CN111087454B - 一种热休克转录因子1显性负效应突变体及其应用 - Google Patents
一种热休克转录因子1显性负效应突变体及其应用 Download PDFInfo
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- CN111087454B CN111087454B CN202010121694.XA CN202010121694A CN111087454B CN 111087454 B CN111087454 B CN 111087454B CN 202010121694 A CN202010121694 A CN 202010121694A CN 111087454 B CN111087454 B CN 111087454B
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Abstract
本发明公开了一种热休克转录因子1显性负效应突变体dn‑Hsf1及其应用,属于生物技术领域。通过同源比对人类和黄曲霉(Aspergillus flavus)中的热休克转录因子1 HSF1蛋白,将黄曲霉Hsf1蛋白C末端的第576位至第788位共213个氨基酸残基删除,获得黄曲霉中热休克转录因子1显性负效应突变体dn‑Hsf1蛋白的氨基酸序列如SEQ ID NO.1所示,编码核苷酸序列如SEQ ID NO.2所示。该显性负效应突变体dn‑Hsf1能抑制正常Hsf1在真菌体内发挥功能,从而抑制真菌的生长,应用于防控真菌的污染方面。
Description
技术领域
本发明属于生物技术领域,具体涉及一种热休克转录因子1显性负效应突变体及其应用。
背景技术
真菌是一类广泛分布于自然界的真核生物,对人类的生活有益处也有坏处。许多真菌为我们提供了食物来源,作为发酵工业和食品加工业的重要菌种,它们所产生的多种次级代谢产物在工业生产和医学上具有重要应用价值。也有许多真菌对人类的健康和社会经济造成极大危害。不少真菌,如烟曲霉(Aspergillus fumigatus)和白念珠菌(Cadida albicans),能够侵染人体,造成局部组织器官或全身性的真菌感染甚至死亡。许多真菌还可以寄生于动植物及其加工制品中,并产生剧毒的真菌毒素污染,严重危害人畜健康,如黄曲霉和寄生曲霉(Aspergillus paraciticus)能够产生黄曲霉毒素,镰刀菌(Fusarium graminearum)能够产生伏马毒素和呕吐毒素,烟曲霉能产生胶霉毒素。我国每年因农产品及其制品被真菌毒素污染而造成的损失巨大。因此,如果能做到提前干预,防止或降低真菌污染,不仅能有效的减少农业和食品加工业的损失,也能够更好的保障食品安全和公共健康,具有重大的社会和经济意义。
自然界中, 热休克反应是一种广泛存在于细菌、真菌、植物和动物中的机体保护机制,其主要作用是在生长发育过程中以及各种应激条件下调控机体内相关基因的转录表达,起到防御和保护机体的作用。哺乳动物的热休克转录因子1(Heat Shocktranscription Factor 1,HSF1)是细胞热休克蛋白表达的主要调控因子。HSF1可以被热应激、氧化压力、化学刺激和生理应激等激活,形成同源三聚体结合到热休克蛋白启动子区的热休克元件上,调控导热休克蛋白合成。HSF1不仅能够调节热休克蛋白的表达, 而且能调控多药耐药基因MDR1的表达。在人肝癌细胞中的研究表明,HSF1的活化促进HepG2细胞的阿霉素耐药性。白念珠菌和大豆疫霉(Phytophthora sojae)的HSF1也与菌株的致病性相关。
在哺乳动物中表达具有显性负效应的突变体dn-cHSF1可抑制正常HSF1在体内发挥功能。然而,目前还未有利用真菌的Hsf1显性负效应突变体来防控真菌污染的应用。因此本发明构建一种真菌来源的Hsf1显性负效应突变体dn-Hsf1来抑制正常Hsf1在真菌体内发挥功能,从而抑制真菌的生长、达到防控真菌污染的目的。
发明内容
本发明的目的在于提供一种热休克转录因子1显性负效应突变体及其应用。将黄曲霉Hsf1蛋白C末端的第576位至第788位共213个氨基酸残基删除获得黄曲霉中热休克转录因子1显性负效应突变体dn-Hsf1。该显性负效应突变体dn-Hsf1能抑制正常Hsf1在真菌体内发挥功能,从而抑制真菌的生长,在防控真菌污染方面具有广阔应用前景。
为实现上述目的,本发明采用如下技术方案:
