CN114107327A - 绿色木霉高温胁迫响应关键酶基因TvHSP70、重组表达载体、工程菌及其应用 - Google Patents
绿色木霉高温胁迫响应关键酶基因TvHSP70、重组表达载体、工程菌及其应用 Download PDFInfo
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Abstract
本发明涉及微生物技术领域，具体涉及一种绿色木霉高温胁迫响应关键酶基因TvHSP70、重组表达载体、工程菌及其应用。所述绿色木霉高温胁迫响应关键酶基因TvHSP70，其特征在于，所述基因的CDS的核苷酸序列为如下(a1)～(a3)任意一种：(a1)如SEQ ID NO.19所示的核苷酸序列；(a2)如SEQ ID NO.19所示的核苷酸序列经一个或多个核苷酸的替换、缺失或插入形成的序列；(a3)在严格条件下能够与如(a1)或(a2)所述的核苷酸序列杂交并编码相同功能蛋白的核苷酸序列。本发明构建筛选具有较强抗逆性的木霉工程菌株，有效提高了促进木霉的生长性能，提高了抗逆性、对病原菌的拮抗能力，以及木霉对植物的促生能力。
Description
技术领域
本发明涉及微生物技术领域，具体涉及一种绿色木霉高温胁迫响应关键酶基因TvHSP70、重组表达载体、工程菌及其应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
木霉(Trichoderma spp.)属于半知菌亚门、丝孢纲、丝孢目、丛梗孢科、木霉属，是一种重要的多功能的丝状真菌，是工业上重要的酶制剂生产菌株，是土壤环境中有毒物质的降解菌株，是农业生产中重要的植病生防菌株，也是复合微生物有机肥和全元生物有机肥中重要的微生物菌种之一，在工农业生产及环境保护领域都有着广泛的应用。
在生产、保存、运输以及工农业应用中，木霉不可避免的要面对一些不利环境，这直接影响木霉生存，进而影响其工农业生产能力。以木霉菌生物有机肥为例，木霉作为功能菌株，需要先适应所处的环境并在其周围大量定殖才能发挥促生、增产、防病等多重作用。但是在实验筛选获得的优良木霉菌株却出现田间应用效果不稳定的情况。尤其是一些温度过高的土壤环境条件下，木霉存活率低、定殖能力差，进而影响施肥效果，这也成为木霉菌生物有机肥料的大规模商品化生产的重要制约因子之一。因此，提高木霉对不利环境的适应能力具有重要意义。
发明内容
针对现有技术中存在的问题，本发明的目的是提供一种绿色木霉高温胁迫响应关键酶基因TvHSP70、重组表达载体、工程菌及其应用，木霉产生的次级代谢产物种类丰富，生物活性多样，很多代谢产物参与调控木霉对不利环境的适应能力，本发明利用基因工程技术对木霉进行遗传改良，构建筛选具有较强抗逆性的木霉工程菌株，有效提高了促进木霉的生长性能，提高了抗逆性、对病原菌的拮抗能力，以及木霉对植物的促生能力，对促进木霉菌在工农业生产中广泛应用具有重要的意义。
为了实现上述目的，本发明的技术方案如下所述：
在本发明的第一方面，提供一种绿色木霉高温胁迫响应关键酶基因TvHSP70，所述基因的CDS的核苷酸序列为如下(a1)～(a3)任意一种：
(a1)如SEQ ID NO.19所示的核苷酸序列；
(a2)如SEQ ID NO.19所示的核苷酸序列经一个或多个核苷酸的替换、缺失或插入形成的序列；
(a3)在严格条件下能够与如(a1)或(a2)所述的核苷酸序列杂交并编码相同功能蛋白的核苷酸序列。
在本发明的第二方面，提供一种绿色木霉热休克蛋白TvHSP70，其氨基酸序列为如下(b1)或(b2)：
(b1)SEQ ID NO.20所示的氨基酸序列组成的蛋白质；
(b2)将(b1)经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同生物活性的由其衍生的蛋白质。
在本发明的第三方面，提供一种重组表达载体，所述重组表达载体含有第一方面中所述的绿色木霉高温胁迫响应关键酶基因TvHSP70；
优选地，所述重组表达载体由绿色木霉高温胁迫响应关键酶基因TvHSP70有效地连接到表达载体上获得；
优选地，所述表达载体为病毒载体、质粒、噬菌体、噬菌粒、黏粒、F黏粒、噬菌体或人工染色体中的任意一种或多种；进一步优选为真菌质粒；更进一步优选为pBARGPE1-Hygro质粒。
进一步优选地，重组表达载体pBARGPE1-Hygro-TvHSP70大小为8014bp，包含木霉启动子gpdA、终止子TrpC、抗性筛选基因HygR以及TvHSP70基因表达盒的完整序列。
在本发明的第四方面，提供一种工程菌，所述工程菌中含有第一方面所述的绿色木霉高温胁迫响应关键酶基因TvHSP70或第四方面所述的重组表达载体；所述TvHSP70基因在工程菌中过表达；
所述工程菌包括但不限于细菌和真菌，优选的，所述工程菌为木霉属，优选的为绿色木霉，进一步优选为绿色木霉(Trichoderma viride)Tv-1511，已于2018年12月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，保藏号为：CGMCC NO.16800。该菌株已在专利号为ZL201910626525.9的专利中公开。
在本发明中，除重组表达载体和工程菌之外的其他含有所述TvHSP70基因的生物材料也在本发明的保护范围之内；
所述生物材料包括表达盒、转座子、宿主细胞或转基因细胞系。
本发明中，所述载体和宿主细胞可理解为本领域技术人员在转基因过程中所使用的载体和宿主细胞。但随着科技发展，所述载体和宿主细胞的选择也许会发生变化，或在非转基因目的的应用领域，也同样涉及载体和宿主细胞的利用，但只要含有本发明所述基因或本发明所述的载体，均在本发明的保护范围之内。
本发明的第五个方面，提供上述绿色木霉热休克蛋白基因TvHSP70、重组表达载体和工程菌的获得方法；
