CN116286404A - 一株高效生产木霉菌素的紫杉木霉菌株、构建方法及应用 - Google Patents
一株高效生产木霉菌素的紫杉木霉菌株、构建方法及应用 Download PDFInfo
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- CN116286404A CN116286404A CN202310158326.6A CN202310158326A CN116286404A CN 116286404 A CN116286404 A CN 116286404A CN 202310158326 A CN202310158326 A CN 202310158326A CN 116286404 A CN116286404 A CN 116286404A
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Abstract
本发明公开了一株高效生产木霉菌素的紫杉木霉(Trichodermataxi)基因工程菌株,所述紫杉木霉基因工程菌株是过表达了乙酰基转移酶基因Tri3的紫杉木霉基因工程菌株。本发明基因工程菌株过表达乙酰基转移酶基因Tri3,大大提高了木霉菌素产量,紫杉木霉基因工程菌株S1的木霉菌素产量可以达到1.2g/L,相比宿主菌株的0.9g/L提高了33.3%。本发明提高了菌株发酵效价,增加了稳定性,为微生物发酵法制备木霉菌素提供了优良菌种。
Description
技术领域
本发明属于生物工程技术领域,尤其是一株高效生产木霉菌素的紫杉木霉菌株、构建方法及应用。
背景技术
木霉菌素属于典型的单端孢霉烯类化合物,最早发现于20世纪60年代,是绿色木霉(Trichoderma viride)、紫杉木霉(Trichodermataxi)和拟枝孢镰刀菌(Fusariumsprotrichoioides)等真菌的代谢产物,普遍应用于生物防治领域,也有希望成为新型抗肿瘤药物。在生物防治领域,木霉菌素能够影响真核生物蛋白合成的延伸与终止过程,是一种优良的蛋白合成抑制剂,对多种植物病原真菌具有很强的抑制作用,可以用于新型农药的生产。木霉菌素是紫杉木霉的主要生物防治功能因子,具有开发成为一种良好的新型生防制剂的潜力。但是,木霉菌素存在合成网络复杂、调控机制不清晰、合成水平低等问题。
目前,木霉菌素生产途经主要是微生物发酵法。微生物发酵法以可利用再生碳源等优点日益受到重视,但是野生型紫杉木霉菌株发酵效价较低、稳定性较差,严重限制了发酵法生产木霉菌素的工业化进程。因此构建一种高效生产木霉菌素的紫杉木霉菌株,以提高木霉菌素的生产效率十分必要。
通过检索,尚未发现与本发明专利申请相关的公开文献。
发明内容
本发明的目的在于克服现有技术上存在的问题,提供一株高效生产木霉菌素的紫杉木霉菌株、构建方法及应用。
本发明解决技术问题所采用的技术方案是:
一株高效生产木霉菌素的紫杉木霉(Trichodermataxi)基因工程菌株,所述紫杉木霉基因工程菌株是过表达了乙酰基转移酶基因Tri3的紫杉木霉基因工程菌株。
进一步地,所述乙酰基转移酶基因Tri3的基因序列为NCBI-locus_tag为KY860616.1;所述乙酰基转移酶基因Tri3编码的氨基酸序列为SEQ ID NO.2。
如上所述的高效生产木霉菌素的木霉菌素基因工程菌株的构建方法,包括如下步骤:
(1)构建乙酰基转移酶基因Tri3过表达载体
以野生型紫杉木霉TrichodermataxiZJUF0986基因组为模板,通过PCR反应扩增获得基因Tri3的序列片段,回收PCR产物获得目的片段;将基因Tri3的序列片段克隆到载体pLH509(北京华大基因合成,SEQ ID NO.4),构建基因Tri3过表达质粒pLH1541;
(2)乙酰基转移酶基因Tri3过表达菌株的获得
将所述质粒pLH1541转化至紫杉木霉宿主菌株S2530中,经转化子筛选和潮霉素抗性基因重组,获得紫杉木霉乙酰基转移酶基因Tri3过表达菌株S1。
进一步地,所述紫杉木霉宿主菌株S2530的构建方法包括如下步骤:
将Tet-on调控Cre表达的由北京华大基因合成的质粒pLH472(Tet-on-cre,hph,SEQ ID NO.3)转化至紫杉木霉野生型菌株ZJUF0986([1]章初龙,王国平,郑必强,陈绍瑗,林福呈.紫杉木霉ZJUF0986抗真菌活性代谢产物分离提取及其活性研究[J].菌物学报,2008(03):439-446.)中,经转化子筛选和潮霉素抗性基因重组,获得高效生产木霉菌素的紫杉木霉基因工程菌株的宿主菌S2530。因此可以实现对loxP-hph-loxP元件中潮霉素抗性基因hph的重组去除,从而可以反复使用hphmarker实现连续的对多个基因的过表达或敲除。
进一步地,所述紫杉木霉宿主菌株S2530基因组内整合了外源cre基因,该基因受Tet-on系统的调控表达;当以所述菌株S2530为出发菌进行遗传改造,并以loxP-hph-loxP为筛选标记时,能够通过强力霉素启动Tet-on系统表达Cre重组酶,实现对loxP-hph-loxP元件的重组,从而实现应用一个hph marker进行连续的基因过表达或敲除且实现最终目的工程菌株基因组内无外源抗性基因的残留。
如上所述的紫杉木霉基因工程菌株在生产木霉菌素方面中的应用。
利用如上所述的基因工程菌株发酵生产木霉菌素的方法,包括如下步骤:
将紫杉木霉基因工程菌株接种在MEA培养基上,于30℃培养五天,至菌体铺满平板,收集菌丝并将菌丝悬液接种于种子培养基中,28℃恒温180rpm培养48h,将培养物以10%(V/V)的接种量接入发酵培养基,在25℃恒温150rpm培养7天,即得木霉菌素。
进一步地,所述MEA培养基的成分及配制方法为:
葡萄糖10g/L,胰蛋白胨4g/L,酵母浸出物3g/L,麦芽浸出物10g/L,溶剂为水,121℃高压灭菌20min。固体培养基加入1.5%琼脂。
所述种子培养基的成分及配制方法为:
蔗糖10.8g/L,淀粉20g/L,胰蛋白胨0.93g/L,酵母浸出物0.86g/L,七水硫酸镁0.5125g/L,尿素0.5g/L,无水磷酸氢二铵0.73g/L,无水氯化钾0.17g/L,硝酸钠0.12g/L,碳酸钙0.43g/L,溶剂为水,115℃高压灭菌20min;
所述发酵培养基的成分及配制方法为:
葡萄糖23g/L,淀粉15g/L,胰蛋白胨5g/L,酵母浸出物3g/L,七水硫酸镁0.25g/L,酒石酸铵2g/L,无水磷酸二氢钾1g/L,硝酸钙0.4g/L,溶剂为水,115℃高压灭菌20min。
进一步地,所述方法得到的木霉菌素产量为1.2g/L,相比于出发菌株提高了33.3%。
本发明取得的有益效果为:
1、本发明基因工程菌株过表达乙酰基转移酶基因Tri3,大大提高了木霉菌素产量,本发明大大提高了木霉菌素产量,减少了下游分离纯化木霉菌素过程中分离成本,为工业化生产紫杉木霉提供了优良菌株。本发明显著地提高了紫杉木霉发酵生产木霉菌菌素的产量,提高了菌株发酵效价,增加了稳定性,为微生物发酵法制备木霉菌素提供了优良菌种。本发明克服了现有技术中现有紫杉木霉发酵生产木霉菌素过程中,菌株发酵效价较低,稳定性较差,造成后续木霉菌素纯化工艺成本提高且无法进行工业化生产。
2、本发明紫杉木霉菌株能够应用在木霉菌素生产方面中,该菌株于摇瓶发酵条件下7天,通过HPLC分析得知tri3基因过表达菌株S1的木霉菌素产量为1.2g/L,相比宿主菌株的0.9g/L提高了33.3%。
3、本发明中将Tet-on调控Cre表达的由北京华大基因合成的质粒pLH472(Tet-on-cre,hph,SEQ IDNO.3)转化至紫杉木霉野生型菌株ZJUF0986,经转化子筛选和潮霉素抗性基因重组,获得可以用强力霉素诱导表达Cre的高效生产木霉菌素的紫杉木霉基因工程菌株的宿主菌S2530。因此可以实现对loxP-hph-loxP元件中潮霉素抗性基因hph的重组去除,从而可以反复使用hphmarker实现连续的对多个基因的过表达或敲除。
4、本发明所用的宿主菌株是紫杉木霉菌株S2530,高效生产木霉菌素的紫杉木霉基因工程菌株S1是在紫杉木霉菌株S2530的基础上共过表达了乙酰基转移酶基因tri3的紫杉木霉菌株。
5、本发明以紫杉木霉为底盘细胞,通过分析木霉菌素合成网络中的代谢关键点,利用基因组编辑技术和大片段DNA组装技术对木霉菌素合成相关功能模块进行强化及调控,构建高产木霉菌素的细胞工厂,实现木霉菌素的高效绿色生物制造。
附图说明
图1为本发明中使用的含Tet-on-cre元件的质粒pLH472图谱;
图2为本发明中构建的tri3基因过表达质粒pLH1541图谱;
图3为本发明中构建的tri3基因过表达质粒pLH1541双酶切验证图;其中,M为DNAMarker,N为阴性对照,1为EcoRⅠ和KpnⅠ双酶切验证载体;
图4为本发明中Cre-loxP系统PCR验证图,引物P1和P2验证tet-on+cre基因;其中,M为DNA Marker,N为阴性对照,P为阳性对照,1-4为成功过表达cre重组酶紫杉木霉转化子基因组;
图5为本发明中tri3基因PCR验证图,引物P3和P4验证tri3基因;其中,M为DNAMarker,N为阴性对照,P为阳性对照,1-4为成功过表达tri3基因紫杉木霉转化子基因组;
图6为本发明中构建的工程菌株于摇瓶发酵中的木霉菌素产量图;其中,S2530为出发菌株在第7天的木霉菌素产量,S1为tri3基因过表达菌株在第7天的木霉菌素产量。
具体实施方式
为更好理解本发明,下面结合实施例对本发明做进一步地详细说明,但是本发明要求保护的范围并不局限于实施例所表示的范围。
本发明中所使用的原料,如无特殊说明,均为常规市售产品,本发明中所使用的方法,如无特殊说明,均为本领域常规方法,本发明所使用的各物质质量均为常规使用质量。
一株高效生产木霉菌素的紫杉木霉(Trichodermataxi)基因工程菌株,所述紫杉木霉基因工程菌株是过表达了乙酰基转移酶基因Tri3的紫杉木霉基因工程菌株。
