CN117467676B - 一种利用平菇MADS-box基因提高酿酒酵母多重胁迫抗性的应用 - Google Patents
一种利用平菇MADS-box基因提高酿酒酵母多重胁迫抗性的应用 Download PDFInfo
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Abstract
本发明公开了一种利用平菇MADS‑box基因提高酿酒酵母多重胁迫抗性的应用，属于基因工程技术领域，编码基因序列如SEQ ID NO.1所示，用于提高酿酒酵母多重胁迫抗性。本发明采用PEG/LiAc法，将过表达重组载体转入酿酒酵母中，获得酿酒酵母转基因菌株；通过超表达平菇Po‑MADS1的转基因酿酒酵母菌株pY816‑Po‑MADS1与对照pY816相比，在抗高温、抗低温、抗氧化胁迫等方面具有明显的作用，说明所获得的转录因子Po‑MADS1参与抗逆境胁迫的调控，在实际生产中应用有助于提高酿酒酵母的抗胁迫能力。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种利用平菇MADS-box基因提高酿酒酵母多重胁迫抗性的应用。
背景技术
酿酒酵母(Saccharomyces cerevisiae)是人类了解最早的一种酵母，因其生长周期短、发酵能力强、规模化生产性能好等优点，成为微生物发酵生产的主要底盘细胞之一。目前在发酵食品、饮料、药物、生物酶等食品和化学品生产中应用广泛。但在工业生产过程中，受环境的变化或工艺操作的影响，酿酒酵母会受到多种胁迫因素的影响，导致生产效率下降。这些胁迫主要包括啤酒、葡萄酒和白酒工业生产所带来的氧化应激、食醋酿造生产中高温糖化所带来的温度冲击等。因此，提高这些耐受性对于酿酒酵母的工业应用至关重要。近年来，许多研究专注于探究酿酒酵母耐受机制与相关调控网络，并利用基因工程等技术开发出耐受性更强的酿酒酵母工业菌株，为酿酒酵母在恶劣工业条件下的应用奠定了基础。
MADS-box转录因子是真核生物中一类在进化上高度保守的基因家族，其名称分别来自酿酒酵母MCM1基因、拟南芥AGAMOUS基因、金鱼草DEFICIENS基因和人血清应答因子SRF4基因的首字母，在这些基因编码的蛋白质中均含有由50-60个氨基酸组成的高度保守区域，被称为MADS盒。MADS-box转录因子作为重要的调控因子参与调控植物生长发育、非生物胁迫等许多方面。例如在大白菜中发现有13个MADS-box转录因子响应冷胁迫，有8个响应干旱胁迫，有6个响应盐胁迫；番茄中，一种MADS-box类基因SlMADS23-like的沉默降低了植株的抗冷胁迫能力，因而SlMADS23-like基因在抗冷胁迫中起正调控作用；在水稻中超表达OsMADS87基因可提高水稻的抗热性，表明OsMADS87可以作为提高水稻生殖发育过程中热弹性潜在的目标基因。因此，将MADS-box转录因子通过基因工程技术应用于酿酒酵母中，对提高酿酒酵母对各种胁迫的耐受性具有重大意义，然而相关的研究非常有限，尤其是将大型真菌中的MADS-box基因应用于酿酒酵母中。
发明内容
基于上述不足，本发明的目的是提供一种利用平菇MADS-box类转录因子Po-MADS1提高酿酒酵母多重胁迫抗性的用途，所述的平菇MADS-box类转录因子Po-MADS1编码基因的核苷酸序列如SEQ ID NO.1所示，用于提高酿酒酵母在多重胁迫下的抗性。
进一步的，所述的用途，将所述编码基因转入酿酒酵母中，并在转基因菌株中超量表达，能使酿酒酵母抗高温、抗低温和抗氧化胁迫的能力提高。
本发明的另一目的是提供一种转基因菌株的构建方法，采用PEG/LiAc法，将过表达重组载体转入酿酒酵母中，所述的过表达重组载体包含平菇MADS-box类转录因子Po-MADS1编码基因的cDNA全长核苷酸序列，所述的平菇MADS-box类转录因子Po-MADS1编码基因的核苷酸序列如SEQ ID NO.1所示，筛选获得酿酒酵母转基因菌株，所述的酿酒酵母转基因菌株与对照相比，抗高温、抗低温和抗氧化胁迫的能力提高。
本发明的优点及有益效果：本发明的过表达平菇Po-MADS1基因的酿酒酵母菌株可以显著提高抗高温、低温和抗氧化胁迫的能力，在实际生产中应用有助于提高酿酒酵母对多种胁迫的耐受性，该基因可作为重要的基因资源，在酿酒酵母工业生产中得到应用。
