CN114592034A - 一种紫红曲霉YJX-8 cDNA文库的构建方法及应用 - Google Patents
一种紫红曲霉YJX-8 cDNA文库的构建方法及应用 Download PDFInfo
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Abstract
本发明属于微生物技术领域，具体涉及一种紫红曲霉YJX‑8 cDNA文库的构建方法及利用该文库进行酯合成酶LIP05互作蛋白的筛选应用。本发明公开的紫红曲霉YJX‑8 cDNA文库的构建方法，利用依据此方法构建的文库筛选获得LIP05互作蛋白，即细胞核酸结合蛋白和真核翻译起始因子，可在转录和翻译水平上调控LIP05的表达。本发明构建的cDNA文库具有筛选互作蛋白的应用前景和实际价值。
Description
技术领域
本发明属于微生物技术领域，尤其涉及一种紫红曲霉YJX-8 cDNA文库的构建方法及利用该文库进行酯合成酶LIP05互作蛋白的筛选应用。
背景技术
白酒的主体物质为水和乙醇，含有的少量酯、酸、酮类化合物等风味物质，决定产品香型。其中，己酸乙酯和辛酸乙酯是浓香型白酒品质的重要表征物质，而酒体中己酸乙酯等酯类物质含量作为产品品质的重要衡量标准已在业内达成广泛共识。白酒酿造过程中产酯生香周期长，酯合成量低且不稳定，导致优质浓香型白酒出酒率低，严重影响酒质和产量。究其原因是发酵过程中酿酒微生物中酯合成酶催化己酸乙酯合成效率不稳定。探究己酸乙酯等重要酯类物质的合成机制，对从根本上保障浓香型白酒品质和产量的稳定性，提供实际应用价值。中国传统浓香型白酒酿造过程独特而复杂，不同种属的多种功能微生物均可以产生酯合成酶进而生成己酸乙酯等酯类风味物质。研究表明，浓香型白酒中紫红曲霉YJX-8来源酯合成酶LIP05对己酸乙酯的高效合成具有显著贡献。然而到目前为止，紫红曲霉来源高效催化己酸乙酯和辛酸乙酯合成的酯合成酶调控机制尚未见报道。揭示调控酯合成酶LIP05催化己酸乙酯合成的分子机理，有利于根源性解决该科学问题。构建紫红曲霉YJX-8 cDNA文库，以LIP05为诱饵，利用酵母双杂交系统筛选其互作蛋白以全面系统深度解析LIP05合成己酸乙酯过程中所受的调控机制。该文库的构建方法和应用，为高品质白酒商业化大规模生产提供重要理论参考。
发明内容
本发明的目的在于提供一种紫红曲霉YJX-8 cDNA文库的构建方法及利用该文库进行酯合成酶LIP05互作蛋白的筛选应用。
本发明是通过以下技术方案来实现：
本发明公开了一种紫红曲霉YJX-8 cDNA文库的构建方法。
本发明还公开了利用紫红曲霉YJX-8 cDNA文库进行酯合成酶LIP05互作蛋白的筛选应用。
本发明还公开了利用紫红曲霉YJX-8 cDNA文库筛选到酯合成酶LIP05的互作蛋白为细胞核酸结合蛋白和真核翻译起始因子。
与现有技术相比，本发明具有以下有益的技术效果：
本发明利用分子生物学技术，提取紫红曲霉YJX-8总RNA，逆转录成cDNA，利用试剂盒进行cDNA文库的构建。
本发明提供的紫红曲霉YJX-8 cDNA文库的构建方法及利用该文库进行酯合成酶LIP05互作蛋白的筛选应用，为全面系统深度探究LIP05合成己酸乙酯过程中所受的调控机制和白酒酿造工艺持续发展提供参考。
附图说明
图1为紫红曲霉YJX-8 cDNA文库稀释液SD/-Leu平板涂布，(a)稀释10000倍涂布于SD/-Leu培养基；(b)稀释100倍涂布于SD/-Leu培养基
图2为pGBKT7-LIP05的毒性检测
图3为pGBKT7-LIP05的转录自激活活性检测
图4为SD/-Leu/-Trp/X-α-Gal/AbA培养基上阳性克隆筛选，(a)初次筛选；(b)二次筛选；(c)三次筛选
图5为酵母双杂交实验验证LIP05互作蛋白
具体实施方式
下面结合具体的附图和实施例对本发明做进一步的详细说明，所述是对本发明的解释而不是限定。下述实施例中未详细述及的操作步骤或条件，均按照本领域常规技术、条件实现。
实施例1紫红曲霉YJX-8 cDNA文库的构建
1.紫红曲霉YJX-8培养
将紫红曲霉YJX-8接种于PDA斜面培养基，在30℃条件下静置培养72h，使菌丝体布满培养基。利用无菌水洗涤孢子，孢子悬浮液置于30℃条件下，180rpm培养5d。
2.紫红曲霉YJX-8总RNA提取
DEPC水冲洗菌丝，滤纸吸干，迅速置于预冷的研钵中，加入液氮充分研磨成粉末，利用试剂盒提取样品的总RNA，1％琼脂糖凝胶电泳检测RNA完整性。
3.紫红曲霉YJX-8 cDNA第一链合成
以紫红曲霉YJX-8总RNA为模板，依据
Gold酵母双杂系统(Clontech公司)说明书合成cDNA第一条链。
离心管中加入RNA 2μL，Oligo-dT Primer 2μL，ddH2O 1μL，反应条件为72℃，2min；冰浴2min，14000×g离心10s。离心管中加入产物4μL，5×First-Strand Buffer 2μL，DTT(100mM)1μL，dNTP Mix(10mM)1μL，SMART MMLV ReverseTranscriptase 1μL，反应条件为25℃，10min；42℃，10min。加入1μL SMART III-modifed oligo，混匀，反应条件：42℃，1h；75℃，10min。加入1μL RNase H，反应条件为37℃，10min，获得cDNA第一链。