一种热休克转录因子1 显性负效应突变体dn-Hsf1:
(1) 所述dn-Hsf1蛋白的氨基酸序列如SEQ ID NO.1所示;或
(2) 具有(1)所限定的序列,但在上述序列之外通过一个或几个氨基酸残基的取代、缺失或添加而形成的序列,且基本具有(1)所限定的dn-Hsf1的功能的由(1)衍生的dn-Hsf1。
优选的氨基酸残基的取代、缺失或添加的氨基酸为1-20个或1-15或1-3个或1个。
更优选的,缺失如SEQ ID NO.1所示氨基酸序列中第364位至第378位共15个氨基酸残基。
优选的,所述dn-Hsf1来源于黄曲霉(Aspergillus flavus)。
在进一步优选的实施方式中,所述dn-Hsf1的氨基酸序列与SEQ ID NO:1所示氨基酸序列具有80%以上,优选85%以上,更优选90%以上,更优选95%以上,更优选96%、97%、98%、99%的同源性。
在第二方面,本发明提供第一方面所述的dn-Hsf1的编码核苷酸序列。
优选的,所述的dn-Hsf1的编码核苷酸序列如SEQ ID NO.2所示。
在第三方面,本发明提供包含第一方面所述的dn-Hsf1的编码核苷酸序列的表达载体。
上述包含dn-Hsf1的编码核苷酸序列的表达载体的构建方法为:以黄曲霉NRRL3357菌株基因组DNA为模板,PCR扩增Hsf1自身启动子P hsf1 和Hsf1终止子T hsf1 的核苷酸片段;以黄曲霉NRRL3357菌株cDNA为模板扩增dn-Hsf1编码核苷酸片段dn-hsf1;将P hsf1 、 dn-hsf1、和T hsf1 共3个片段用融合PCR法连接到一起构建包含hsf1自身启动子P hsf1 、dn-hsf1编码核苷酸和Hsf1终止子终止子T hsf1 的融合核苷酸片段P hsf1 -dn-hsf1-T hsf1 ,并将其***pPTR I载体中,经转化,筛选阳性转化子,测序验证,获得表达载体质粒pPTR I-dn-hsf1。
在第四方面,本发明提供包含第一方面所述的dn-Hsf1的编码核苷酸序列的融合核苷酸片段。
所述包含dn-Hsf1的编码核苷酸序列的融合核苷酸片段的构建方法:以黄曲霉NRRL3357菌株基因组DNA为模板,PCR的扩增dn-Hsf1上游重组片段up;从烟曲霉AF293菌株基因组DNA中用PCR的方法扩增pyrG筛选标记基因片段;从产黄青霉NRRL1951菌株基因组DNA中用PCR扩增木糖诱导启动子P xylP 片段;从质粒pPTR I-dn-hsf1中用PCR法扩增约dn- hsf1-T hsf1 核苷酸片段;将上游重组片段up、pyrG筛选标记基因片段、木糖诱导启动子P xylP 片段和dn-hsf1-T hsf1 核苷酸片段,共4个片段用融合PCR法连接到一起构建融合核苷酸片段up-pyrG-P xylP -dn-hsf1-T hsf1 。
上述表达载体及融合核苷酸片段的构建中所用引物序列见表1.
表1 引物序列表
在第五方面,本发明提供第一方面所述的dn-Hsf1、或第二方面所述编码核苷酸序列、或第三方面所述的表达载体、或第四方面所述融合核苷酸片段在防控真菌污染方面的应用。
将热休克转录因子1 Hsf1显性负效应突变体dn-Hsf1;或者热休克转录因子1Hsf1显性负效应突变体dn-Hsf1的编码核苷酸序列;或者携带热休克转录因子1 Hsf1显性负效应突变体dn-Hsf1的编码核苷酸序列的载体,或者热休克转录因子1 Hsf1显性负效应突变体dn-Hsf1的编码核苷酸序列的融合核苷酸片段导入真菌细胞中,使真菌细胞表达dn-Hsf1,抑制细胞内正常的Hsf1发挥功能,抑制真菌的生长。
本发明的应用和优点:
(1) 本发明提供了一种来源于真菌的dn-Hsf1,与人源的dn-cHSF1的同源性很低,该dn-Hsf1能抑制真菌、特别是霉菌的生长;
(2) 本发明提供的dn-Hsf1能够抑制真菌细胞内正常Hsf1的功能,进而抑制真菌的生长,因此dn-Hsf1在应用时无需考虑真菌细胞自身的hsf1基因是否存在以及存在的拷贝数;
(3) 使用本发明提供的dn-Hsf1来抑制真菌的生长更环保、更安全,可避免使用化学农药抑菌伴随的环境污染和人体毒副作用,在防控霉菌等真菌的污染方面具有广阔的应用前景。
附图说明
图1. 黄曲霉dn-Hsf1表达载体的构建。A:融合PCR法扩增黄曲霉P hsf1 -dn-hsf1- T hsf1 核苷酸片段的琼脂糖凝胶电泳图谱;B:表达载体pPTR I-dn-hsf1阳性大肠杆菌转化子菌落PCR扩增P hsf1 -dn-hsf1-T hsf1 片段的琼脂糖凝胶电泳图谱。