其中，所述绿色木霉热休克蛋白基因TvHSP70的获得方法可以是人工合成或者以上述绿色木霉Tv-1511基因组DNA或者cDNA为模板，用引物扩增得到，优选的，扩增引物为SEQ ID NO.21-SEQ ID NO.24。
所述重组表达载体的获得方法为上述基因或其基因表达盒有效地连入质粒，优选的，基因表达盒的构建中扩增引物为SEQ ID NO.23、SEQ ID NO.24，酶切连接位点为BamHI和EcoRI；优选的，质粒的构建中采用pBARGPE-Hygro线性载体，将TvHSP70基因表达盒连入经BamHI和EcoRI双酶切的pBARGPE1-Hygro线性载体中即得。
所述工程菌的构建方法为将上述重组表达载体转入宿主细胞原生质体中；优选的，所述宿主细胞原生质体为木霉原生质体，或者转入方法包括生物学上可接受的直接转化法(包括基因枪法、电击法、超声波法、显微注射法、PEG法)或者间接转化法(包括DNA病毒载体介导法、农杆菌介导法)，优选的，利用PEG-CaCl2法；优选的，所述木霉为绿色木霉(Trichoderma viride)Tv-1511。
本发明的第六个方面，提供上述绿色木霉热休克蛋白基因TvHSP70、绿色木霉热休克蛋白TvHSP70、重组表达载体和/或工程菌在如下任意一种或多种中的应用：
(c1)促进木霉的生长；
(c2)提高木霉抗逆性；
(c3)提高木霉对病原菌的拮抗能力；
(c4)提高木霉对植物的促生能力。
其中，所述木霉为绿色木霉Tv-1511。
(c2)应用中，逆境环境包括但不限于高温、高盐和干旱；
(c3)应用中，病原菌包括：尖孢镰刀菌(Fusarium oxysporum)、串珠镰孢菌(F.moniliforme)、灰葡萄孢菌(Botrytis cinerea)和葡萄座腔菌(Botryosphaeriadothidea)。
(c4)应用中，促生能力为提高植物种子萌发率、促进种胚生长或促进幼苗生长，优选的，所述植物包括大豆和黄瓜，进一步的，所述黄瓜为津研四号，大豆为TZX-805。
本发明的第七方面，提供一种基于转录组数据和基因表达分析来筛选绿色木霉Tv-1511响应高温胁迫关键酶基因的方法，所述方法为：
(d1)基于转录组数据筛选绿色木霉Tv-1511中与高温胁迫相关的功能基因。
采用Trizol法分别提取高温处理和未处理的绿色木霉Tv-1511的总RNA，纯化后进行转录组文库构建和Illumina HiSeq测序，获得原始测序序列，使用DESeq R软件包(1.18.0)进行差异基因表达分析，并采用Benjamini-Hochberg方法校正P值，当校正后的P值小于0.05时，该基因被认为是差异表达。
优选地，所述高温处理是将Tv-1511孢子液置于46℃温度下热激24h，然后接种于PD液体培养基中28℃恒温培养；
(d2)基于(d1)中筛选出来的功能基因对高温胁迫的表达响应分析来筛选绿色木霉Tv-1511中响应高温胁迫的关键基因。
采用Trizol法分别提取高温处理和未处理的绿色木霉Tv-1511的总RNA，反转录成cDNA为模板，根据(d1)中测序结果中的关键酶基因序列，设计特异性引物，进行半定量RT-PCR和实时荧光定量PCR反应。
优选地，所述高温处理是将绿色木霉Tv-1511孢子液分别置于28℃，37℃和46℃温度下热激24h，然后接种于PD液体培养基中28℃培养。
基于所述转录组测序序列设计基因特异性引物，序列为SEQ ID NO.1-SEQ IDNO.16；绿色木霉Tv-1511内参基因扩增的引物序列为SEQ ID NO.17和SEQ ID NO.18。
本发明的具体实施方式具有以下有益效果：
1)本发明基于转录组数据和基因表达分析，对绿色木霉Tv-1511响应高温胁迫的关键酶基因进行筛选，获得的关键基因为木霉抗逆功能菌的选育提供基因资源和参考依据。
2)本发明首次在绿色木霉Tv-1511中克隆得到热休克蛋白基因TvHSP70，并利用表达载体和原生质体融合的方法构建了TvHSP70过表达的木霉工程菌株TvHSP70-OE。经试验证实了TvHSP70基因过表达可以增强绿色木霉菌抵御自然环境及工业发酵中不利生长环境的能力，提高绿色木霉对植物的抗病促生效果。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
图1是Tv-1511响应高温胁迫关键基因的RT-PCR分析及保守结构域；其中：M:DL2000 marker；1，2，3表示28℃、37℃和46℃温度处理。
图2是Tv-1511响应高温胁迫关键基因的qRT-PCR分析；
图3是重组载体pBARGPE1-Hygro-TvHSP70的质粒图谱；
图4是TvHSP70基因过表达工程菌的鉴定；其中：WT为绿色木霉Tv-1511原始菌株；1-12为阳性转化子；*和**分别表示阳性转化子与原始菌株(WT)在P<0.05和P<0.01水平的显著差异性；
图5是TvHSP70-OE工程菌株的鉴定；其中：WT为绿色木霉Tv-1511原始菌株；OE-5、OE-7和OE-11为TvHSP70过表达工程菌株；柱上不同字母代表不同处理间在P<0.05和P<0.05水平的显著差异性；
图6是TvHSP70-OE工程菌株的耐高温能力分析；其中：WT为绿色木霉Tv-1511原始菌株；OE-5、OE-7和OE-11为TvHSP70过表达工程菌株。
图7是TvHSP70-OE工程菌株的耐盐能力分析；其中：WT为绿色木霉Tv-1511原始菌株；OE-5、OE-7和OE-11为TvHSP70过表达工程菌株；
图8是TvHSP70-OE工程菌株拮抗病原菌能力分析；其中：WT为绿色木霉Tv-1511原始菌株；OE-5、OE-7和OE-11为TvHSP70过表达工程菌株；柱上不同字母代表不同处理间在P<0.05和P<0.05水平的显著差异性；
图9是TvHSP70-OE工程菌株对大豆种子萌发的影响；其中：CK是以无菌水为发芽溶液；T1是加入终浓度为105cfu/mL的木霉原始菌株(WT)孢子液；T2是加入终浓度为105cfu/mL的TvHSP70-OE工程菌株孢子液；柱上不同字母代表不同处理间在P<0.05和P<0.05水平的显著差异性；
