CN117264776A - 一株产黄酮醇的拟棘孢曲霉菌及其基因编辑体系的构建方法 - Google Patents
一株产黄酮醇的拟棘孢曲霉菌及其基因编辑体系的构建方法 Download PDFInfo
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- CN117264776A CN117264776A CN202310865477.5A CN202310865477A CN117264776A CN 117264776 A CN117264776 A CN 117264776A CN 202310865477 A CN202310865477 A CN 202310865477A CN 117264776 A CN117264776 A CN 117264776A
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Abstract
本发明公开了一种拟棘孢曲霉(Aspergillusaculeatinus)SF1菌株,该菌株已于2023年7月7日保藏于中国典型培养物保藏中心保藏,保藏编号:CCTCCNO.M20231229。本发明还公开了利用上述拟棘孢曲霉SF1菌株制备黄酮醇。本发明还公开了上述拟棘孢曲霉SF1菌株基因编辑体系的构建方法。本发明提供的拟棘孢曲霉菌SF1的培养周期短,培养基成分简单,成本低,能够在短时间内快速高效发酵生产黄酮类(黄酮醇)物质。本发明提供的拟棘孢曲霉菌SF1也可成功制备出原生质体且能通过CRISPR基因编辑技术进行菌株改造。
Description
技术领域
本发明涉及微生物技术领域,具体涉及一株产黄酮醇的拟棘孢曲霉菌及其基因编辑体系的构建方法。
背景技术
黄酮代表一类高度多样化的次生植物代谢物,迄今为止已鉴定出约9000种结构。这些化合物存在于所有维管植物以及一些苔藓中。即使在相同的物种中,也可能会出现许多不同的类黄酮。已经确定黄酮对植物生物学的各个方面都有重大影响。它们在生理学、生物化学和生态学中表现出广泛的功能,例如紫外线防护、花色、种间相互作用和植物防御。
某些黄酮的其他非常显着的特性是它们的营养价值和对人类的药用价值,其中包括抗氧化或抗癌活性。如黄酮醇,黄酮醇类化合物可能是水果和蔬菜中最常见和最大的黄酮亚群,主要包括山奈酚、槲皮素、杨梅素、芦丁等,槲皮素是黄酮醇的典型代表,广泛存在于水果、蔬菜和谷物等植物中。槲皮素在山楂、苹果、梨、柑橘水果中和可作为中药的槐花、银杏叶等较为丰富。茶和红酒也是黄酮醇的来源。流行病学和动物研究表明,饮食中大量摄入黄酮醇可能与降低患几种癌症(例如肺癌和结肠癌)、冠心病、慢性炎症和骨质疏松症的风险有关。
曲霉类真菌是工业微生物与合成生物学的重要底盘细胞与研究模式生物,曲霉属可用于生产纤维素酶,糖类,小分子物质,以及高价值的生物碱,黄酮,紫杉醇前体等物质。虽然专利号为CN102277304B的专利中公开了棘孢曲霉菌产5,7,8,4'-四羟基异黄酮,但其为异黄酮,不是黄酮醇。异黄酮类化合物是黄酮类化合物的一个独特的亚群,异黄酮的分布十分有限,几乎局限于大豆和一些豆科植物中。
然而除了少数曲霉类模式真菌,大多数曲霉菌仍然难以通过CRISPR基因编辑技术进行菌株改造,大大限制了该菌种的潜在经济价值,而进行基因编辑改造的前提条件是能够高质量的获取真菌原生质体,真菌的细胞壁成分复杂,结构坚固,难以获得高质量原生质体,而研究中普遍使用的sigma公司产的L1412溶壁酶已停产,急需新的原生质体酶解液制备方法。