较优地,所述乙酰基转移酶基因Tri3的基因序列为NCBI-locus_tag为KY860616.1;所述乙酰基转移酶基因Tri3编码的氨基酸序列为SEQ ID NO.2。
如上所述的高效生产木霉菌素的木霉菌素基因工程菌株的构建方法,包括如下步骤:
(1)构建乙酰基转移酶基因Tri3过表达载体
以野生型紫杉木霉TrichodermataxiZJUF0986基因组为模板,通过PCR反应扩增获得基因Tri3的序列片段,回收PCR产物获得目的片段;将基因Tri3的序列片段克隆到载体pLH509(北京华大基因合成,SEQ ID NO.4),构建基因Tri3过表达质粒pLH1541;
(2)乙酰基转移酶基因Tri3过表达菌株的获得
将所述质粒pLH1541转化至紫杉木霉宿主菌株S2530中,经转化子筛选和潮霉素抗性基因重组,获得紫杉木霉乙酰基转移酶基因Tri3过表达菌株S1;
较优地,所述紫杉木霉宿主菌株S2530的构建方法包括如下步骤:
将Tet-on调控Cre表达的由北京华大基因合成的质粒pLH472(Tet-on-cre,hph,SEQ ID NO.3)转化至紫杉木霉野生型菌株ZJUF0986([1]章初龙,王国平,郑必强,陈绍瑗,林福呈.紫杉木霉ZJUF0986抗真菌活性代谢产物分离提取及其活性研究[J].菌物学报,2008(03):439-446.)中,经转化子筛选和潮霉素抗性基因重组,获得高效生产木霉菌素的紫杉木霉基因工程菌株的宿主菌S2530。因此可以实现对loxP-hph-loxP元件中潮霉素抗性基因hph的重组去除,从而可以反复使用hphmarker实现连续的对多个基因的过表达或敲除。
较优地,所述紫杉木霉宿主菌株S2530基因组内整合了外源cre基因,该基因受Tet-on系统的调控表达;当以所述菌株S2530为出发菌进行遗传改造,并以loxP-hph-loxP为筛选标记时,能够通过强力霉素启动Tet-on系统表达Cre重组酶,实现对loxP-hph-loxP元件的重组,从而实现应用一个hphmarker进行连续的基因过表达或敲除且实现最终目的工程菌株基因组内无外源抗性基因的残留。
如上所述的紫杉木霉基因工程菌株在生产木霉菌素方面中的应用。
利用如上所述的基因工程菌株发酵生产木霉菌素的方法,包括如下步骤:
将紫杉木霉基因工程菌株接种在MEA培养基上,于30℃培养五天,至菌体铺满平板,收集菌丝并将菌丝悬液接种于种子培养基中,28℃恒温180rpm培养48h,将培养物以10%(V/V)的接种量接入发酵培养基,在25℃恒温150rpm培养7天,即得木霉菌素。
较优地,所述MEA培养基的成分及配制方法为:
葡萄糖10g/L,胰蛋白胨4g/L,酵母浸出物3g/L,麦芽浸出物10g/L,溶剂为水,121℃高压灭菌20min。固体培养基加入1.5%琼脂。
所述种子培养基的成分及配制方法为:
蔗糖10.8g/L,淀粉20g/L,胰蛋白胨0.93g/L,酵母浸出物0.86g/L,七水硫酸镁0.5125g/L,尿素0.5g/L,无水磷酸氢二铵0.73g/L,无水氯化钾0.17g/L,硝酸钠0.12g/L,碳酸钙0.43g/L,溶剂为水,115℃高压灭菌20min;
所述发酵培养基的成分及配制方法为:
葡萄糖23g/L,淀粉15g/L,胰蛋白胨5g/L,酵母浸出物3g/L,七水硫酸镁0.25g/L,酒石酸铵2g/L,无水磷酸二氢钾1g/L,硝酸钙0.4g/L,溶剂为水,115℃高压灭菌20min。
较优地,所述方法得到的木霉菌素产量为1.2g/L,相比于出发菌株提高了33.3%。
具体地,相关的制备及检测如下:
实施例1:Cre-loxP遗传操作系统的构建
本实施例包括以下步骤:
以野生型紫杉木霉ZJUF0986为出发菌株,通过PEG介导转化质粒pLH472(北京华大基因合成,SEQ ID NO.3),将由Tet-on调控表达的cre基因整合到野生型紫杉木霉ZJUF0986基因组并将筛选标记hphMarker重组掉后得到菌株S2530。提取S2530基因组,设计P1/P2(表1)为验证引物,经PCR验证Tet-on-cre元件是否整合到基因组,扩增结果满足Tet-on+Cre扩增呈阳性(如图1、图4)。S2530即为构建高效生产木霉菌素的紫杉木霉基因工程菌株的宿主菌。
S2530菌株中Tet-on-cre元件的成功整合,使得S2530菌株在含有强力霉素(doxycycline)的培养基中能够诱导表达Cre重组酶,Cre重组酶可以识别loxP位点,从而将loxP-hph-loxP中的hphmarker重组掉,实现hphmarker的反复使用,进而连续应用同一个hphmarker实现多个基因的过表达或敲除。
所述诱导重组方法为:将约2000个Cre-loxP遗传操作系统过表达克隆的原生质体与4mLSTCBuffer和4mL上层培养基混合均匀后均匀涂布到含有150μg/mL多西环素的下层培养基上,30℃培养至长出单克隆,然后随机挑取100个克隆转接至MEA培养基上30℃培养24h,随后将克隆一一对应地转接到含有潮霉素的MEA培养基上30℃培养72h,最后通过表型观察筛选抗性标记诱导成功的转化子,即在含潮霉素的MEA培养基上不能生长而在MEA培养基上能正常生长的克隆即为即为hphmarker成功诱导重组的转化子。选择其中一个正确的克隆,命名为S2530。该S2530即为含有Cre-loxP遗传操作系统的紫杉木霉基因工程菌株的宿主菌。
实施例2:高效生产木霉菌素的紫杉木霉基因工程菌株的构建
本实施例包括以下步骤:
(1)tri3基因过表达载体的构建:
为扩增tri3基因序列片段,以紫杉木霉基因组为模板设计扩增引物tri3-F和tri3-R,通过PCR扩增回收得到tri3基因序列片段,然后应用诺唯赞C113-ClonExpress-MultiSOneStepCloningKit试剂盒将tri3基因序列同时与经EcoRⅠ/KpnⅠ双酶切线性化后的出发载体pLH509(北京华大基因合成,SEQIDNO.4)进行连接,将连接产物经化转至大肠杆菌JM109感受态细胞中,均匀的涂布在含有100ug/mL卡那霉素抗性的LB固体培养基中,37℃倒置过夜培养,挑取单克隆经菌落PCR验证和提质粒双酶切验证(图3),获得成功连接tri3基因序列片段的载体pLH1541,其图谱如图2所示。
所述扩增引物序列见表1:
表1.所用引物序列
*下划线序列表示酶切位点。
所述tri3基因的氨基酸序列为SEQ ID NO.2,氨基酸个数为519个。
所述含有卡那霉素抗性的LB固体培养基成分为:胰胰蛋白胨10g/L、酵母浸出物5g/L、氯化钠10g/L、琼脂粉15g/L。121℃灭菌20min。灭菌完毕冷却至50℃左右时加入卡那霉素至终浓度100μg/mL。
(2)tri3基因过表达菌株的获得:
首先从平板用棉签将紫杉木霉宿主菌株S2530菌丝体洗下,接种到N15种子培养基中(100μg/mL Amp+100μg/mLKan+0.1%吐温80和5颗玻璃珠),180rpm,28℃培养2d,制得种子液;然后收集种子液中的菌丝体,在无菌研钵中轻轻研磨菌球,取2药匙接种50mLN15液体种子培养基中,180rpm,28℃培养12h后用miracloth过滤获得菌丝体,每瓶菌丝体转移至含有10mL酶解液(15mg/mL崩溃酶+30mg/mL蜗牛酶+30mg/mL溶壁酶)进行酶解,100rpm,30℃酶解两小时;加入等体积STbuffer,用枪头轻轻上下吹打,用2层灭菌纱布过滤掉未酶解的菌丝球,获得原生质体溶液至50mL无菌离心管中,3000rpm,4℃离心10min;弃部分上清后,再用20mLSTbuffer轻轻重悬原生质体沉淀,3000rpm,4℃离心10min;弃部分上清后,加入STCbuffer轻轻重悬原生质体沉淀,3000rpm,4℃离心10min;弃部分上清后将原生质体混悬液转移至10mL离心管中,3000rpm,4℃离心10min;弃上清至200ul-300ul,用枪头轻轻吹打重悬,获得原生质悬液,取200μL原生质体至新的50mLEP管后加入20μg线性化pLH1541质粒片段和680μLPEG溶液,混合均匀后冰浴20min;向其中加入2mLPEG溶液,上下颠倒,室温放置20min;最后向其中加入提前预热混好的4mLSTC和4mL上层培养基,铺板于含有相应抗性的下层培养基上(hyg浓度为40μg/mL)进行培养直至长出转化子,对转化子进行筛选,转化子表型为潮霉素抗性不敏感,对此类转化子提基因组进行验证,设计验证引物P3/P4(表1),扩增结果满足PpkiA+Ttrpc扩增呈阳性(如图5),挑取其中一个正确的且产量最高的tri3过表达克隆进行抗性标记潮霉素的诱导重组从而获得不具有潮霉素抗性的tri3基因过表达菌株,获得紫杉木霉乙酰基转移酶基因Tri3过表达菌株S1。
所述诱导重组方法为:将约2000个tri3基因过表达克隆的原生质体与4mLSTCBuffer和4mL上层培养基混合均匀后均匀涂布到含有150μg/mL多西环素的下层培养基上,30℃培养至长出单克隆,然后随机挑取100个克隆转接至MEA培养基上30℃培养24h,随后将克隆一一对应地转接到含有潮霉素的MEA培养基上30℃培养72h,最后通过表型观察筛选抗性标记诱导成功的转化子,即在含潮霉素的MEA培养基上不能生长而在MEA培养基上能正常生长的克隆即为即为hphmarker成功诱导重组的转化子。
(3)所述基因过表达的转化方法为原生质体介导法。
其中,所述PEG溶液的配置方法为:称取PEG6000120g,加适量蒸馏水溶解后加入提前配置好的1M/L的Tris-Hcl(pH=7.5)2mL和1M/L的CaCl2·2H2O4mL,充分溶解后定容至200mL,121℃灭菌20min。
所述STBuffer溶液的配置方法为:称取D-山梨醇182.18g,加适量蒸馏水溶解后加入提前配置好的1M/L的Tris-Hcl(pH=7.5)10mL,充分溶解后定容至1L,调节pH为7.5后,121℃灭菌20min。