图10是TvHSP70-OE工程菌株对黄瓜幼苗生长的影响；其中：CK是以无菌水为发芽溶液；T1是加入终浓度为105cfu/mL的木霉原始菌株(WT)孢子液；T2是加入终浓度为105cfu/mL的TvHSP70-OE工程菌株孢子液。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本申请提供进一步的说明。除非另有指明，本申请使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
为了使得本领域技术人员能够更加清楚地了解本公开的技术方案，以下将结合具体的实施例与对比例详细说明本公开的技术方案。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
生物材料来源：
绿色木霉Tv-1511为本实验室筛选鉴定的木霉菌株，在中国普通微生物菌种保藏中心的保藏号为CGMCC No.16800；
大肠杆菌DH5α、T4连接酶试剂盒、反转录试剂盒、高保真Taq酶、pMD19-T载体等购自于南京诺唯赞公司；
无内毒素质粒大提试剂盒、DNA纯化回收试剂盒购自于天根生化科技有限公司；
真菌RNA提取试剂盒、真菌DNA提取试剂盒购自于OMEGA公司；
过表达载体构建质粒pBARGPE1-Hygro购自于Addgene公司；
限制性内切酶KpnI和EcoRI购自于NEB公司；
氨苄、潮霉素B和溶菌酶购自于Sigma公司；
LB培养基、PDA培养基和PDB培养基等购自于青岛海博公司。
实施例1
绿色木霉Tv-1511耐热关键基因的筛选和表达分析
1.1绿色木霉Tv-1511响应高温胁迫关键基因的筛选
(1)实验处理
以Tv-1511孢子液为实验材料，进行分组处理。实验组：将绿色木霉Tv-1511孢子液置于46℃温度下热激24h，然后接种于PD液体培养基中28℃培养；对照组：将绿色木霉Tv-1511孢子液置于28℃温度下24h，然后接种于PD液体培养基中28℃培养。每个处理重复3次。再处理3d后收集菌丝，立即置于液氮中进行速冻处理。
(2)有参转录组测序获得Tv-1511响应高温胁迫的功能基因
采用Trizol法提取不同处理组的Tv-1511总RNA，进行浓度、纯度以及完整性的检测；取3μg RNA进行文库构建，具体操作方法参考
UltraTMII RNA文库制备试剂盒说明书；文库构建成功后，用Agilent 2100进行检测；将质检合格的文库上机测序；获得原始测序序列后，通过统计读段长度、GC含量、Q20、Q30以及碱基数等指标对原始数据进行质量评估；选用TopHat v2.0.12将测序获得的高质量数据与绿色木霉Tv-1511基因组(GenBank Accession No.VCEC00000000；BioProject:PRJNA543939；BioSample:SAMN11791795)进行比对；本试验中的样品有生物学重复，因此使用DESeq R软件包(1.18.0)进行差异基因表达分析，并采用Benjamini-Hochberg方法校正P值，当校正后的P值小于0.05时，该基因被认为是差异表达。
最终，从Tv-1511中筛选出8个与高温胁迫相关的功能基因，分别为A6376、A4009、A5978、A3581、A1421、A1326、A2155、A0852。
1.2绿色木霉Tv-1511响应高温胁迫关键基因的表达分析
(1)半定量RT-PCR
将Tv-1511孢子液分别置于28℃，37℃和46℃热激24h，然后接种于PD液体培养基中28℃培养，收集菌丝；采用Trizol法提取样品总RNA，进行浓度、纯度以及完整性的检测；取1μg RNA，按照反转录试剂盒说明书进行反转录，获得cDNA；根据转录组测序结果中的基因序列，设计半定量RT-PCR引物，序列为SEQ ID NO.1-SEQ ID NO.16；
SEQ ID NO.1：A6376-F TCCGAGGTCCAGGCTGATATGAAG；
SEQ ID NO.2：A6376-R GTCTCACGCATCTTGGTCAGGATC；
SEQ ID NO.3：A1326-F ATCAAGAAGCACTCCGAGTTCATCAG；
SEQ ID NO.4：A1326-R GGTCTCCTCCTCAGCATCCTCATC；
SEQ ID NO.5：A2155-F GCCTAAGAAACAACCTGCCGACTC；
SEQ ID NO.6：A2155-R TTGACACGAGAGGGAAAGTTGAAAGAG；
SEQ ID NO.7：A4009-F GACCAGTCCATCACCATTGCTTCC；
SEQ ID NO.8：A4009-R TACTTCTCGGAGTCCTCAACCATCTG；
SEQ ID NO.9：A5978-F ATTCTCGGCAAGATGAAGGAGGTTG；
SEQ ID NO.10：A5978-R GTTGAAGTAGGCAGGGACAGTAACG；
SEQ ID NO.11：A3581-F ACCTGGAGCACTCACTATACGATACG；
SEQ ID NO.12：A3581-R TTGGACAATTCCTGGTTGAAGTAGAGC；
SEQ ID NO.13：A1421-F ACCAACACCACTATAACAGACACAGAC；
SEQ ID NO.14：A1421-R GAGAGAGGTCGCAGATGTCGTTTG；
SEQ ID NO.15：A0852-F AGCGAGGATTTACAGCGACCAAAG；
SEQ ID NO.16：A0852-R CTTTTCCCTGAGACCGTGGATGAC；
选择绿色木霉β-Actin基因作为内参基因，扩增引物为SEQ ID NO.17和SEQ IDNO.18；
SEQ ID NO.17：Actin-F TTCGCTCTTCCTCACGC；