如名为“Two novel species of Aspergillus section Nigri from Thaicoffee beans”文章公开了拟棘孢曲霉菌是单列种,形态与棘孢曲霉菌(双列种)相似,但产的分生孢子较小(2~5μm)。本发明拟得到一种能产黄酮醇且能通过CRISPR基因编辑技术进行菌株改造的拟棘孢曲霉菌。
发明内容
鉴于目前存在的上述不足,本发明提供一株产黄酮醇的拟棘孢曲霉菌及其基因编辑体系的构建方法,本发明的拟棘孢曲霉菌能产黄酮醇且能通过CRISPR基因编辑技术进行菌株改造。
为了达到上述目的,本发明提供了一种拟棘孢曲霉(Aspergillus aculeatinus)SF1菌株,该菌株的保藏编号是CCTCC NO.M 20231229。
基于同一发明构思,本发明还提供了一种如上述拟棘孢曲霉的应用,所述应用为利用上述拟棘孢曲霉制备黄酮醇。
依照本发明的一个方面,述应用包括以下步骤:
步骤1:将拟棘孢曲霉SF1菌液加入到液体培养基中培养,获得发酵液;
步骤2:将发酵液置于细胞破碎仪上破碎,得到菌体完全破碎开的发酵液;
步骤3:将破碎处理后的发酵液采用有机溶剂提取、浓缩,获得黄酮醇浓缩液。
依照本发明的一个方面,在步骤1中,拟棘孢曲霉SF1菌液的加入量为液体培养基的1-2wt%,液体培养基为PDB液体培养基,培养时间为7-10d,培养温度为28-30℃,培养的转速为180-200rpm。
依照本发明的一个方面,在步骤2中,破碎的温度为28-30℃,破碎采用的变幅杆的末端直径为6mm,破碎总时长为20min,其中,每超声2s,间隔3s。
依照本发明的一个方面,所述步骤3具体为:
步骤31:将破碎处理后的发酵液与乙酸乙酯混合,静置放置后,分离得到上层有机相,备用;
步骤32:将初提取后分离了上层有机相的发酵液再次与乙酸乙酯混合,静置后取上层有机相,将两次提取收集到的有机相混合;
步骤33:将提取收集到的有机相浓缩,得到黄酮醇浓缩液。
基于同一发明构思,本发明还提供了一种基于上述拟棘孢曲霉SF1菌株基因编辑体系的构建方法,包括以下步骤:
步骤a:将拟棘孢曲霉SF1采用组合酶酶解液酶解,制备成原生质体;其中,组合酶酶解液为3%溶壁酶+2%yatalase酶溶液+1%纤维素酶酶解液;
步骤b:向原生质体中加入质粒p19ACas9g,经混匀、静置、加PEG缓冲液、混匀、静置;其中,质粒p19ACas9g由pUC19质粒与真菌质粒克隆片段AMA1加Cas9基因与sgRNA片段构建而成;
步骤c:加入STC溶液后,再将体系与上层等渗筛选MM培养基混匀,铺在含有潮霉素的下层等渗筛选MM培养基平板上,待上层培养基凝固后,用封口膜封口,37℃恒温静置培养2天。
依照本发明的一个方面,步骤a具体为:
步骤a1:将拟棘孢曲霉SF1接种到100mL PDB液体培养基中,30℃培养5-7天;
步骤a2:使用0.1%吐温80溶液冲洗菌体表面,收集孢子,然后离心,用无菌水洗涤2次;接种孢子至100ml PDB液体培养基,控制孢子浓度为108,37℃,180rpm,6.5h;
步骤a3:待孢子发芽至体积2至3倍长度,8000rpm,4℃,15min,离心收集置于无菌三角锥瓶中,悬浮于经过滤除菌的10mL 3%溶壁酶+2%yatalase酶溶液+1%纤维素酶酶解液中,进行酶解;
步骤a4:将上述溶液放于恒温震荡培养箱中,设置30℃培养,转速为90rpm,培养2.5-4h后,每隔半小时观察一次;当在显微镜下观察到原生质体酶解好后,使用1.2M山梨醇溶液冲洗,并将上述溶液用falcon细胞滤网过滤,除去残余的菌丝体,收集原生质体液;