附图说明
图1是平菇Po-MADS1基因的PCR扩增电泳图，M：DL2000 DNA marker；1：Po-MADS1基因cDNA全长扩增结果；
图2是平菇Po-MADS1蛋白三级结构预测图；
图3是转平菇Po-MADS1基因的酿酒酵母菌株菌液PCR检测图，M：DL5000DNAmarker；1：阳性对照；2：阴性对照；3-5：酿酒酵母转化子检测；
图4是转基因酿酒酵母菌株与对照菌株在高温50℃下胁迫2h后的生长情况图；
图5是转基因酿酒酵母菌株与对照菌株在低温-20℃下胁迫48h后的生长情况图；
图6是转基因酿酒酵母菌株与对照菌株在20mM过氧化氢胁迫24h后的生长情况图。
具体实施方式
本发明提供了一种平菇MADS-box类转录因子Po-MADS1，以平菇菌株RNA反转录的cDNA为模版，通过设计引物、基因扩增、序列测定，获得了Po-MADS1基因全长序列，并确定了它的核苷酸序列和氨基酸序列；进一步将Po-MADS1基因构建到表达载体pY816中，并转化酿酒酵母感受态，鉴定Po-MADS1的功能。下面举例对本发明做进一步的说明：1、平菇Po-MADS1基因的克隆
对平菇(Pleurotus ostreatus)菌种采用PDA培养基进行活化，25℃培养5～8d，收集平菇菌丝体，并使用RNAprep Pure植物总RNA提取试剂盒对平菇菌丝体总RNA进行提取，经检测合格的RNA样品，按照Reverse Transcriptase M-MLV(RNase H)试剂盒说明书将RNA样品反转录为cDNA，置于-20℃保存备用。
根据平菇基因组数据分析、筛选得到Po-MADS1基因，根据基因全长序列设计一对克隆引物：正向引物序列F1为：5′-ATG GCC ACT TTT CGC AGA GTC-3′；反向引物序列R1为：5′-TCA CCT GCG CCC TCC ATA ATG-3′。以上述-20℃保存的平菇cDNA为模板，采用100μL体系进行PCR扩增，扩增程序为：95℃预变性5min；95℃变性30s，55℃退火40s，72℃延伸40s，共35个循环；最后72℃延伸10min。PCR扩增产物如图1所示，与预测大小相符，PCR产物经胶回收纯化后分别与pMD18-T载体(TaKaRa，大连)连接，转化大肠杆菌DH5α，挑选阳性克隆，进行测序。结果表明，Po-MADS1含有一个1245bp的开放阅读框(ORF)，编码414个氨基酸。
2、平菇Po-MADS1转录因子的生物信息学分析
采用ExPASy服务器上的ProtParam tool软件对Po-MADS1基因编码的氨基酸序列进行分子量大小、等电点等理化性质分析，结果显示其分子量为44.01kDa，等电点为9.01；对该蛋白的保守结构域预测显示Po-MADS1蛋白具有MADS superfamily保守结构域；采用ExPASy服务器上的ProtScale软件对氨基酸的亲/疏水性分析，Po-MADS1蛋白中亲水性氨基酸数量多于疏水性氨基酸，属于亲水性蛋白；分别使用在线工具TMHMM Server v.2.0和SignalP 5.0server对蛋白的跨膜区和信号肽进行分析，结果表明Po-MADS1蛋白不具跨膜结构，不含信号肽；通过ExPASy服务器上的GOR在线软件对Po-MADS1蛋白进行二级结构预测，表明Po-MADS1蛋白的主要组成部分包括9.9％的α-螺旋、10.9％的延伸链和74.2％的无规卷曲；通过生物信息软件Expasy对平菇Po-MADS1蛋白进行三级结构预测，使用SWISS-MODEL数据库预测构建平菇Po-MADS1转录因子蛋白的三级结构模型(图2)。通过NCBI网站上的BLASTP功能，将得到的cDNA序列进行同源性比对，并通过MEGA11.0软件建立该基因的系统进化树，从系统进化树中可以看出，该基因与红平菇(Pleurotus djamor)的MADS-box基因具有很高的同源性。
3、平菇Po-MADS1转录因子提高酿酒酵母多重胁迫的功能验证
(1)含有Po-MADS1基因的重组载体构建