图2. pPTR I-dn-Hsf1表达载体转化黄曲霉。WT表示出发菌株,“-”表示没有转化核酸的原生质体,“+ pPTR I”表示转化pPTR I空载体,“+ pPTR I-dn-hsf1”表示转化pPTRI-dn-hsf1质粒,“+Pyrithiamine”表示培养基中添加了吡啶硫胺,“-Pyrithiamine”表示培养基中未添加吡啶硫胺。
图3.诱导型dn-Hsf1表达片段整合黄曲霉基因组的构建策略示意图。A:通过融合PCR方法构建包含上游重组序列up、来源于烟曲霉(Aspergillus fumigatus)的pyrG营养筛选标记基因、来源于产黄青霉的P xylP 木糖诱导表达启动子,dn-Hsf1的编码核苷酸及终止子T hsf1 的融合片段,即P xylP -dn-hsf1-T hsf1 片段,转化并整合入黄曲霉基因组,即得到诱导型P xylP -dn-hsf1 菌株;B:dn-Hsf1Var:dn-Hsf1蛋白的中间第364位至第378位共15个氨基酸残基删除。
图4. dn-Hsf1及其活性片段dn-Hsf1Var的诱导表达抑制黄曲霉的生长。图中T1、T2表示不同的转化子,YGT和GMM为含葡萄糖的非诱导培养基,YXT和XMM为含木糖和木聚糖的诱导培养基,图中所示为37℃培养3天后的菌落生长情况。
具体实施方式
发明人经过广泛而深入的研究,发现将黄曲霉热休克转录因子1Hsf1蛋白C末端第576位至第788位的共213个氨基酸残基删除,能够获得具有显性负效应的突变体dn-Hsf1,其氨基酸序列如SEQ ID NO.1所示。进一步研究发现,再将dn-Hsf1蛋白的第364位至第378位共15个氨基酸残基删除后的dn-Hsf1蛋白片段仍然具有显性负效应的活性。它们都能够抑制正常Hsf1在真菌体内发挥功能,显著抑制真菌的生长,具有防控真菌污染的应用价值。
如本文所用,“分离的”是指物质从其原始环境中分离出来(如果是天然的物质,原始环境即是天然环境)。如活体细胞内的天然状态下的多聚核苷酸和多肽是没有分离纯化的,但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质中分开,则为分离纯化的。
本文所用的术语“自然状态”指的是在微生物中多肽处于未修饰状态的活性,即自然状态下的活性。
关于本发明,dn-Hsf1可以是如SEQ ID NO:1所示氨基酸序列对应的蛋白,也可以不是SEQ ID NO:1所示的氨基酸序列对应的蛋白;换言之,本发明的dn-Hsf1可以与SEQ IDNO:1所示的氨基酸序列具有较高的同源性,也可以具有较低的同源性。本发明的dn-Hsf1可以来源于各种物种,包括但不限于黄曲霉(Aspergillus flavus)、米曲霉(Aspergillus oryzae)、寄生曲霉(Aspergillus parasiticus)、黑曲霉(Aspergillus niger)、土曲霉(Aspergillus terreus)、烟曲霉(Aspergillus fumigatus)、棒曲霉(Aspergillus clavatus)、构巢曲霉(Aspergillus nidulans)、粗糙脉孢菌(Neurospora crassa)、禾谷镰孢菌(Fusarium graminearum),只要其具有dn-Hsf1的活性,能够抑制正常Hsf1在真菌体内发挥功能从而抑制真菌的生长的,也包括在本发明的范围内。
在具体的实施方式中,所述同源性或序列相同性可以是80%以上,优选85%以上,更优选90%以上,更优选95%以上,更优选96%、97%、98%、99%的同源性。因此,与本发明的具体dn-Hsf1有80%以上,优选85%以上,更优选90%以上,更优选95%以上,更优选96%、97%、98%、99%的序列相同性或同源性的dn-Hsf1也包括在本发明的保护范围内。
本文所用的术语“活性片段”与本领域技术人员常规理解的含义相同或相似,均是指该片段的氨基酸序列是完整蛋白或多肽的氨基酸序列的一部分,但该片段具备与完整蛋白或多肽相同或相似的功能或活性。具体地说,在本发明中,“活性片段”表示具有dn-Hsf1活性的任意氨基酸序列。