SEQ ID NO.18：Actin-R GATGTCACGGACGATTTC；
RT-PCR反应程序为普通PCR程序，退火温度为60℃，循环次数为26；反应结束后，取PCR产物进行电泳检测。
结果显示：8个与高温胁迫相关的功能基因之间表达量存在差异，其中在高温处理下也保持较高表达量的基因是A6376、A1326和A2155，这三个基因都含有与高温胁迫响应相关的保守结构域，进一步分析发现，A6376为HSP70基因，A1326为HSP83基因，A2155为sHSP基因。
(2)实时荧光定量PCR
将Tv-1511孢子液分别置于28℃，37℃和46℃温度下热激0h，12h和24h，然后接种于PD液体培养基中28℃培养，收集菌丝；提取样品总RNA，反转录成cDNA；根据HSP70(A6376)，HSP83(A1326)和sHSP(A2155)基因序列，设计特异性引物，引物为SEQ ID NO.1-SEQ ID NO.6；选择绿色木霉β-Actin基因作为内参基因，扩增引物为SEQ ID NO.17和SEQID NO.18；在定量PCR仪中进行扩增反应，反应结束后，采用2-ΔΔCT法分析数据。
结果显示：在37℃和46℃处理后，HSP70、HSP83和sHSP基因的表达量明显增加，其中一直保持较高表达量的基因是HSP70，因此，绿色木霉Tv-1511中响应高温胁迫的关键基因是HSP70。
实施例2
Tv-1511响应高温胁迫关键基因的克隆和表达载体的构建
2.1TvHSP70基因的克隆
按照真菌DNA提取试剂盒说明书进行Tv-1511基因组DNA的提取；根据转录组测序结果中的A6376基因序列，设计扩增全长引物，引物为SEQ ID NO.1和SEQ ID NO.2；以基因组DNA为模板，利用高保真PCR聚合酶进行PCR扩增，获得HSP70基因表达盒；利用DNA回收试剂盒对扩增产物进行回收与纯化；将回收产物与T载体进行连接，并转化至大肠杆菌DH5a中，获得阳性克隆单菌落，送公司测序。
结果显示：分离得到了绿色木霉Tv-1511热休克蛋白70基因，将其命名为TvHSP70。TvHSP70序列长为2022bp(SEQ ID NO.19)，该序列上游有起始密码子ATG，下游有终止密码子TAA，包括一个编码一个由673个氨基酸组成的蛋白(SEQ ID NO.20)。
TvHSP70基因的CDS的核苷酸序列如下：
SEQ ID NO.19：
ATGTCTATGGGACCAGCCGTCGGTATCGACCTGGGTACTACGTACTCTTGCGTCGGTATCTTCCGTGAGGACCGATGTGATATCATCGCCAACGACCAGGGTAACCGAACGACCCCTTCGTTCGTTGCCTTCACCGACACCGAGCGTCTGATCGGTGATGCGGCCAAGAACCAGGTCGCCATGAACCCCCAGAACACCGTCTTCGACGCCAAGCGATTGATCGGACGCAAGTTTGCCGACTCCGAGGTCCAGGCTGATATGAAGCACTTCCCCTTCAAGATCATCGACAAGGGCTCCAAGCCCGTCGTCGAGGTTGAGTTCAAGGGCGAGAAGAAGACCTTCACCCCCGAGGAGATCTCCTCCATGATCCTGACCAAGATGCGTGAGACTGCCGAGTCCTACCTCGGAACCACCGTCAACAACGCCGTCATCACTGTCCCCGCCTACTTCAACGACAGCCAGCGTCAGGCTACCAAGGACGCCGGTCTCATTGCCGGTCTCAACGTCCTGCGTATCATCAACGAGCCCACCGCTGCTGCCATCGCCTACGGCCTTGACAAGAAGGTCGAGGGTGAGCGCAACGTCCTCATCTTCGATCTCGGTGGTGGTACCTTCGATGTCTCTCTCCTGACCATTGAGGAGGGTATCTTCGAGGTCAAGTCCACTGCCGGTGACACTCACTTGGGTGGTGAAGATTTCGACAACCGTCTGGTCAACCACTTTGTTAACGAGTTTAAGCGCAAGAACAAGAAGGTTAGTTTACCCACAAGCGATTCCCCCCAGAACCAGAACCAGCACATGCTAACATCTCACCAGGATCTGTCCACCAACGCTCGTGCTCTTCGCCGTCTCCGCACTGCCTGCGAGCGTGCTAAGCGAACTCTGTCTTCGTCTGCCCAGACCTCCATTGAGATCGACTCTCTCTTCGAGGGTATCGACTACTACACCTCCATCACTCGTGCCCGTTTCGAGGAGCTGTGCCAGGATCTCTTCCGATCCACCATCCAGCCCGTCGACCGTGTCCTTGCCGATGCCAAGATCGACAAGTCCCAGGTCCACGAGATTGTCCTCGTCGGTGGCTCTACCCGTATCCCCCGTATCCAGAAGCTCATCACCGACTACTTCAACGGCAAGGAGCCCAACAAGAGCATCAACCCCGATGAGGCCGTCGCCTACGGTGCTGCCGTCCAGGCCGCCATTCTGTCTGGTGACACCTCCTCCAAGTCCACCAACGAGATCCTGCTTCTCGATGTCGCTCCCCTGTCTCTCGGTATCGAGACTGCTGGTGGCATGATGACCAAGCTCATCCCCCGCAACACCACCATCCCCACCAAGAAGTCCGAGGTCTTCTCCACCTTCTCTGACAACCAGCCTGGTGTGCTCATCCAGGTCTACGAGGGTGAGCGTCAGCGCACCAAGGACAACAACCTGCTCGGCAAGTTCGAGCTCACTGGCATTCCCCCTGCCCCCCGTGGTGTTCCCCAGATTGAGGTCACCTTCGACGTCGATGCCAACGGTATCATGAACGTCTCCGCCGTCGAGAAGGGCACTGGCAAGTCCAACAAGATTGTCATCACCAACGACAAGGGCCGCCTGTCCAAGGAGGAGATTGAGCGCATGCTGTCTGACGCCGAGAAGTACAAGGAGGAGGACGAGGCTGAGGGCCGCCGTGTCTCTGCCAAGAACGGCCTTGAGTCCTACGCCTACTCTCTCCGCAACACCCTCAACGACCCCAAGGTCGACGAGAAGATTGAGGCCGCCGACAAGGAGACCCTCAAGACCGAGATCGACAAGATCGTCCAGTGGCTCGATGAAAACCAGCAGGCCACTCGTGAGGAGTACGAGGAGCACCAGAAGGAGCTCGAGGGTGTCGCCAACCCCATCATGATGAAGTTCTACGGAGCTGGTGGTGAGGGTGGCATGCCCGGTGGCATGCCCGGCGGCCCTGGTGGCTTCCCCGGCGCTGGCGGCGCTCCCCACGCTGGTGGCGATGACGGCCCCACCGTCGAGGAGGTCGACTAA
绿色木霉热休克蛋白TvHSP70的氨基酸序列如下：
SEQ ID NO.20：
MSMGPAVGIDLGTTYSCVGIFREDRCDIIANDQGNRTTPSFVAFTDTERLIGDAAKNQVAMNPQNTVFDAKRLIGRKFADSEVQADMKHFPFKIIDKGSKPVVEVEFKGEKKTFTPEEISSMILTKMRETAESYLGTTVNNAVITVPAYFNDSQRQATKDAGLIAGLNVLRIINEPTAAAIAYGLDKKVEGERNVLIFDLGGGTFDVSLLTIEEGIFEVKSTAGDTHLGGEDFDNRLVNHFVNEFKRKNKKVSLPTSDSPQNQNQHMLTSHQDLSTNARALRRLRTACERAKRTLSSSAQTSIEIDSLFEGIDYYTSITRARFEELCQDLFRSTIQPVDRVLADAKIDKSQVHEIVLVGGSTRIPRIQKLITDYFNGKEPNKSINPDEAVAYGAAVQAAILSGDTSSKSTNEILLLDVAPLSLGIETAGGMMTKLIPRNTTIPTKKSEVFSTFSDNQPGVLIQVYEGERQRTKDNNLLGKFELTGIPPAPRGVPQIEVTFDVDANGIMNVSAVEKGTGKSNKIVITNDKGRLSKEEIERMLSDAEKYKEEDEAEGRRVSAKNGLESYAYSLRNTLNDPKVDEKIEAADKETLKTEIDKIVQWLDENQQATREEYEEHQKELEGVANPIMMKFYGAGGEGGMPGGMPGGPGGFPGAGGAPHAGGDDGPTVEEVD*
2.2TvHSP70过表达载体的构建
以Tv-1511基因组DNA为模板，利用高保真PCR聚合酶进行PCR扩增，克隆获得带有酶切连接位点(BamHI、EcoRI)的TvHSP70基因表达盒，引物序列为SEQ ID NO.21-SEQ IDNO.24；对扩增产物进行回收与纯化、T载体连接，测序验证序列的正确性；参考无内毒素质粒大提试剂盒说明书，将测序正确的TvHSP70基因和表达载体pBARGPE1-Hygro分别进行质粒提取；用限制性内切酶BamHI和EcoRI分别对基因和载体质粒进行双酶切；酶切产物经回收纯化、T4 DNA连接酶连接后，转化至大肠杆菌DH5a中；提取质粒，进行酶切验证。
SEQ ID NO.21：TvHSP70-F ATGTCTATGGGACCAGCCGT；
SEQ ID NO.22：TvHSP70-R TTAGTCGACCTCCTCGACGG；
SEQ ID NO.23：TvHSP70-BamHI-F CGGGATCC ATGTCTATGGGACCAGCCGT；
SEQ ID NO.24：TvHSP70-EcoRI-R CGGAATTC TTAGTCGACCTCCTCGACGG；
结果显示：获得的重组载体pBARGPE1-Hygro-TvHSP70大小为8014bp，包含木霉启动子gpdA、终止子TrpC、抗性筛选基因HygR以及TvHSP70基因表达盒的完整序列。
实施例3
TvHSP70基因过表达工程菌的获得及鉴定
3.1TvHSP70基因过表达工程菌的获得
(1)绿色木霉Tv-1511原生质体的制备
配置溶液A：1.2M D-Sorbito、0.1M KH2PO4，pH 5.6；溶液B：1M D-Sorbito、50mMCaCl2、10mM Tris-HCl，pH 7.5；裂解液：取0.24g溶菌酶溶于5.7mL溶液A中，过滤除菌，加入0.3mL PBS溶液，pH 5.8。
将绿色木霉Tv-1511孢子液接种于生长培养基(在液体培养基PD中加入1.5％的酵母提取物)，于28℃、180rpm培养10h；离心去上清，用25mL溶液A洗涤3次；加入6mL裂解液重悬，于30℃、60rpm裂解3h；用装有4层纱布的1.5mL管过滤上述混合液，保留下层滤液，向滤液中加入20mL溶液A，4℃、3800rpm离心5min，弃上清，保留底部的原生质体，加15mL溶液B洗涤2次，离心去上清；加1mL的溶液B(4℃预冷)重悬，冰上得到原生质体；用血球计数板观察计数，稀释原生质体至107个/mL。
(2)PEG-CaCl2介导的原生质体转化
取150μL原生质体悬浮液和150μL质粒载体，混匀，冰浴20min；逐滴加入1.5mL PEG溶液(25％PEG600、50mM CaCl2，10mM Tris HCl，pH 7.5)，混匀，冰浴20min，室温放置20min；取100μL转化液均匀涂布到含1M蔗糖PDA平板上，28℃培养24h；将含抗生素的PDA平板(潮霉素300μg/mL)，平铺在原平板上，观察转化子的出现。
