gatcaaccca cttttaccct caacttctcc 960
aaaggttcta gtcaagtaat tgctcagtac tatcaactca ttcgcttggg ttttgaggga 1020
tacaagaaca taatggagaa ttgccaagaa aatgcaatga tgcttaaaga aggattggag 1080
aagacaggac ggttcgacat agtgtccaag gacaatggag tcccactagt ggccttctca 1140
ctgaaagaca acagctgtca caacgaattc gaagtgtcag acttgttgcg ccgctttgga 1200
tggatcgtcc ccgcctacac catgcccccc gacgcccaac acataaccgt gctgcgagtt 1260
gtcattaggg aagacttctc gcgcaccctt gcagagcgcc ttgttagtga catccagaaa 1320
gtcttgctcg agctagatag cctccctgca agggttagtg ccaagatggc tgtagtccaa 1380
gagtcagttg tcaagaaaac tgatcttgag gtgcagaggg agatcactga tgcttggaaa 1440
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Glu Ala Ile Met Leu Ala Gly Leu Ala Phe Lys Arg Lys Trp Gln Asn
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Lys Met Lys Ala Leu Gly Lys Pro Tyr Asp Asn Pro Asn Ile Val Thr
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Pro Ile His Val Asp Ala Ala Ser Gly Gly Phe Ile Ala Pro Phe Leu
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Lys Gly Ser Ser Gln Val Ile Ala Gln Tyr Tyr Gln Leu Ile Arg Leu
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Gly Phe Glu Gly Tyr Lys Asn Ile Met Glu Asn Cys Gln Glu Asn Ala
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Met Met Leu Lys Glu Gly Leu Glu Lys Thr Gly Arg Phe Asp Ile Val
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Lys Lys Thr Asp Leu Glu Val Gln Arg Glu Ile Thr Asp Ala Trp Lys
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Claims (1)
1.一种提高乳酸菌胁迫能力的方法，其特征在于，将GAD基因连接到表达载体上获得重组质粒，向乳酸菌中引入上述重组质粒，通过乳酸菌过量表达合成λ-氨基丁酸的谷氨酸脱羧酶CsGAD以提高乳酸菌耐低温胁迫能力和耐酸胁迫能力；
所述GAD基因序列如SEQIDNO.l所示；
所述GAD基因编码的蛋白质氨基酸序列如SHQJDN0.2所示。
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