所述STCBuffer溶液的配置方法为:称取D-山梨醇182.18g,加适量蒸馏水溶解后加入提前配置好的1M/L的Tris-Hcl(pH=7.5)10mL和1M/L的CaCl2·2H2O20mL,充分溶解后定容至1L,调节pH为7.5,121℃灭菌20min。
所述上层培养基的配置方法为:称取麦芽提取物15g,胰胰蛋白胨2.5g,D-山梨醇91.09g,加适量蒸馏水充分溶解后定容至500ml,调节pH为7.5后分装于锥形瓶中,瓶中加入1.5%琼脂,121℃灭菌20min。
所述下层培养基的配置方法为:称取麦芽提取物15g,胰胰蛋白胨2.5g,D-山梨醇91.09g,加适量蒸馏水充分溶解后定容至500ml,调节pH为7.5后分装于锥形瓶中,瓶中加入1.5%琼脂,121℃灭菌20min,灭菌完毕冷却至50℃左右时加入潮霉素至终浓度40μg/mL。
所述验证引物序列见表1。
实施例3:工程菌株发酵生产木霉菌素的应用
利用本发明构建的紫杉木霉tri3基因过表达菌株S1于摇瓶发酵产生木霉菌素的方法,其具体步骤如下:
首先,将得到的工程菌株接种在MEA培养平板上并置于30℃培养箱中倒置培养5天直至菌体布满培养平板;
所述MEA培养基的组成为:葡萄糖10g/L,胰蛋白胨4g/L,酵母浸出物3g/L,麦芽浸出物10g/L,溶剂为水,121℃高压灭菌20min。固体培养基加入1.5%琼脂。
然后,收集菌株S1的菌丝体并接种于N15种子培养基中,在28℃,180rpm培养2天。
最后,将5mL种子培养液接种至木霉菌素发酵培养基中,将摇瓶放置于25℃、150rpm条件下培养7天。
所述N15种子培养基的组成为:蔗糖10.8g/L,淀粉20g/L,胰蛋白胨0.93g/L,酵母浸出物0.86g/L,七水硫酸镁0.5125g/L,尿素0.5g/L,无水磷酸氢二铵0.73g/L,无水氯化钾0.17g/L,硝酸钠0.12g/L,碳酸钙0.43g/L,溶剂为水,115℃高压灭菌20min。
所述木霉菌素发酵培养基的组成为:葡萄糖23g/L,淀粉15g/L,胰蛋白胨5g/L,酵母浸出物3g/L,七水硫酸镁0.25g/L,酒石酸铵2g/L,无水磷酸二氢钾1g/L,硝酸钙0.4g/L,溶剂为水,115℃高压灭菌20min。
最后,收集发酵产物,制备检测样品,样品经HPLC测定其中木霉菌素的含量。结果显示,木霉菌素产量明显提高,于出发菌相比有了大大的提高,结果见图6。出发菌S2530木霉菌素产量为0.9g/L,tri3基因过表达菌株S1木霉菌素产量为1.2g/L,相比出发菌株提高了33.3%,有了显著性的提高。
所述检测样品制备方法:吸取振荡摇匀的发酵液2mL,离心后取上清液,用甲醇稀释10倍,经0.22μm有机滤膜过滤后存放于液相小瓶中用于HPLC检测。
所述HPLC检测木霉菌素的方法为:安捷伦高效液相色谱仪UV检测器,COSMOSILC18分析柱(250mm×4.6mm),流动相:乙腈:水=3:2。流速1mL/min,柱温30℃,波长193nm,进样体积为20μL。
本发明的研究成果大大提高了紫杉木霉发酵生产木霉菌素的产量,木霉菌素的产量从宿主菌的0.9g/L提升到了菌株S1的1.2g/L,提高了菌株发酵效价,增加了发酵稳定性,为实现工业化发酵生产木霉菌素奠定了一定的基础并提供了优良菌株。
另外,王国平团队以紫杉木霉野生型菌株ZJUF0986进行紫外诱变获得突变体菌株UL60-11,其木霉菌素产量为632.16mg/L([1]王国平,郑必强,周转忠,章初龙.紫杉木霉突变株UL60-11产木霉菌素的发酵条件优化[J].中国生物防治,2010,26(04):486-491.DOI:10.16409/j.cnki.2095-039x.2010.04.018.),为国内已知最高发酵水平;而本发明中紫杉木霉野生型菌株ZJUF0986进行利用基因组编辑技术和大片段DNA组装技术获得的菌株S1,其木霉菌素产量为1.2g/L,相较于菌株UL60-11提高了89.9%,有着显著的提升。
本发明中使用到的序列如下:
乙酰基转移酶基因Tri3的基因序列:SEQ ID NO.1:
ATGGGCAGCAAACTTCCTGAGCTCCCAAAATTATGCCCAGAGAAGCACAGATGGGAGAAGTCAAAGGTGAATCCACACGTCCTGCAGCGTCAAGGCATAGGCTCCGAGGCTATTGTGGGACTGGAAAAGAGTAATCGGAGAGGACAATACGATCTATATCTCCTCGCAACTCTCCGTACAGCTGATATTTCAACTTCAACGCCGTTGTCTCTTTCGTACTTGAAAGAGAAGTTGGAGTTAGCTTTAATAGTGACGAGATTTGAGCACCCAGAGTGTGCTTGTACCGTGACGTGGGACGACCAGATTTTTCCTATTATACAATACGCGTCTCCTGGGAATGATGAAGACGCTGTTACATGGGCTAAAAGCTCTGTGCATATTCGGACTACTACGAAAACAGGTTTCGATATTCGAAATGAGATAGAAGAAAGACGACTAGAGCTTGACCAGGATGATATGGAGCCAGCCCGCCCGGTTGTGATATACCTTATCTCGGACGTCACCACTGAGAATACACAGCTGAGTAGCGGAGCTACGATTGACGTATTACTACACATGAATCATCTGTTTTGGGATGGAATCAGTTCACGGATGTTTACAGGAGATCTGTTTCGGCAAATGAATAAGCTCATAGTCTCTCCTGAGCAAGAACCACTGAAATTGCATTGGGGAGCAGAAACATCGAATCTAAGCGTTCCTGTACTGGATGCTATGAAAATTAATGTCGAAGAGTTGCGGGAAGAGTTTGATGCCACCAGCGATCAATATGTCAAAGCATTATATGAGAACTACGGAGGCTGGGGTCTCGAGTTCAAATACGGACTGGGATTACCTCGTACAGATATACTTACCTTTACCGCTGTTGAGAGTAAAGCAATTATTAATGCTGTCAAAACTCGCCTTGGGCATCAGTATACCATTTCTCATTTGGCACAGGCGGCAGTCGTGATTGCGATGCTGGAAATCATTCAACCATCCAACCTTTCAGACAAAGACGCTTTTGTGTCACCAATGCCTGTAAATGGCCGAAGATGGTTGAAGGATGAATTAGCAGACAACCATTACAGCATATGCGAAACTGGAGCAGTTATCCGAATCGAAAATATCAAATCTCTTGTCCTCAACAGTAACGATGACAAGAAGACTGTTATTGAGGCGATAAAGAGAGCATGCGAAGATGTTAAAAAGTCCTTTGATAGGTGGATCGGTAACCCTTATCAATTGGCTCTTGGTCTCGCGGTTCACACTTTAGAAGCATCATTCTTAACTGCGAATCCAATGCCCTTTGACAAGGTCGCCGCGCCATTTTTTATAAGCGACGGGAAAAACGAACGATTCATCCCTGCGAGTATCACCTCTGCGACTGGAGAAACCCTTATGACAGTTGATCACTTTTACTTCTTCTTGAACCAGTGTTTGCCGTATTTGGCCATCCGACTCGAGAGCTGGAAGGACGCTTCCACTCTGTCTGTCTGCTACAACGAAGCAAATTATAGTCAAGAAGAGGCAACCAAATTTCTTAAATCTGTCGCCAGCTATATGTTAATCTTCTCCCAATGA
乙酰基转移酶基因Tri3编码的氨基酸序列:SEQ ID NO.2:
MetGlySerLysLeuProGluLeuProLysLeuCysProGluLysHisArgTrpGluLysSerLysValAsnProHisValLeuGlnArgGlnGlyIleGlySerGluAlaIleValGlyLeuGluLysSerAsnArgArgGlyGlnTyrAspLeuTyrLeuLeuAlaThrLeuArgThrAlaAspIleSerThrSerThrProLeuSerLeuSerTyrLeuLysGluLysLeuGluLeuAlaLeuIleValThrArgPheGluHisProGluCysAlaCysThrValThrTrpAspAspGlnIlePheProIleIleGlnTyrAlaSerProGlyAsnAspGluAspAlaValThrTrpAlaLysSerSerVaHisIleArgThrThrThrLysThrGlyPheAspIleArgAsnGluIleGluGluArgArgLeuGluLeuAspGlnAspAspMetGluProAlaArgProValValIleTyrLeuIleSerAspValThrThrGluAsnThrGlnLeuSerSerGlyAlaThrIleAspValLeuLeuHisMetAsnHisLeuPheTrpAspGlyIleSerSerArgMetPheThrGlyAspLeuPheArgGlnMetAsnLysLeuIleValSerProGluGlnGluProLeuLysLeuHisTrpGlyAlaGluThrSerAsnLeuSerValProValLeuAspAlaMetLysIleAsnValGluGluLeuArgGluGluPheAspAlaThrSerAspGlnTyrValLysAlaLeuTyrGluAsnTyrGlyGlyTrpGlyLeuGluPheLysTyrGlyLeuGlyLeuProArgThrAspIleLeuThrPheThrAlaValGluSerLysAlaIleIleAsnAlaValLysThrArgLeuGlyHisGlnTyrThrIleSerHisLeuAlaGlnAlaAlaValValIleAlaMetLeuGluIleIleGlnProSerAsnLeuSerAspLysAspAlaPheValSerProMetProValAsnGlyArgArgTrpLeuLysAspGluLeuAlaAspAsnHisTyrSerIleCysGluThrGlyAlaValIleArgIleGluAsnIleLysSerLeuValLeuAsnSerAsnAspAspLysLysThrValIleGluAlaIleLysArgAlaCysGluAspValLysLysSerPheAspArgTrpIleGlyAsnProTyrGlnLeuAlaLeuGlyLeuAlaValHisThrLeuGluAlaSerPheLeuThrAlaAsnProMetProPheAspLysValAlaAlaProPhePheIleSerAspGlyLysAsnGluArgPheIleProAlaSerIleThrSerAlaThrGlyGluThrLeuMetThrValAspHisPheTyrPhePheLeuAsnGlnCysLeuProTyrLeuAlaIleArgLeuGluSerTrpLysAspAlaSerThrLeuSerValCysTyrAsnGluAlaAsnTyrSerGlnGluGluAlaThrLysPheLeuLysSerValAlaSerTyrMetLeuIlePheSerGln