3.2TvHSP70基因过表达工程菌的筛选及鉴定
挑取转化子，在含潮霉素B(300μg/mL)的PDA平板连续转接三代，获得稳定遗传的阳性转化子12株(图4A)，进行单孢分离培养并保存菌株，进一步进行分子鉴定。
将Tv-1511原始菌株和阳性转化子的孢子液分别置于46℃热激24h，然后接种于PD液体培养基中28℃培养，收集菌丝；利用1.2中所述的实时荧光定量PCR法检测TvHSP70转录表达。结果发现，与原始菌株(WT)相比，12株阳性转化子的基因表达量均显著增加，其中有3株基因表达差异达到显著水平(P＜0.05)，有9株基因表达差异达到极显著水平(P＜0.01)。因此，成功获得TvHSP70基因过表达工程菌(TvHSP70-OE)12株。
实施例4
TvHSP70-OE工程菌株生长和抗逆特性分析
4.1生长性能鉴定
将木霉原始菌株(WT)和TvHSP70过表达工程菌株(TvHSP70-OE)接种于PDA平板上进行活化，28℃培养60-72h，使原始菌株和过表达工程菌株长势均一，用打孔器制备大小一致的菌块，接种于PDA平板上，置于培养箱培养，每天测定木霉生长圈的直径、扫描生长圈的形态。结果显示：TvHSP70-OE菌株的生长速率显著高于WT(P＜0.05)，说明TvHSP70可以显著促进绿色木霉的生长(图5)。
4.2耐热性分析
制备木霉原始菌株(WT)和TvHSP70过表达工程菌株(TvHSP70-OE)的菌块，接种于PDA平板上，置于35℃和37℃培养箱中培养，观察木霉生长圈的形态和大小。结果发现，TvHSP70-OE菌株在35℃和37℃温度下的生长速率显著高于原始菌株(图6A)。
进一步，将WT和TvHSP70-OE的菌块接种于PDA平板上，置于28℃培养24h，再置于40℃、43℃、46℃和49℃的高温下热激24h，然后置于28℃培养箱中培养，观察木霉生长圈的形态和大小。结果显示：TvHSP70-OE能够在高温热激后尽快恢复生长，具有较强的抗高温性能(图6B)。以上结果表明TvHSP70可以显著提高绿色木霉对高温的耐受能力。
4.3耐盐性分析
在不同盐浓度(0、100、200、300和400mM NaCl)的平板上接种WT和TvHSP70-OE的菌块，置于28℃培养，观察木霉生长圈的形态和大小。结果显示：盐胁迫处理下，TvHSP70-OE菌株的生长速率显著高于WT(P＜0.05)，其中100、200、300和400mM NaCl处理96h后的菌丝生长半径是WT的1.09、1.22、1.19、1.41和2.07倍，表明TvHSP70可以显著促进绿色木霉对盐胁迫的耐受性(图7)。
实施例5
TvHSP70-OE工程菌株在拮抗病原真菌中的应用
选用四种植物病原真菌，包括尖孢镰刀菌(Fusarium oxysporum)、串珠镰孢菌(F.moniliforme)、灰葡萄孢菌(Botrytis cinerea)和葡萄座腔菌(Botryosphaeriadothidea)。用打孔器制备大小一致的菌块，在培养皿两侧对称位置分别接种病原真菌和绿色木霉(WT、TvHSP70-OE)，以只接病原菌的平板为对照。置于恒温培养箱培养，记录病原菌的生长半径，计算抑菌率。
抑菌率(％)＝(对照病原菌菌落半径-处理病原菌菌落半径)/对照病原菌菌落半径×100％。
结果表明，与WT相比，TvHSP70-OE对尖孢镰刀菌、串珠镰孢菌)、灰葡萄孢菌和葡萄座腔菌具有更强的抑制作用，抑菌率分别为79.2％、77.4％、68.1％和62.2％，分别是WT的1.15、1.12、1.14和1.04倍(图8)。因此，TvHSP70-OE工程菌具有更强的生物防治能力。
实施例6
TvHSP70-OE工程菌株对植物促生能力的分析
6.1种子萌发
供试植物：大豆(TZX-805)和黄瓜(津研四号)。
挑选健康饱满的种子先用75％酒精消毒30s，再用2％的NaClO消毒3min，无菌水冲洗4-5次。培养皿中放3层灭菌滤纸，每皿16粒种子，5mL相应发芽溶液。以无菌水为发芽溶液的为对照组(CK)，以加入WT孢子液(终浓度为105cfu/mL)为处理组(T1)，以加入TvHSP70-OE孢子液(终浓度为105cfu/mL)为处理组(T2)，每个处理设置4个重复，分别置于28℃和35℃培养箱中培养，统计种子的发芽率，并对胚根长等指标进行测定。
结果表明：绿色木霉菌Tv-1511原始菌株和TvHSP70-OE工程菌株均能有效促进黄瓜和大豆种子的发芽率以及胚的生长，其中以TvHSP70-OE工程菌的促进效果更为显著。在28℃生长条件下，TvHSP70-OE工程菌处理的黄瓜的胚根长、胚芽长、侧根数目和发芽率分别是原始菌株WT的1.11、1.10、1.25和1.05倍，而在35℃高温胁迫下，相应的指标是WT的1.47、1.39、1.44和1.32倍(表1)。此外，28℃生长条件下，TvHSP70-OE工程菌处理的大豆的发芽率和胚根长与原始菌株WT相比分别增加了18.2％和22.3％，而在35℃高温胁迫下，发芽率和胚根长分别增加了25.0％和44.8％(图9)。以上结果表明，TvHSP70可以有效促进植物种子的萌发，且在高温胁迫下促进效果更为明显。
表1TvHSP70-OE工程菌株对黄瓜种子萌发的影响
6.2幼苗生长
供试植物：黄瓜(津研四号)。
挑选长势一致的幼苗移至水培装置，以1/2Hoagland营养液为水培溶液的为对照组(CK)，以加入WT孢子液(终浓度为105cfu/mL)为处理组(T1)，以加入TvHSP70-OE孢子液(终浓度为105cfu/mL)为处理组(T2)，每个处理设置4个重复。
结果显示：绿色木霉菌Tv-1511原始菌株和TvHSP70-OE工程菌株处理的黄瓜均呈现一定的生长优势，且以高温胁迫下TvHSP70-OE工程菌株处理组的生长优势最为明显。因此，TvHSP70-OE工程菌株可以促进黄瓜幼苗的生长，且在高温胁迫下促生效果更为明显。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 山东省科学院生物研究所、中国科学院遗传与发育生物学研究所、山东师范大学