此外,本领域普通技术人员也不难知晓,在多肽的某些区域,改变少数氨基酸残基基本上不会改变生物活性,例如,适当替换某些氨基酸得到的序列并不会影响其活性(可参见Watson等,Molecular Biology of The Gene,第四版,1987,The Benjamin/CummingsPub.Co .P224)。因此,本领域普通技术人员能够实施这种替换(包括残基的取代、缺失、***、添加)并且确保所得多肽仍保持其初始功能。因此,对本发明的dn-Hsf1作进一步突变而得到仍具备dn-Hsf1的功能和活性的进一步突变体是显而易见的。例如,发明人将dn-Hsf1蛋白的第364位至第378位氨基酸残基删除后的dn-Hsf1蛋白片段仍然具有显性负效应的活性。
基于本发明的教导和本发明具体得到的dn-Hsf1,本领域技术人员不难得到具有相同或相似活性或功能的活性片段,这样的活性片段当然应该落在本发明的保护范围内。
任意氨基酸序列与SEQ ID NO:1所示氨基酸序列的对应于位置可以通过氨基酸序列之间的比对确定。本领域普通技术人员公知的测定序列同源性或相同性的方法包括但不限于:计算机分子生物学(Computational Molecular Biology),Lesk,A .M .编,牛津大学出版社,纽约,1988;生物计算:信息学和基因组项目(Biocomputing: Informatics andGenome Projects),Smith,D.W.编,学术出版社,纽约,1993;序列数据的计算机分析(Computer Analysis of Sequence Data),第一部分,Griffin,A.M .和Griffin,H.G.编,Humana Press,新泽西,1 9 94 ;分子生物学中的序列分析(Sequence Analysis inMolecular Biology),von Heinje,G.,学术出版社,1987和序列分析引物(SequenceAnalysis Primer),Gribskov,M.与Devereux,J.编M Stockton Press,纽约,1991和Carillo,H .与Lipman,D.,SIAM J. Applied Math.,48:1073(1998)。测定相同性的优选方法要在测试的序列之间得到最大的匹配。测定相同性的方法编译在公众可获得的计算机程序中。优选的测定两条序列之间相同性的计算机程序方法包括但不限于:GCG程序包(Devereux,J .等,1984)、BLASTP、BLASTN和FASTA(Altschul,S,F.等,1990)。公众可从NCBI和其它来源得到BLASTX程序(BLAST手册,Altschul,S.等,NCBI NLM NIH Bethesda,Md.20894;Altschul,S.等,1990)。熟知的Smith Waterman算法也可用于测定相同性。
本发明还提供了编码本发明多肽的多核苷酸(如SEQ ID No.2所示)。术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸,也可以是还包括附加编码和/或非编码序列的多核苷酸。dn-hsf1的编码核苷酸可以是在严格的条件下与SEQ ID NO.2所示核苷酸序列(例如与SEQ ID NO.2所示核苷酸序列的部分或者整个序列互补的序列)制备的探针杂交的DNA,只要保持其初始功能即可。所述“严格的条件”是指可形成所谓的特异性杂交且未形成非特异性杂交的条件。例如,同源性较高的DNA,例如具有80%以上同源性的DNA彼此杂交,同源性低于80%的DNA彼此不杂交的条件,或者通常的Southern杂交的清洗条件,即在相当于60℃、1*SSC、0 .1%SDS、优选60℃、0 .1*SSC、0 .1%SDS,更优选68℃、0 .1*SSC、0.1%SDS的盐浓度及温度下清洗1次、优选2-3次的条件。
此外,由于密码子的简并性因宿主而异,dn-hsf1的编码核苷酸中的任意密码子可以用相应的等价密码子替换,也就是说,dn-hsf1的编码核苷酸可以是由于遗传密码的简并性而在任何dn-hsf1的编码核苷酸上面的突变。例如,dn-hsf1的编码核苷酸可以是经修饰的核苷酸,使得其具有根据要使用的宿主中密码子频率的最佳密码子。
本文所用的术语“宿主”是具有本领域普通技术人员通常理解的含义,即,能够产生本发明dn-Hsf1的宿主。换言之,本发明可以应用于任何宿主,只要本发明的dn-Hsf1能在该宿主中表达。