4.ds cDNA合成
利用LD-PCR方法扩增上述cDNA第一链，合成ds cDNA，反应体系如表1所示。
表1 ds cDNA合成反应体系
PCR反应程序如表2所示。
表2 PCR反应程序
1.2％琼脂糖凝胶电泳检测PCR产物。采用CHROMASPIN+TE-400纯化柱纯化dscDNA。
5.紫红曲霉YJX-8 cDNA文库质量检测
统计SD/-Leu培养基平板中菌斑数量(图1)，计算文库库容量和滴定度。
实施例2紫红曲霉YJX-8酯合成酶LIP05互作蛋白的筛选
1.pGBKT7-LIP05诱饵载体的构建
以去除信号肽后的LIP05序列为模板进行引物设计，引物序列如下：
LIP05-F：5’-ATGGAGGCCGAATTCCTCCCCCTAACACCC-3’；
LIP05-R：5’-GATCCCCGGGAATTCTCACGATGAAGCAGC-3’。
以含有LIP05基因的质粒为模板进行PCR扩增以获取目的基因片段，利用EcoR I单酶切诱饵载体pGBKT7获取目的载体片段，上述片段经胶回收纯化后，连接转化，成功获得重组载体pGBKT7-LIP05。
2.诱饵载体pGBKT7-LIP05毒性及转录自激活活性检测
取1μL pGBKT7-LIP05和pGBKT7质粒分别转化Y2HGold酵母菌株，分别涂布于缺陷型固体培养基SD/-Trp、SD/-Trp/X-α-Gal和SD/-Trp/-X-α-Gal/AbA，30℃条件下倒置培养3-5d。
(1)毒性检测：生长于SD/-Trp平板中pGBKT7-LIP05和pGBKT7两种菌落的长势和形态基本一致(图2)，说明pGBKT7-LIP05无毒性。
(2)转录自激活活性检测：观察涂布于SD/-Trp/X-α-Gal和SD/-Trp/-X-α-Gal/AbA平板中pGBKT7-LIP05菌落生长情况，SD/-Trp/X-α-Gal平板中未出现蓝色单克隆，SD/-Trp/-X-α-Gal/AbA中无菌落生长(图3)，说明pGBKT7-LIP05无转录自激活活性。
3.酵母双杂交技术筛选LIP05互作蛋白
pGBKT7-LIP05与cDNA文库酵母共培养，涂布于筛选培养基SD/-Leu/-Trp/X-α-Gal/AbA，培养结果如图4a所示。平板中蓝色单克隆330个；挑取划线于SD/-Ade/-His/-Leu/-Trp/X-α-Gal/AbA平板，培养3d后蓝斑减少至120个，如图4b所示。继续划线，筛选培养。蓝斑减少至11个，如图4c所示，初步确认其为候选互作蛋白。
4.LIP05互作蛋白验证
将pGBKT7-LIP05分别与筛选获得的11个候选质粒共转化至Y2HGold酵母细胞，涂布于SD/-Ade/-His/-Leu/-Trp/X-α-Gal/AbA筛选培养基，30℃倒置培养5d后生长出2个蓝色菌斑(图5)，对其进行序列测定，经阵列比对，最终确定编号为112和122的蛋白分别为细胞核酸结合蛋白(cellular nucleic acid binding protein，CNBP)和真核翻译起始因子(eukaryotic translation initiation factor 1A，eIF1A)。
细胞核酸结合蛋白广泛存在于真核生物中，具有高度保守性，行使基础且重要的生物学作用。CNBP可以与DNA和RNA结合，在转录和翻译水平上表现出双重调节基因表达的能力。紫红曲霉YJX-8体内的CNBP与LIP05互作形成蛋白复合体，可在转录水平和翻译水平上调控LIP05的表达，提升LIP05催化合成己酸乙酯的能力。
真核翻译起始因子可与核糖体、信使核糖核酸等组成动态翻译起始复合体，执行真核生物的翻译起始的功能。真核生物体内的翻译过程复杂，eIF1A种类较多，在eIF1A之间、与核糖体之间都存在大量的相互作用，构成的互作网络有利于提升翻译水平。相互作用形成稳定的蛋白复合物参与翻译起始进程；同样也存在动态的相互作用，即在特殊的环境条件下形成蛋白复合体以调控翻译水平，促进产物的合成量。紫红曲霉YJX-8体内的eIF1A与LIP05互作形成蛋白复合体，调控LIP05的翻译水平，提高其表达量，促进LIP05快速高效地表达，催化相应底物合成大量的己酸乙酯。
Claims (3)
1.一种紫红曲霉YJX-8 cDNA文库的构建方法，其特征在于，所述紫红曲霉YJX-8经培养后进行总RNA的提取和cDNA的第一链合成，扩增上述第一链cDNA，合成ds cDNA，完成紫红曲霉YJX-8 cDNA文库的构建。
2.由权利要求1所述构建方法创制而成紫红曲霉YJX-8 cDNA文库的应用，其特征在于，可借助酵母双杂交技术应用于紫红曲霉YJX-8来源酯酶LIP05互作蛋白的筛选与鉴定。
3.如权利要求2所述紫红曲霉YJX-8 cDNA文库的应用，利用酵母双杂交技术筛选出与LIP05互作的细胞核酸结合蛋白(cellular nucleic acid binding protein，CNBP)和真核翻译起始因子(eukaryotic translation initiation factor 1A，eIF1A)。
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