对Po-MADS1基因编码区进行克隆，以平菇cDNA为模板，根据Po-MADS1编码区设计引物并引入pY816载体同源臂，
Po-MSpY-F：5’-AGGGAATATTAAGCTATGGCCACTTTTCGCAGAGTC-3’
Po-MSpY-R：5’-CCCCCATGGTAAGCTTCACCTGCGCCCTCCATAATG-3’
其中划横线的字母为引入的载体同源臂。
将pY816质粒(实验室保存)用限制性核酸内切酶HindⅢ进行单酶切后切胶回收，与经PCR扩增得到的带有载体同源臂的Po-MADS1纯化产物采用试剂盒进行同源重组，转化大肠杆菌TOP 10感受态细胞，挑选阳性克隆，提取质粒进行测序，测序结果正确，即获得了重组载体，标记为pY816-Po-MADS1。
(2)重组载体pY816-Po-MADS1转化酿酒酵母
将重组载体pY816-Po-MADS1，采用PEG/LiAc法转化酿酒酵母INVSc1感受态细胞，主要步骤为：取100μl冰上融化的INVSc1感受态细胞，依次加入预冷的目的质粒0.5-2μg，Carrier DNA(95℃，5min，快速冰浴，重复一次)10μl，PEG/LiAc 500μl并吸打几次混匀，30℃水浴30min(每15min翻转6-8次混匀)；再放入42℃水浴15min(每7.5min翻转6-8次混匀)；5000rpm离心2min弃上清，用100μl无菌ddH2O重悬，涂布于尿嘧啶缺陷培养基SD-Ura板上，30℃避光倒置培养48-96h。同时将空pY816载体转入INVSC1，作为对照，标记为INVSC1(pY816)。随机挑取转化的酿酒酵母单菌落(含重组质粒pY816-Po-MADS1)扩大培养，提取酵母DNA，以引物T7：5’-TAATACGACTCACTATAGGG-3’和引物Ter：5’-GTGACATAACTAATTACATGATG-3’进行PCR扩增，1％琼脂糖凝胶电泳检测，结果如图3所示，扩增片段大小与预期相符，表明目的基因已成功转入酿酒酵母INVSc1中。
(3)酿酒酵母胁迫处理
挑取酿酒酵母(pY816-Po-MADS1)和酿酒酵母(含有空载pY816，作为对照)单克隆细胞在SD-Ura液体培养基(含有2％葡萄糖)中，30℃，180rpm振荡培养至OD600＝0.5，离心收集菌体，用含有2％半乳糖的SC-Ura液体培养基(诱导培养基)调整OD600＝0.4，30℃诱导表达24h。测量并调整OD600，使酿酒酵母(pY816-Po-MADS1)和酿酒酵母(pY816)OD600都为1.0，分别离心收集菌体用于高温胁迫(50℃，2h)、低温胁迫(-20℃，48h)、氧胁迫(过氧化氢20mM，24h)处理，每个处理重复三次，其中高温和低温胁迫后，在30℃条件下恢复生长9h。将处理后的菌液作10×稀释，取2μL点在SD-Ura固体培养基上，30℃培养48h后观察酿酒酵母生长情况(图4、图5、图6)。通过试验对比发现本发明的酿酒酵母转基因菌株pY816-Po-MADS1与对照pY816相比，抗高温、抗低温和抗氧化胁迫的能力提高，说明所获得的转录因子Po-MADS1参与抗逆境胁迫的调控，在实际生产中应用有助于提高酿酒酵母的抗胁迫能力。
Claims (2)
1.一种利用平菇MADS-box类转录因子Po-MADS1提高酿酒酵母多重胁迫抗性的用途，所述的多重胁迫为高温、低温和氧化胁迫，所述的平菇MADS-box类转录因子Po-MADS1编码基因的核苷酸序列如SEQ ID NO.1所示，将所述编码基因转入酿酒酵母中，并在转基因菌株中超量表达，能使酿酒酵母抗高温、抗低温和抗氧化胁迫的能力提高。
2.一种转基因菌株的构建方法，采用PEG/LiAc法，将过表达重组载体转入酿酒酵母中，所述的过表达重组载体包含平菇MADS-box类转录因子Po-MADS1编码基因的cDNA全长核苷酸序列，所述的平菇MADS-box类转录因子Po-MADS1编码基因的核苷酸序列如SEQ ID NO.1所示，筛选获得酿酒酵母转基因菌株，其特征在于，所述的酿酒酵母转基因菌株与对照相比，抗高温、抗低温和抗氧化胁迫的能力提高。
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