<120> 绿色木霉高温胁迫响应关键酶基因TvHSP70、重组表达载体、工程菌及其应
用
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ttccgtgagg accgatgtga tatcatcgcc aacgaccagg gtaaccgaac gaccccttcg 120
ttcgttgcct tcaccgacac cgagcgtctg atcggtgatg cggccaagaa ccaggtcgcc 180
atgaaccccc agaacaccgt cttcgacgcc aagcgattga tcggacgcaa gtttgccgac 240
tccgaggtcc aggctgatat gaagcacttc cccttcaaga tcatcgacaa gggctccaag 300
cccgtcgtcg aggttgagtt caagggcgag aagaagacct tcacccccga ggagatctcc 360
tccatgatcc tgaccaagat gcgtgagact gccgagtcct acctcggaac caccgtcaac 420
aacgccgtca tcactgtccc cgcctacttc aacgacagcc agcgtcaggc taccaaggac 480
gccggtctca ttgccggtct caacgtcctg cgtatcatca acgagcccac cgctgctgcc 540
atcgcctacg gccttgacaa gaaggtcgag ggtgagcgca acgtcctcat cttcgatctc 600
ggtggtggta ccttcgatgt ctctctcctg accattgagg agggtatctt cgaggtcaag 660
tccactgccg gtgacactca cttgggtggt gaagatttcg acaaccgtct ggtcaaccac 720
tttgttaacg agtttaagcg caagaacaag aaggttagtt tacccacaag cgattccccc 780
cagaaccaga accagcacat gctaacatct caccaggatc tgtccaccaa cgctcgtgct 840
cttcgccgtc tccgcactgc ctgcgagcgt gctaagcgaa ctctgtcttc gtctgcccag 900
acctccattg agatcgactc tctcttcgag ggtatcgact actacacctc catcactcgt 960
gcccgtttcg aggagctgtg ccaggatctc ttccgatcca ccatccagcc cgtcgaccgt 1020
gtccttgccg atgccaagat cgacaagtcc caggtccacg agattgtcct cgtcggtggc 1080
tctacccgta tcccccgtat ccagaagctc atcaccgact acttcaacgg caaggagccc 1140
aacaagagca tcaaccccga tgaggccgtc gcctacggtg ctgccgtcca ggccgccatt 1200
ctgtctggtg acacctcctc caagtccacc aacgagatcc tgcttctcga tgtcgctccc 1260
ctgtctctcg gtatcgagac tgctggtggc atgatgacca agctcatccc ccgcaacacc 1320
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ctgtctgacg ccgagaagta caaggaggag gacgaggctg agggccgccg tgtctctgcc 1680
aagaacggcc ttgagtccta cgcctactct ctccgcaaca ccctcaacga ccccaaggtc 1740
gacgagaaga ttgaggccgc cgacaaggag accctcaaga ccgagatcga caagatcgtc 1800
cagtggctcg atgaaaacca gcaggccact cgtgaggagt acgaggagca ccagaaggag 1860
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atgcccggtg gcatgcccgg cggccctggt ggcttccccg gcgctggcgg cgctccccac 1980
gctggtggcg atgacggccc caccgtcgag gaggtcgact aa 2022
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Claims (10)
1.一种绿色木霉高温胁迫响应关键酶基因TvHSP70，其特征在于，所述基因的CDS的核苷酸序列为如下(a1)～(a3)任意一种：
(a1)如SEQ ID NO.19所示的核苷酸序列；
(a2)如SEQ ID NO.19所示的核苷酸序列经一个或多个核苷酸的替换、缺失或插入形成的序列；
(a3)在严格条件下能够与如(a1)或(a2)所述的核苷酸序列杂交并编码相同功能蛋白的核苷酸序列。
2.一种绿色木霉热休克蛋白TvHSP70，其特征在于，其氨基酸序列为如下(b1)或(b2)：
(b1)SEQ ID NO.20所示的氨基酸序列组成的蛋白质；
(b2)将(b1)经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同生物活性的由其衍生的蛋白质。