质粒pLH472:SEQ ID NO.3:
GGGACTAGTCGGAGAATATGGAGCTTCATCGAATCACCGGCAGTAAGCGAAGGAGAATGTGAAGCCAGGGGTGTATAGCCGTCGGCGAAATAGCATGCCATTAACCTAGGTACAGAAGTCCAATTGCTTCCGATCTGGTAAAAGATTCACGAGATAGTACCTTCTCCGAAGTAGGTAGAGCGAGTACCCGGCGCGTAAGCTCCCTAATTGGCCCATCCGGCATCTGTAGGGCGTCCAAATATCGTGCCTCTCCTGCTTTGCCCGGTGTATGAAACCGGAAAGGCCGCTCAGGAGCTGGCCAGCGGCGCAGACCGGGAACACAAGCTGGCAGTCGACCCATCCGGTGCTCTGCACTCGACCTGCTGAGGTCCCTCAGTCCCTGGTAGGCAGCTTTGCCCCGTCTGTCCGCCCGGTGTGTCGGCGGGGTTGACAAGGTCGTTGCGTCAGTCCAACATTTGTTGCCATATTTTCCTGCTCTCCCCACCAGCTGCTCTTTTCTTTTCTCTTTCTTTTCCCATCTTCAGTATATTCATCTTCCCATCCAAGAACCTTTATTTCCCCTAAGTAAGTACTTTGCTACATCCATACTCCATCCTTCCCATCCCTTATTCCTTTGAACCTTTCAGTTCGAGCTTTCCCACTTCATCGCAGCTTGACTAACAGCTACCCCGCTTGAGCAGACATCACCACCATGTCTAGACTGGACAAGAGCAAAGTCATAAACGGCGCTCTGGAATTACTCAATGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGATGCCCTGCCAATCGAGATGCTGGACAGGCATCATACCCACTTCTGCCCCCTGGAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATTCCGCTGTGCTCTCCTCTCACATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGAAACAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTGGAGAACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAGGAACAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCGATTCTATGCCCCCACTTCTGAGACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCCGAACCTGCCTTCCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGCGAAAGCGGCGGGCCGGCCGACGCCCTTGACGATTTTGACTTAGACATGCTCCCAGCCGATGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGACGCTCTTGACGATTTTGACCTTGACATGCTCCCCGGGTAACTAAGTAAACAGCAGAAGAATCTCTCTCCGCTGTTGCTTCAGTGTCTGCCATGCATTAACTTCATCCTACTGTCCTACCCGCAGTACCCATTCACATTTGCGGCAGATACCAGGTTGTTTTCTATTCCCTCGGTTCTTCAGTTCTTCAGATATTACATTACTCGAGAAATTGGCGGCGATGCAGGAGTTTGGTTGGTTTTAATTGTTTCAGTCTCCTTGACCTGATTTGTATGACATGCAATGTTTCGACGGAAGACTATCTCGGTGAATATACGTCATGAATCATGCGTATCACGAGGCCCTTTCGTCTTCACTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGCTCCCCATCTTCAGTATATTCATCTTCCCATCCAAGAACCTTTATTTCCCCTAAGTAAGTACTTTGCTACATCCATACTCCATCCTTCCCATCCCTTATTCCTTTGAACCTTTCAGTTCGAGCTTTCCCACTTCATCGCAGCTTGACTAACAGCTACCCCGCTTGAGCAGACATCACCGAATTCATGTCCAACCTGTTGACCGTCCATCAGAACTTGCCTGCTTTGCCTGTTGACGCTACTTCTGACGAAGTTCGCAAGAACCTCATGGACATGTTCCGCGATAGACAGGCTTTCTCTGAGCATACCTGGAAGATGCTGCTGTCTGTCTGTAGATCTTGGGCTGCTTGGTGTAAGCTGAACAACCGTAAGTGGTTCCCTGCTGAACCTGAAGATGTCAGAGATTACCTGCTGTACTTGCAGGCTAGAGGTTTGGCTGTCAAGACCATCCAGCAACATCTTGGCCAGCTGAACATGCTGCATAGACGTTCTGGTCTGCCTCGTCCTTCTGATTCTAACGCTGTCTCCCTGGTTATGCGTCGTATTCGTAAAGAGAACGTTGACGCTGGAGAAAGAGCTAAGCAAGCTCTCGCTTTCGAGAGAACTGACTTCGATCAGGTCCGCTCTTTGATGGAAAACTCCGATCGTTGCCAGGATATCCGCAATTTGGCTTTCCTGGGTATCGCTTACAACACCCTGTTGCGTATCGCTGAAATTGCTCGCATCCGCGTTAAGGATATCTCTCGTACTGACGGAGGCAGAATGCTTATCCATATCGGCAGAACCAAGACCTTGGTCTCTACTGCTGGCGTTGAAAAGGCTCTGTCTTTGGGAGTCACCAAGTTGGTCGAACGTTGGATCTCTGTCTCTGGAGTTGCTGACGATCCTAACAACTACCTGTTCTGCCGCGTTCGTAAAAACGGAGTTGCTGCTCCTTCTGCTACCTCTCAATTGTCTACTCGCGCTCTGGAAGGTATCTTCGAGGCTACCCATAGACTGATCTACGGAGCTAAGGACGATTCTGGTCAGCGTTACTTGGCTTGGTCTGGTCATTCTGCTCGCGTTGGAGCTGCTCGCGATATGGCTAGAGCTGGAGTCTCTATCCCTGAGATCATGCAAGCTGGAGGTTGGACTAACGTCAACATCGTCATGAACTACATCCGCAACCTGGACTCTGAAACCGGAGCTATGGTCAGACTCTTGGAAGACGGAGATTAACCATGGATAACTTCGTATAATGTATGCTATACGAAGTTATGTCGACGTTAACTGATATTGAAGGAGCATTTTTTGGGCTTGGCTGGAGCTAGTGGAGGTCAACAATGAATGCCTATTTTGGTTTAGTCGTCCAGGCGGTGAGCACAAAATTTGTGTCGTTTGACAAGATGGTTCATTTAGGCAACTGGTCAGATCAGCCCCACTTGTAGCAGTAGCGGCGGCGCTCGAAGTGTGACTCTTATTAGCAGACAGGAACGAGGACATTATTATCATCTGCTGCTTGGTGCACGATAACTTGGTGCGTTTGTCAAGCAAGGTAAGTGGACGACCCGGTCATACCTTCTTAAGTTCGCCCTTCCTCCCTTTATTTCAGATTCAATCTGACTTACCTATTCTACCCAAGCATCCAAATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAGTTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTCCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCATGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCCTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGCCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGAGTAGATGCCGACCGGGAACCAGTTAACGTCGAATAACTTCGTATAATGTATGCTATACGAAGTTATTCTAGAACTAGTGGGCCCCTGCAGGATCCGGTGCAGGCTGCGCAACAGTATCAGAAGGAGGGGTGGGAAGCCTACCTGGCCCGTGTCGGCGGAAACTAATACTATAAAAGGAGGATCGAAGTTCTGATGGTTATGAATGATATAGAAATGCAACTTGCCGCAACGGATACGGAAGCGGAAACGGACCAATGTCGAGCACGGGTAGTCAGACTGCGGCATCGGATGTCCAAACGGTATTGATCCTGCAGGCTACTATGGTGTGGCACAAGGATCAATGCGGTACGACGATTTGATGCAGATAAGCAGGCTGCGAAGTAGTAACTCTTGCGTAGAGAAAATGGCGACGGGTGGCTGATAAGGGCGGTGATAAGCTTAATTGTCATCGCAGATAAGCACTGCTGTCTTGCATCCAAGTCAGCGTCAGCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGCTAGAGCAGCTTGAGCTTGGATCAGATTGTCGTTTCCCGCCTTCAGTTTAAACTATCAGTGTTTGACAGGATATATTGGCGGGTAAACCTAAGAGAAAAGAGCGTTTATTAGAATAACGGATATTTAAAAGGGCGTGAAAAGGTTTATCCGTTCGTCCATTTGTATGTGCATGCCAACCACAGGGTTCCCCTCGGGATCAAAGTACTTTGATCCAACCCCTCCGCTGCTATAGTGCAGTCGGCTTCTGACGTTCAGTGCAGCCGTCTTCTGAAAACGACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCCTGCCCTTTTCCTGGCGTTTTCTTGTCGCGTGTTTTAGTCGCATAAAGTAGAATACTTGCGACTAGAACCGGAGACATTACGCCATGAACAAGAGCGCCGCCGCTGGCCTGCTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTGACCAACCAACGGGCCGAACTGCACGCGGCCGGCTGCACCAAGCTGTTTTCCGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGATGCTTGACCACCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGACCGCCTGGCCCGCAGCACCCGCGACCTACTGGACATTGCCGAGCGCATCCAGGAGGCCGGCGCGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGACACCACCACGCCGGCCGGCCGCATGGTGTTGACCGTGTTCGCCGGCATTGCCGAGTTCGAGCGTTCCCTAATCATCGACCGCACCCGGAGCGGGCGCGAGGCCGCCAAGGCCCGAGGCGTGAAGTTTGGCCCCCGCCCTACCCTCACCCCGGCACAGATCGCGCACGCCCGCGAGCTGATCGACCAGGAAGGCCGCACCGTGAAAGAGGCGGCTGCACTGCTTGGCGTGCATCGCTCGACCCTGTACCGCGCACTTGAGCGCAGCGAGGAAGTGACGCCCACCGAGGCCAGGCGGCGCGGTGCCTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCGGCCGCCGAGAATGAACGCCAAGAGGAACAAGCATGAAACCGCACCAGGACGGCCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGATGATCGCGGCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTGCGGCTGCATGAAATCCTGGCCGGTTTGTCTGATGCCAAGCTGGCGGCCTGGCCGGCCAGCTTGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAAGGTGATGTGTATTTGAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTATATGATGCGATGAGTAAATAAACAAATACGCAAGGGGAACGCATGAAGGTTATCGCTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCGCAACCCATCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGTTCTGTTAGTCGATTCCGATCCCCAGGGCAGTGCCCGCGATTGGGCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGACCGCGACGTGAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTGTGTCCGCGATCAAGGCAGCCGACTTCGTGCTGATTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGCCGACCTGGTGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAAGCGGCCTTTGTCGTGTCGCGGGCGATCAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTGAGTCCCGTATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCAGAACCCGAGGGCGACGCTGCCCGCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAGGTAAAGAGAAAATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGCAACGTTGGCCAGCCTGGCAGACACGCCAGCCATGAAGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACCAAGCTGAAGATGTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAATACATCGCGCAGCTACCAGAGTAAATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAGAACAACCAGGCACCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAGGCGTAAGCGGCTGGGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCCCCAAGCCCGAGGAATCGGCGTGACGGTCGCAAACCATCCGGCCCGGTACAAATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGCAGGCCGCCCAGCGGCAACGCATCGAGGCAGAAGCACGCCCCGGTGAATCGTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAATCCCGGCAACCGCCGGCAGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTTCGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCGCAGCATCATGGACGTGGCCGTTTTCCGTCTGTCGAAGCGTGACCGACGAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTTCCGCAGGGCCGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGGCGGTTTCCCATCTAACCGAATCCATGAACCGATACCGGGAAGGGAAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTACTCAAGTTCTGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTTAAACACCACGCACGTTGCCATGCAGCGTACGAAGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCTTGATTAGCCGCTACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAGTACATCGAGATCGAGCTAGCTGATTGGATGTACCGCGAGATCACAGAAGGCAAGAACCCGGACGTGCTGACGGTTCACCCCGATTACTTTTTGATCGATCCCGGCATCGGCCGTTTTCTCTACCGCCTGGCACGCCGCGCCGCAGGCAAGGCAGAAGCCAGATGGTTGTTCAAGACGATCTACGAACGCAGTGGCAGCGCCGGAGAGTTCAAGAAGTTCTGTTTCACCGTGCGCAAGCTGATCGGGTCAAATGACCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCAGGCTGGCCCGATCCTAGTCATGCGCTACCGCAACCTGATCGAGGGCGAAGCATCCGCCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGCAGGGGAAAAAGGTCGAAAAGGTCTCTTTCCTGTGGATAGCACGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGAACCCGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGTCACACATGTAAGTGACTGATATAAAAGAGAAAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAAACTCTTAAAACCCGCCTGGCCTGTGCATAACTGTCTGGCCAGCGCACAGCCGAAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTGCGCTCCCTACGCCCCGCCGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCGCTCAAAAATGGCTGGCCTACGGCCAGGCAATCTACCAGGGCGCGGACAAGCCGCGCCGTCGCCACTCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGCATTCTAGGTACTAAAACAATTCATCCAGTAAAATATAATATTTTATTTTCTCCCAATCAGGCTTGATCCCCAGTAAGTCAAAAAATAGCTCGACATACTGTTCTTCCCCGATATCCTCCCTGATCGACCGGACGCAGAAGGCAATGTCATACCACTTGTCCGCCCTGCCGCTTCTCCCAAGATCAATAAAGCCACTTACTTTGCCATCTTTCACAAAGATGTTGCTGTCTCCCAGGTCGCCGTGGGAAAAGACAAGTTCCTCTTCGGGCTTTTCCGTCTTTAAAAAATCATACAGCTCGCGCGGATCTTTAAATGGAGTGTCTTCTTCCCAGTTTTCGCAATCCACATCGGCCAGATCGTTATTCAGTAAGTAATCCAATTCGGCTAAGCGGCTGTCTAAGCTATTCGTATAGGGACAATCCGATATGTCGATGGAGTGAAAGAGCCTGATGCACTCCGCATACAGCTCGATAATCTTTTCAGGGCTTTGTTCATCTTCATACTCTTCCGAGCAAAGGACGCCATCGGCCTCACTCATGAGCAGATTGCTCCAGCCATCATGCCGTTCAAAGTGCAGGACCTTTGGAACAGGCAGCTTTCCTTCCAGCCATAGCATCATGTCCTTTTCCCGTTCCACATCATAGGTGGTCCCTTTATACCGGCTGTCCGTCATTTTTAAATATAGGTTTTCATTTTCTCCCACCAGCTTATATACCTTAGCAGGAGACATTCCTTCCGTATCTTTTACGCAGCGGTATTTTTCGATCAGTTTTTTCAATTCCGGTGATATTCTCATTTTAGCCATTTATTATTTCCTTCCTCTTTTCTACAGTATTTAAAGATACCCCAAGAAGCTAATTATAACAAGACGAACTCCAATTCACTGTTCCTTGCATTCTAAAACCTTAAATACCAGAAAACAGCTTTTTCAAAGTTGTTTTCAAAGTTGGCGTATAACATAGTATCGACGGAGCCGATTTTGAAACCGCGGTGATCACAGGCAGCAACGCTCTGTCATCGTTACAATCAACATGCTACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTAACATGAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATCGAGTGGTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGGCTGGCTGGTGGCAGGATATATTGTGGTGTAAACAAATTGACGCTTAGACAACTTAATAACACATTGCGGACGTTTTTAATGTACTGAATTAACGCCGAATTAATTCGGGGGATCTGGATTTTAGTACTGGATTTTGGTTTTAGGAATTAGAAATTTTATTGATAGAAGTATTTTACAAATACAAATACATACTAAGGGTTTCTTATATGCTCAACACATGAGCGAAACCCTATAGGAACCCTAATTCCCTTATCTGGGAACTACTCACACATTATTATGGAGAAACTCGAGGTACCC
载体pLH509:SEQ ID NO.4:
CTAGACTAGTAAATGGAAGAGAAAACCTCCGAGTACTTACTTAGGGTCCCTGTCTACTGACCAGAGTCTCGTCCTCATTACTATGATTAATTACCCACTGGACAAAAAAATAAAATAAAATAAAAATAAAAAGGGAGACAGCTTCTCCATAACTGGCAACTGGGTCCGTCCGAGCAGAGCAAAATTCAGCCTTATGGGTTCCGATGGAGTCAGGGAAATAGTTCTTGCGAAGGGCATTGGGCTTTTTTGCGAGGAGAAAATTCAGCACCGACAAAGCATCCAAATCCACCTCGCTAGGAGAGAATGGATCCGCGACGATGTGGGGTCAACTGGACAGAGTGAGAGGGTATCATGTGGTCCTGCCAGATACTTCGCAGAATGTTGTGTGGGTGTCTGATTGTGGCTTGGGCGTGAATTGCTTTTGGTCTTCCCAACCAATTATTATTGCATGCGGCGTATGAATGCCTGAGATGCGCGGAGGGAAGGTGCCTGAGGATGTAGTGGACAAATGCTGCTGATCGCTGGGCGGAAACCCTTGGCTGACCAGTGAAAAGAGCGGACGGAGGCAGCAGGTGTATCTACGATCAAAGAATAGTAGCAAAGCAGTGAAAGGTGGATCACCCAGCAAATAATTGAGTTTTGATACCCAGCGATAGTGCCGGGGGGGAGAAAAAGTCATTAATAATGGGAATTATGTAGGCGATGGGAAGTGTGATTGTAACTACTCCGTAGCTGGAGGCACAACTAACAAGCCAGCTCTCAACCCGCGGGGAACCGACCGACAGAAAAAAGCGTCCCAAAGCAGGAATCCCACCAAAAAGGGCCGATCCAGCCAATCACCGCCGCCAACATTTTTCCTTCCCGGGCACCCCTCCTCTAGTCCACCATCTCTCTCTTCTCTCGCTCACCGGCCCCGTCTTTTCCTTCCCTATTATCTCTCCCTCTTCTCCTCCCTTCTCTCCCTCCATTCTTTCTCCCATCTTCATCAATCCCTTCTCTTCTGTCTTCCCCCCCGGTTCAGTAGAGATCAATCATCCGTCAAGATGGAATTCAATTCGAGCTCGGTACCCTGCAGAGATCTGGATCCACTTAACGTTACTGAAATCATCAAACAGCTTGACGAATCTGGATATAAGATCGTTGGTGTCGATGTCAGCTCCGGAGTTGAGACAAATGGTGTTCAGGATCTCGATAAGATACGTTCATTTGTCCAAGCAGCAAAGAGTGCCTTCTAGTGATTTAATAGCTCCATGTCAACAAGAATAAAACGCGTTTTCGGGTTTACCTCTTCCAGATACAGCTCATCTGCAATGCATTAATGCATTGACTGCAACCTAGTAACGCCTTNCAGGCTCCGGCGAAGAGAAGAATAGCTTAGCAGAGCTATTTTCATTTTCGGGAGACGAGATCAAGCAGATCAACGGTCGTCAAGAGACCTACGAGACTGAGGAATCCGCTCTTGGCTCCACGCGACTATATATTTGTCTCTAATTGTACTTTGACATGCTCCTCTTCTTTACTCTGATAGCTTGACTATGAAAATTCCGTCACCAGCNCCTGGGTTCGCAAAGATAATTGCATGTTTCTTCCTTGAACTCTCAAGCCTACAGGACACACATTCATCGTAGGTATAAACCTCGAAATCANTTCCTACTAAGATGGTATACAATAGTAACCATGCATGGTTGCCTAGTGAATGCTCCGTAACACCCAATACGCCGGCCGAAACTTTTTTACAACTCTCCTATGAGTCGTTTACCCAGAATGCACAGGTACACTTGTTTAGAGGTAATCCTTCTTTCTAGACCCGGGGGGCCCTACGTAATAACTTCGTATAATGTATGCTATACGAAGTTATGTCGACGTTAACTGATATTGAAGGAGCATTTTTTGGGCTTGGCTGGAGCTAGTGGAGGTCAACAATGAATGCCTATTTTGGTTTAGTCGTCCAGGCGGTGAGCACAAAATTTGTGTCGTTTGACAAGATGGTTCATTTAGGCAACTGGTCAGATCAGCCCCACTTGTAGCAGTAGCGGCGGCGCTCGAAGTGTGACTCTTATTAGCAGACAGGAACGAGGACATTATTATCATCTGCTGCTTGGTGCACGATAACTTGGTGCGTTTGTCAAGCAAGGTAAGTGGACGACCCGGTCATACCTTCTTAAGTTCGCCCTTCCTCCCTTTATTTCAGATTCAATCTGACTTACCTATTCTACCCAAGCATCCAAATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAGTTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTCCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCATGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCCTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGCCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGAGTAGATGCCGACCGGGAACCAGTTAACGTCGAATAACTTCGTATAATGTATGCTATACGAAGTTATAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGCTAGAGCAGCTTGAGCTTGGATCAGATTGTCGTTTCCCGCCTTCAGTTTAAACTATCAGTGTTTGACAGGATATATTGGCGGGTAAACCTAAGAGAAAAGAGCGTTTATTAGAATAACGGATATTTAAAAGGGCGTGAAAAGGTTTATCCGTTCGTCCATTTGTATGTGCATGCCAACCACAGGGTTCCCCTCGGGATCAAAGTACTTTGATCCAACCCCTCCGCTGCTATAGTGCAGTCGGCTTCTGACGTTCAGTGCAGCCGTCTTCTGAAAACGACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCCTGCCCTTTTCCTGGCGTTTTCTTGTCGCGTGTTTTAGTCGCATAAAGTAGAATACTTGCGACTAGAACCGGAGACATTACGCCATGAACAAGAGCGCCGCCGCTGGCCTGCTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTGACCAACCAACGGGCCGAACTGCACGCGGCCGGCTGCACCAAGCTGTTTTCCGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGATGCTTGACCACCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGACCGCCTGGCCCGCAGCACCCGCGACCTACTGGACATTGCCGAGCGCATCCAGGAGGCCGGCGCGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGACACCACCACGCCGGCCGGCCGCATGGTGTTGACCGTGTTCGCCGGCATTGCCGAGTTCGAGCGTTCCCTAATCATCGACCGCACCCGGAGCGGGCGCGAGGCCGCCAAGGCCCGAGGCGTGAAGTTTGGCCCCCGCCCTACCCTCACCCCGGCACAGATCGCGCACGCCCGCGAGCTGATCGACCAGGAAGGCCGCACCGTGAAAGAGGCGGCTGCACTGCTTGGCGTGCATCGCTCGACCCTGTACCGCGCACTTGAGCGCAGCGAGGAAGTGACGCCCACCGAGGCCAGGCGGCGCGGTGCCTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCGGCCGCCGAGAATGAACGCCAAGAGGAACAAGCATGAAACCGCACCAGGACGGCCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGATGATCGCGGCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTGCGGCTGCATGAAATCCTGGCCGGTTTGTCTGATGCCAAGCTGGCGGCCTGGCCGGCCAGCTTGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAAGGTGATGTGTATTTGAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTATATGATGCGATGAGTAAATAAACAAATACGCAAGGGGAACGCATGAAGGTTATCGCTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCGCAACCCATCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGTTCTGTTAGTCGATTCCGATCCCCAGGGCAGTGCCCGCGATTGGGCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGACCGCGACGTGAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTGTGTCCGCGATCAAGGCAGCCGACTTCGTGCTGATTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGCCGACCTGGTGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAAGCGGCCTTTGTCGTGTCGCGGGCGATCAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTGAGTCCCGTATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCAGAACCCGAGGGCGACGCTGCCCGCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAGGTAAAGAGAAAATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGCAACGTTGGCCAGCCTGGCAGACACGCCAGCCATGAAGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACCAAGCTGAAGATGTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAATACATCGCGCAGCTACCAGAGTAAATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAGAACAACCAGGCACCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAGGCGTAAGCGGCTGGGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCCCCAAGCCCGAGGAATCGGCGTGACGGTCGCAAACCATCCGGCCCGGTACAAATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGCAGGCCGCCCAGCGGCAACGCATCGAGGCAGAAGCACGCCCCGGTGAATCGTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAATCCCGGCAACCGCCGGCAGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTTCGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCGCAGCATCATGGACGTGGCCGTTTTCCGTCTGTCGAAGCGTGACCGACGAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTTCCGCAGGGCCGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGGCGGTTTCCCATCTAACCGAATCCATGAACCGATACCGGGAAGGGAAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTACTCAAGTTCTGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTTAAACACCACGCACGTTGCCATGCAGCGTACGAAGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCTTGATTAGCCGCTACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAGTACATCGAGATCGAGCTAGCTGATTGGATGTACCGCGAGATCACAGAAGGCAAGAACCCGGACGTGCTGACGGTTCACCCCGATTACTTTTTGATCGATCCCGGCATCGGCCGTTTTCTCTACCGCCTGGCACGCCGCGCCGCAGGCAAGGCAGAAGCCAGATGGTTGTTCAAGACGATCTACGAACGCAGTGGCAGCGCCGGAGAGTTCAAGAAGTTCTGTTTCACCGTGCGCAAGCTGATCGGGTCAAATGACCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCAGGCTGGCCCGATCCTAGTCATGCGCTACCGCAACCTGATCGAGGGCGAAGCATCCGCCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGCAGGGGAAAAAGGTCGAAAAGGTCTCTTTCCTGTGGATAGCACGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGAACCCGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGTCACACATGTAAGTGACTGATATAAAAGAGAAAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAAACTCTTAAAACCCGCCTGGCCTGTGCATAACTGTCTGGCCAGCGCACAGCCGAAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTGCGCTCCCTACGCCCCGCCGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCGCTCAAAAATGGCTGGCCTACGGCCAGGCAATCTACCAGGGCGCGGACAAGCCGCGCCGTCGCCACTCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGCATTCTAGGTACTAAAACAATTCATCCAGTAAAATATAATATTTTATTTTCTCCCAATCAGGCTTGATCCCCAGTAAGTCAAAAAATAGCTCGACATACTGTTCTTCCCCGATATCCTCCCTGATCGACCGGACGCAGAAGGCAATGTCATACCACTTGTCCGCCCTGCCGCTTCTCCCAAGATCAATAAAGCCACTTACTTTGCCATCTTTCACAAAGATGTTGCTGTCTCCCAGGTCGCCGTGGGAAAAGACAAGTTCCTCTTCGGGCTTTTCCGTCTTTAAAAAATCATACAGCTCGCGCGGATCTTTAAATGGAGTGTCTTCTTCCCAGTTTTCGCAATCCACATCGGCCAGATCGTTATTCAGTAAGTAATCCAATTCGGCTAAGCGGCTGTCTAAGCTATTCGTATAGGGACAATCCGATATGTCGATGGAGTGAAAGAGCCTGATGCACTCCGCATACAGCTCGATAATCTTTTCAGGGCTTTGTTCATCTTCATACTCTTCCGAGCAAAGGACGCCATCGGCCTCACTCATGAGCAGATTGCTCCAGCCATCATGCCGTTCAAAGTGCAGGACCTTTGGAACAGGCAGCTTTCCTTCCAGCCATAGCATCATGTCCTTTTCCCGTTCCACATCATAGGTGGTCCCTTTATACCGGCTGTCCGTCATTTTTAAATATAGGTTTTCATTTTCTCCCACCAGCTTATATACCTTAGCAGGAGACATTCCTTCCGTATCTTTTACGCAGCGGTATTTTTCGATCAGTTTTTTCAATTCCGGTGATATTCTCATTTTAGCCATTTATTATTTCCTTCCTCTTTTCTACAGTATTTAAAGATACCCCAAGAAGCTAATTATAACAAGACGAACTCCAATTCACTGTTCCTTGCATTCTAAAACCTTAAATACCAGAAAACAGCTTTTTCAAAGTTGTTTTCAAAGTTGGCGTATAACATAGTATCGACGGAGCCGATTTTGAAACCGCGGTGATCACAGGCAGCAACGCTCTGTCATCGTTACAATCAACATGCTACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTAACATGAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATCGAGTGGTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGGCTGGCTGGTGGCAGGATATATTGTGGTGTAAACAAATTGACGCTTAGACAACTTAATAACACATTGCGGACGTTTTTAATGTACTGAATTAACGCCGAATTAATTCGGGGGATCTGGATTTTAGTACTGGATTTTGGTTTTAGGAATTAGAAATTTTATTGATAGAAGTATTTTACAAATACAAATACATACTAAGGGTTTCTTATATGCTCAACACATGAGCGAAACCCTATAGGAACCCTAATTCCCTTATCTGGGAACTACTCACACATTATTATGGAGAAACTCGAGA
尽管为说明目的公开了本发明的实施例,但是本领域的技术人员可以理解:在不脱离本发明及所附权利要求的精神和范围内,各种替换、变化和修改都是可能的,因此,本发明的范围不局限于实施例所公开的内容。
Claims (9)
1.一株高效生产木霉菌素的紫杉木霉(Trichoderma taxi)基因工程菌株,其特征在于:所述紫杉木霉基因工程菌株是过表达了乙酰基转移酶基因Tri3的紫杉木霉基因工程菌株。
2.根据权利要求1所述的高效生产木霉菌素的紫杉木霉基因工程菌株,其特征在于:所述乙酰基转移酶基因Tri3的基因序列为NCBI-locus_tag为KY860616.1;所述乙酰基转移酶基因Tri3编码的氨基酸序列为SEQ ID NO.2。
3.如权利要求1或2所述的高效生产木霉菌素的木霉菌素基因工程菌株的构建方法,其特征在于:包括如下步骤:
(1)构建乙酰基转移酶基因Tri3过表达载体
以野生型紫杉木霉Trichoderma taxi ZJUF0986基因组为模板,通过PCR反应扩增获得基因Tri3的序列片段,回收PCR产物获得目的片段;将基因Tri3的序列片段克隆到载体pLH509,构建基因Tri3过表达质粒pLH1541;
(2)乙酰基转移酶基因Tri3过表达菌株的获得
将所述质粒pLH1541转化至紫杉木霉宿主菌株S2530中,经转化子筛选和潮霉素抗性基因重组,获得紫杉木霉乙酰基转移酶基因Tri3过表达菌株S1。
4.根据权利要求3所述的构建方法,其特征在于:所述紫杉木霉宿主菌株S2530的构建方法包括如下步骤:
将Tet-on调控Cre表达的质粒pLH472转化至紫杉木霉野生型菌株ZJUF0986中,经转化子筛选和潮霉素抗性基因重组,获得高效生产木霉菌素的紫杉木霉基因工程菌株的宿主菌S2530。
5.根据权利要求3所述的构建方法,其特征在于:所述紫杉木霉宿主菌株S2530基因组内整合了外源cre基因,该基因受Tet-on系统的调控表达;当以所述菌株S2530为出发菌进行遗传改造,并以loxP-hph-loxP为筛选标记时,能够通过强力霉素启动Tet-on系统表达Cre重组酶,实现对loxP-hph-loxP元件的重组,从而实现应用一个hph marker进行连续的基因过表达或敲除且实现最终目的工程菌株基因组内无外源抗性基因的残留。
6.如权利要求1或2所述的紫杉木霉基因工程菌株在生产木霉菌素方面中的应用。
7.利用如权利要求1或2所述的基因工程菌株发酵生产木霉菌素的方法,其特征在于:包括如下步骤:
将紫杉木霉基因工程菌株接种在MEA培养基上,于30℃培养五天,至菌体铺满平板,收集菌丝并将菌丝悬液接种于种子培养基中,28℃恒温180rpm培养48h,将培养物以10%(V/V)的接种量接入发酵培养基,在25℃恒温150rpm培养7天,即得木霉菌素。
8.根据权利要求7所述的方法,其特征在于:
所述MEA培养基的成分及配制方法为:
葡萄糖10g/L,胰蛋白胨4g/L,酵母浸出物3g/L,麦芽浸出物10g/L,溶剂为水,121℃高压灭菌20min;固体培养基加入1.5%琼脂。
所述种子培养基的成分及配制方法为:
蔗糖10.8g/L,淀粉20g/L,胰蛋白胨0.93g/L,酵母浸出物0.86g/L,七水硫酸镁0.5125g/L,尿素0.5g/L,无水磷酸氢二铵0.73g/L,无水氯化钾0.17g/L,硝酸钠0.12g/L,碳酸钙0.43g/L,溶剂为水,115℃高压灭菌20min;
所述发酵培养基的成分及配制方法为:
葡萄糖23g/L,淀粉15g/L,胰蛋白胨5g/L,酵母浸出物3g/L,七水硫酸镁0.25g/L,酒石酸铵2g/L,无水磷酸二氢钾1g/L,硝酸钙0.4g/L,溶剂为水,115℃高压灭菌20min。
9.根据权利要求8或9所述的方法,其特征在于:所述方法得到的木霉菌素产量为1.2g/L,相比于出发菌株提高了33.3%。
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