例如,适用于本发明的宿主来自(但不限于) 黄曲霉(Aspergillus flavus)、米曲霉(Aspergillus oryzae)、寄生曲霉(Aspergillus parasiticus)、黑曲霉(Aspergillus niger)、土曲霉(Aspergillus terreus)、烟曲霉(Aspergillus fumigatus)、棒曲霉(Aspergillus clavatus)、构巢曲霉(Aspergillus nidulans)、粗糙脉孢菌(Neurospora crassa)、禾谷镰孢菌(Fusarium graminearum);更优选黄曲霉、米曲霉、寄生曲霉、黑曲霉、土曲霉、烟曲霉、棒曲霉、构巢曲霉。
本发明所述将制备得到的核苷酸片段或表达载体导入真菌细胞中,其导入方法可以是基于原生质体的遗传转化方法,如是PEG/CaCl2介导的原生质体转化法、脂质体-原生质体融合法、限制性内切酶介导的融合(REMI)法等,也可以是基于物理方法的遗传转化方法,如电穿孔技术转化法、基因枪技术转化法和最近刚刚实现的纳米颗粒喷洒转化法(DOI:10.1101/805036),也可以是利用生物递送的遗传转化方法,如农杆菌(Agrobacterium)介导的遗传转化方法等。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件如Sambrook等人,分子克隆:实验室手册(New York: Cold Spring Harbor LaboratoryPress,1989)中所述的条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数按重量计算。
I.材料和方法
本发明所用菌株信息如下:黄曲霉(Aspergillus flavus)菌株NRRL3357菌株(ATCC 200026)、烟曲霉(Aspergillus fumigatus)AF293菌株(ATCC MYA-4609)、产黄青霉(Penicillium chrysogenum)NRRL1951菌株(ATCC 28089)为本实验室购买;黄曲霉WT菌株(∆ku70)和CA14PTs(∆pyrG, ∆ku70)菌株为美国农业部南方研究中心Perng-KuangChang教授惠赠(DOI:10.1016/j.mimet.2010.03.010.);大肠杆菌DH5α化学感受态细胞购自TransGen公司。
高保真DNA聚合酶KOD Plus购自Toyobo公司(Code No. KOD-201),rTaq DNA聚合酶、限制性内切酶购自Invitrogen公司,吡啶硫胺购自Sigma Aldrich公司,酵母提取物和琼脂糖为Oxiod公司产品,葡萄糖、木糖、木聚糖、氨苄青霉素为Solarbio公司产品,质粒载体pPTR I购自Takara公司(Code No. 3621),RNA提取试剂TRIzol(Code No. 15596026)和反转录试剂盒(Code No. K1622)为Thermo Fisher Scientific公司产品。其他常规化学试剂购自Sangon公司和国药集团。
本实施方式中所使用的各种溶液和培养基具体成分如下:
1000×微量元素母液(100 mL):ZnSO4·7H2O 2.2 g;H3BO3 1.1 g;MnCl2·4H2O0.5 g;FeSO4·7H2O 0.16 g;CoCl2·5H2O 0.16 g;CuSO4·5H2O 0.16 g; (NH4)6Mo7O24·4H2O 0.11 g; Na4EDTA 5 g 溶于去离子水,过滤除菌。
YGT液体培养基(1 L):Yeast extract 5g;Glucose 20 g;1000×微量元素母液1mL。在YGT液体培养基中加入尿苷(Uridine 0.001 g/L)和尿嘧啶(Uracil 0.001 g/L)即为YGTUU液体培养基。在以上两种液体培养基中加入琼脂(Agar 20 g/L)即得固体培养基。将YGT培养基中的葡萄糖(Glucose 20 g/L)替换成木糖和木聚糖(xylose 5g/L,xylan 5 g/L)即得YXT固体培养基。
复苏培养基(1 L):Sucrose 342.3 g;NaNO3 3 g;KCl 0.5 g;MgSO4 0.5 g;K2HPO41 g;FeSO4 0.01 g;Agar 5 g(上层)或15 g(下层)。