3.一种重组表达载体，其特征在于，所述重组表达载体含有权利要求1所述的绿色木霉高温胁迫响应关键酶基因TvHSP70。
4.如权利要求3所述的重组表达载体，其特征在于，所述重组表达载体由绿色木霉高温胁迫响应关键酶基因TvHSP70有效地连接到表达载体上获得；
优选地，所述表达载体为病毒载体、质粒、噬菌体、噬菌粒、黏粒、F黏粒、噬菌体或人工染色体中的任意一种或多种；进一步优选为真菌质粒；更进一步优选为pBARGPE1-Hygro质粒。
5.一种工程菌，其特征在于，所述工程菌中含有权利要求1所述的绿色木霉高温胁迫响应关键酶基因TvHSP70或权利要求3所述的重组表达载体。
6.如权利要求5所述的工程菌，其特征在于，所述工程菌包括细菌和真菌，优选的，所述工程菌为木霉属，进一步优选为绿色木霉，更进一步优选为绿色木霉(Trichodermaviride)Tv-1511，已于2018年12月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，保藏号为：CGMCC NO.16800。
7.权利要求1所述的绿色木霉热休克蛋白基因TvHSP70、权利要求3所述的重组表达载体和权利要求5所述的工程菌的获得方法，其特征在于，方法如下：
所述绿色木霉热休克蛋白基因TvHSP70的获得方法是人工合成或者以绿色木霉Tv-1511基因组DNA或者cDNA为模板，用引物扩增得到；
或者，所述重组表达载体的获得方法为将权利要求1所述的基因有效地连入质粒；
或者，所述工程菌的构建方法为将权利要求3所述重组表达载体转入宿主细胞原生质体中。
8.权利要求1所述的绿色木霉热休克蛋白基因TvHSP70、权利要求2所述的绿色木霉热休克蛋白TvHSP70、权利要求3所述的重组表达载体和/或权利要求5所述的工程菌在如下任意一种或多种中的应用：
(c1)促进木霉的生长；
(c2)提高木霉抗逆性；
(c3)提高木霉对病原菌的拮抗能力；
(c4)提高木霉对植物的促生能力；
其中，所述木霉为绿色木霉Tv-1511。
9.如权利要求8所述的应用，其特征在于，(c2)应用中，逆境环境包括但不限于高温、高盐和干旱；
(c3)应用中，病原菌包括：尖孢镰刀菌、串珠镰孢菌、灰葡萄孢菌和葡萄座腔菌；
(c4)应用中，促生能力为提高植物种子萌发率、促进种胚生长或促进幼苗生长，优选的，所述植物包括大豆和黄瓜，进一步地，所述黄瓜为津研四号，大豆为TZX-805。
10.一种筛选绿色木霉Tv-1511响应高温胁迫关键酶基因的方法，其特征在于，所述方法为：
(d1)基于转录组数据筛选绿色木霉Tv-1511中与高温胁迫相关的功能基因：
(d2)基于(d1)中筛选出来的功能基因对高温胁迫的表达响应分析来筛选绿色木霉Tv-1511中响应高温胁迫的关键基因。
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| CN112004931A (zh) * | 2018-03-26 | 2020-11-27 | 诺维信公司 | 真菌伴侣蛋白 |
| CN112553222A (zh) * | 2020-12-10 | 2021-03-26 | 湖南省蔬菜研究所 | 一种辣椒耐热基因及其应用 |
| AU2021103049A4 (en) * | 2020-12-09 | 2021-07-15 | Biology Institute Of Shandong Academy Of Sciences | Saline-alkali tolerant Trichoderma viride Tv-1511 and bio-organic fertilizer and application thereof |
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- 2021-12-23 ZA ZA2021/10858A patent/ZA202110858B/en unknown
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| CN112004931A (zh) * | 2018-03-26 | 2020-11-27 | 诺维信公司 | 真菌伴侣蛋白 |
| AU2021103049A4 (en) * | 2020-12-09 | 2021-07-15 | Biology Institute Of Shandong Academy Of Sciences | Saline-alkali tolerant Trichoderma viride Tv-1511 and bio-organic fertilizer and application thereof |
| CN112553222A (zh) * | 2020-12-10 | 2021-03-26 | 湖南省蔬菜研究所 | 一种辣椒耐热基因及其应用 |
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| CN115197957A (zh) * | 2022-05-16 | 2022-10-18 | 南京农业大学 | 一种提高贵州木霉njau 4742在逆境下分解农作物秸秆的基因片段及其应用 |
| CN115197957B (zh) * | 2022-05-16 | 2023-10-10 | 南京农业大学 | 一种提高贵州木霉njau 4742在逆境下分解农作物秸秆的基因片段及其应用 |
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| CN114107327B (zh) | 2024-02-23 |
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