GMM固体培养基(1 L):NaNO3 6 g;KCl 0.52 g;MgSO4·7H2O 0.52 g;KH2PO4 1.52g;1000×微量元素母液1 mL;Glucose 10 g;Agar 20 g。将GMM固体培养基配方中的葡萄糖(Glucose 10 g/L)替换成木糖和木聚糖(xylose 5g/L,xylan 5 g/L)即得XMM固体培养基。
吡啶硫胺的工作浓度为0.2 μg/mL,根据实际需要添加进不同培养基中。
黄曲霉基因组DNA提取、总RNA提取与反转录、黄曲霉原生质体制备和转化方法均参考自已公开发表的文献(DOI: 10.1016/j.mimet.2010.03.010; DOI: 10.1021/acs.jafc.6b02199)。
具体实施过程中所用引物见表1。
实施例1、dn-Hsf1表达载体的构建
在哺乳动物中的研究表明,将热休克转录因子1HSF1蛋白的C末端约150个氨基酸残基的催化结构域删除,能够得到具有显性负效应的突变体dn-cHSF1,该突变体可抑制正常HSF1在动物体内发挥功能(DOI: 10.1021/acschembio.5b00740)。因此,本发明人通过构建一种真菌来源的Hsf1显性负效应突变体dn-Hsf1来抑制正常Hsf1在真菌体内发挥功能,从而抑制真菌的生长、达到防控真菌污染的目的。
本发明利用人类热休克转录因子1HSF1蛋白的氨基酸序列通过NCBI BLASTP同源比对方法找到黄曲霉中预测的同源蛋白Hsf1(编号为XP_002374014)及其编码基因(编号为AFLA_025030)。黄曲霉Hsf1蛋白(包含788个氨基酸残基)与人HSF1蛋白(包含529个氨基酸残基)的主要结构域的氨基酸序列相似性仅为21%,全长蛋白的相似性更低。因此,通过Lasergene MegAlign软件比对分析后,发明人将黄曲霉Hsf1蛋白C末端的第576位至第788位共213个氨基酸残基删除,构建得具有显性负效应的突变体dn-Hsf1,其氨基酸序列如SEQID NO.1所示。
接下来,本发明通过融合PCR方法构建黄曲霉Hsf1蛋白C末端删除213个氨基酸残基的截短突变体。具体方法如下:
利用引物SEQ ID NO.3和SEQ ID NO.4;从黄曲霉基因组DNA中用PCR的方法扩增长度为1329 bp的包含hsf1自身启动子P hsf1 的核苷酸片段(Region 11703 to 13031 onAAIH02000003.1,GenBank);
利用引物SEQ ID NO.5和引物SEQ ID NO.6;从黄曲霉cDNA中用PCR的方法扩增1728bp的片段dn-hsf1,该片段为Hsf1蛋白C末端删除213个氨基酸残基(保留第1位到第575位氨基酸残基)的编码核苷酸片段dn-hsf1(如SEQ ID NO.2所示);
利用引物SEQ ID NO.7和引物SEQ ID NO.8;从黄曲霉基因组DNA中用PCR的方法扩增长度为1491 bp的包含hsf1终止子T hsf1 的核苷酸片段(Region 7786 to 9279 onAAIH02000003.1,GenBank);
将上述3个片段用融合PCR法连接到一起构建包含hsf1自身启动子P hsf1 、dn-hsf1编码核苷酸和hsf1终止子的长度为4548 bp的融合核苷酸片段P hsf1 -dn-hsf1-T hsf1 (图1A),通过LIC无缝连接克隆法***pPTR I载体中,转化大肠杆菌DH5感受态细胞,挑取单克隆进行菌落PCR验证筛选阳性转化子并测序,阳性转化子能够扩增出分子量为4548 bp的条带P hsf1 -dn-hsf1-T hsf1 (图1B),再经过测序确认,得到pPTR I-dn-hsf1表达载体质粒。
实施例2、表达载体pPTR I-dn-hsf1导入黄曲霉
将表达载体质粒pPTR I-dn-hsf1导入黄曲霉WT菌株原生质体,在含有吡啶硫胺的复苏培养基上筛选黄曲霉转化子。转化板在37℃培养5天后,结果如图2所示:第一、二块培养板中均为没有转化核酸的原生质体,由于第一块培养板不含吡啶硫胺筛选压力,该培养板上长出许多克隆形成菌苔,说明用于转化的原生质体量是足够的,而第二块培养板添加了吡啶硫胺,没有长出克隆,说明吡啶硫胺筛选压力有效。第三块培养板为转化pPTR I空载体的原生质体,在吡啶硫胺的筛选下长出10个克隆,说明转化效率合适。第四块培养板为转化pPTR I-dn-hsf1质粒的原生质体,在吡啶硫胺的筛选下没有长出克隆,说明dn-Hsf1的表达能够显著抑制黄曲霉的生长。以上结果说明,黄曲霉Hsf1蛋白C末端删除213个氨基酸残基的截短突变体dn-Hsf1是一个具有显性负效应活性的突变体。
实施例3、木糖启动子诱导表达dn-Hsf1对黄曲霉生长的抑制作用
为了确认dn-Hsf1对黄曲霉生长的抑制作用,本发明进一步将dn-Hsf1蛋白的表达由黄曲霉hsf1基因自身启动子P hsf1 控制改为使用木糖诱导启动子P xy1P 来控制(构建策略如图3A所示),通过融合PCR方法构建包含上游同源序列up、来源于烟曲霉的pyrG营养筛选标记基因、来源于产黄青霉P xylP 木糖诱导表达启动子、dn-Hsf1的编码核苷酸和终止子T hsf1 的融合片段,即P xylP -dn-hsf1-T hsf1 片段,转化并整合入黄曲霉基因组,即得到诱导型P xylP -dn- hsf1 菌株。观察dn-Hsf1的不表达和诱导表达两种状态对黄曲霉生长的影响。具体方法如下:
利用引物SEQ ID NO.9和SEQ ID NO.10;从黄曲霉NRRL3357菌株基因组DNA中用PCR的方法扩增长度为1474 bp的上游重组片段up(Region 5751637 to 5753110 onCP044622.1,GenBank);
利用引物SEQ ID NO.11和SEQ ID NO.12从烟曲霉AF293菌株基因组DNA中用PCR的方法扩增长度为1890 bp的pyrG筛选标记基因片段(DOI: 10.1021/acs.jafc.6b02199);
利用引物SEQ ID NO.13和SEQ ID NO.14从产黄青霉NRRL1951 基因组DNA中用PCR的方法扩增长度为1632 bp的木糖诱导启动子P xylP 片段(DOI: 10.1128/aem.66.11.4810-4816.2000);
利用引物SEQ ID NO.15和SEQ ID NO.16从实施例1构建所得表达质粒pPTR I-dn- hsf1中用PCR的方法扩增长度为3219 bp的dn-hsf1-T hsf1 核苷酸片段;
将上述上重游组片段up、pyrG筛选标记基因片段、木糖诱导启动子P xylP 片段和dn- hsf1-T hsf1 核苷酸片段,共4个片段用融合PCR法连接到一起构建长度为8215 bp的融合核苷酸片段up-pyrG-P xylP -dn-hsf1-T hsf1 ,转化黄曲霉CA14PTs菌株,使融合核苷酸片段整合到黄曲霉基因组的hsf1基因座上替换掉一个拷贝的hsf1基因(图3A)。37℃培养5天后筛选鉴定,最后得到5个P xylP -dn-hsf1阳性转化子菌株。
随机取两个上述黄曲霉P xylP -dn-hsf1阳性转化子(T1、T2)的新鲜的孢子,分别接种103个孢子于YGT、YXT、GMM和XMM固体培养基平板的中心点,37 ℃持续培养3天。实验结果如图4A所示,在葡萄糖存在的情况下(YGT和GMM培养基),木糖启动子受到葡萄糖抑制,无法转录表达dn-Hsf1,此时黄曲霉能够正常生长;而当葡萄糖不存在时(YXT和XMM培养基),木糖启动子能够被木糖和木聚糖诱导激活,转录表达dn-Hsf1,黄曲霉的生长反而收到显著抑制,菌落直径显著变小。该结果说明,dn-Hsf1的转录表达对黄曲霉的生长是不利的,dn-Hsf1具有显著的显性负效应活性。
实施例4、dn-Hsf1活性片段对黄曲霉生长的抑制作用
本发明进一步设计将由木糖诱导启动子P xy1P 来控制表达的dn-Hsf1蛋白的中间第364位至第378位共15个氨基酸残基删除,检测dn-Hsf1蛋白片段dn-Hsf1Var是否仍具有显性负效应的活性(构建策略如图3B所示)。具体方法如下:
利用引物SEQ ID NO.9和引物SEQ ID NO.17从上述实施例3所获得的黄曲霉P xylP - dn-hsf1菌株基因组DNA中用PCR的方法扩增长度为6085 bp的包含上游重组片段up、筛选标记基因pyrG、木糖诱导启动子P xylP 和dn-hsf1第1位到第363位氨基酸残基编码核苷酸的片段;利用引物SEQ ID NO.18和SEQ ID NO.16从上述实施例3所获得的黄曲霉P xylP -dn-hsf1菌株基因组DNA中用PCR的方法扩增长度为2085 bp的包含dn-hsf1第379位到第575位氨基酸残基编码核苷酸和T hsf1 的片段;
将上述2个片段用融合PCR法连接到一起构建长度为8170 bp的融合核苷酸片段up-pyrG-P xylP -dn-hsf1Var-T hsf1 -down,转化黄曲霉CA14PTs菌株,最后筛选获得4个P xylP - dn-hsf1Var阳性转化子菌株。
随机取两个上述黄曲霉P xylP -dn-hsf1Var阳性转化子(T1、T2)的新鲜的孢子,分别接种103个孢子于YGT、YXT、GMM和XMM固体培养基平板的中心点,37 ℃持续培养3天。实验结果如图4B所示,在葡萄糖存在的情况下(YGT和GMM培养基),木糖启动子受到葡萄糖抑制,无法转录表达dn-Hsf1Var,此时黄曲霉能够正常生长;而当葡萄糖不存在时(YXT和XMM培养基),木糖启动子能够被木糖和木聚糖诱导激活,转录表达dn-Hsf1Var,此时黄曲霉的生长反而收到显著抑制,菌落直径明显变小。该结果说明,dn-Hsf1Var的转录表达对黄曲霉的生长是不利的,dn-Hsf1蛋白片段dn-Hsf1Var也具有显性负效应活性。
综合以上结果,说明本发明所构建的Hsf1突变体dn-Hsf1及其活性片段dn-Hsf1Var在表达时都能够显著抑制黄曲霉的生长,dn-Hsf1及其活性片段确实具有显性负效应活性,具有抑制真菌生长、防控真菌污染的应用价值。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
SEQUENCE LISTING
<110> 福建农林大学
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Claims (8)
1.一种热休克转录因子1显性负效应突变体dn-Hsf1,其特征在于:
所述dn-Hsf1蛋白的氨基酸序列如SEQ ID NO.1所示;或
如SEQ ID NO.1所示氨基酸序列中缺失第364位至第378位共15个氨基酸残基。
2.编码权利要求1所述一种热休克转录因子1显性负效应突变体dn-Hsf1的核苷酸序列。
3.根据权利要求2所述的核苷酸序列,其特征在于,所述序列如SEQ ID NO.2所示。
4.包含编码权利要求1 所述一种热休克转录因子1显性负效应突变体dn-Hsf1的核苷酸序列的表达载体。
5.权利要求4所述表达载体的构建方法,其特征在于:以黄曲霉NRRL3357菌株基因组DNA为模板,PCR扩增黄曲霉NRRL3357热休克转录因子1 (Hsf1)自身启动子P hsf1 和终止子T hsf1 ;以黄曲霉NRRL3357菌株cDNA为模板扩增编码权利要求1所述dn-Hsf1的核苷酸片段dn-hsf1;将P hsf1 、dn-hsf1、和T hsf1 共3个片段用融合PCR法连接到一起构建包含hsf1自身启动子P hsf1 、dn-hsf1编码核苷酸和Hsf1终止子T hsf1 的融合核苷酸片段P hsf1 -dn-hsf1-T hsf1 ,并将其***pPTR I载体中,经转化,筛选阳性转化子,测序验证,获得表达载体质粒pPTR I-dn-hsf1。
6.包含编码权利要求1 所述一种热休克转录因子1显性负效应突变体dn-Hsf1的核苷酸序列的融合核苷酸片段。
7.权利要求6所述的融合核苷酸片段的构建方法,其特征在于:以黄曲霉NRRL3357菌株基因组DNA为模板,PCR扩增黄曲霉NRRL3357热休克转录因子1的上游重组片段up;从烟曲霉AF293菌株基因组DNA中用PCR方法扩增pyrG筛选标记基因片段;从产黄青霉NRRL1951菌株基因组DNA中用PCR扩增木糖诱导启动子P xylP 片段;从根据权利要求5的方法获得的表达载体质粒pPTR I-dn-hsf1中用PCR法扩增dn-hsf1-T hsf1 核苷酸片段;将上述上游重组片段up、pyrG筛选标记基因片段、木糖诱导启动子P xylP 片段和dn-hsf1-T hsf1 核苷酸片段,共4个片段用融合PCR法连接到一起构建包含上游重组片段、筛选标记基因pyrG、木糖诱导启动子P xylP 和dn-hsf1-T hsf1 片段的融合核苷酸片段up-pyrG-P xylP -dn-hsf1-T hsf1 。
8.如权利要求1所述的dn-Hsf1、或权利要求2所述的编码核苷酸序列、或权利要求4所述的表达载体、或权利要求6所述的融合核苷酸片段在制备防控黄曲霉污染制剂中的应用。
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