步骤a5:将上述原生质体液置于离心机里,5000rpm,室温,10min;离心后,去上清,获得原生质体。
依照本发明的一个方面,所述STC溶液:1.2M山梨醇,50mM氯化钙,50mM Tris-HCl;所述上层等渗筛选MM培养基:1.2M山梨醇,20×Salts溶液2%,1000×trace elements溶液0.1%,葡萄糖1%,琼脂1%,补充100ug/ml潮霉素,pH自然值,115℃灭菌20min;所述下层等渗筛选MM培养基:1.2M山梨醇,20×Salts溶液2%,1000×trace elements溶液0.1%,葡萄糖1%,琼脂1.5%,补充100ug/ml潮霉素,pH自然值,115℃灭菌20min。
依照本发明的一个方面,所述PDB液体培养基:马铃薯提取物20%,葡萄糖2%,pH自然值,115℃灭菌20min。
本发明的有益效果:
(1)本发明提供的拟棘孢曲霉菌Aspergillus aculeatinus SF1培养周期短,培养基成分简单,成本低,能够在短时间内快速高效发酵生产黄酮类(黄酮醇)物质。
(2)本发明提供的拟棘孢曲霉菌Aspergillus aculeatinus SF1菌株可成功制备出原生质体,且能通过CRISPR基因编辑技术进行菌株改造,因此存在潜在经济价值。
附图说明
图1(a)为本发明实施例1所述的拟棘孢曲霉SF1菌株在PDA平板培养基上生长形成的菌落情况;图1(b)本发明实施例2所述的拟棘孢曲霉SF1菌株在PDB液体培养基上生长形成的菌落情况;
图2为本发明实施例1所述的拟棘孢曲霉SF1菌株的系统发育树;
图3为本发明实施例2所述的拟棘孢曲霉菌SF1发酵液萃取后的甲醇洗液的色谱图;
图4为本发明实施例3所述的拟棘孢曲霉SF1菌株的原生质体的电镜图;
图5为本发明实施例3所述的菌落PCR产物的凝胶成像图。
具体实施方式
为使本发明更加容易理解,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,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。除非另有定义,下文所用专业术语和本领域专业技术人员所理解的含义一致;除非特殊说明,本文所涉及的原料、试剂均可从市场购买,或通过公知的方法制得。
术语:
菌落PCR(Colony PCR)可不必提取目的基因DNA,不必酶切鉴定,而是直接以菌体热解后暴露的DNA为模板进行PCR扩增,使用引物来筛选阳性克隆。通常利用此pcr的方法进行筛选插入的目的基因或者DNA测序分析。最后的PCR产物大小是引物之间的片断大小。
实施例1
一种拟棘孢曲霉菌Aspergillus aculeatinus SF1的分离纯化及鉴定
所述的拟棘孢曲霉菌Aspergillus aculeatinus SF1是从湖南省长沙市岳麓山银杏根中分离得到的,具体步骤如下:
取适量湖南省长沙市岳麓山银杏根样品用流水冲洗表面的泥土和杂质,将冲洗干净的银杏根置于超声清洗仪中超声清洗(160W,15min),进一步去除表面残留的泥沙和其他有机物质。在超净工作台内,经无菌水漂洗过的银杏根用75%的酒精消毒30s,用无菌水反复冲洗5次;再用4%次氯酸钠浸泡3min,立即用无菌水冲洗5次。平铺在含有无菌滤纸的平板中,无菌滤纸吸干表面水分。分别取10-4、10-5、10-6三个梯度的稀释液100μL均匀涂布在PDA平板培养基上,所述PDA平板培养基的组成为:马铃薯粉6.0g/L;葡萄糖20.0g/L;氯霉素0.1g/L;琼脂20.0g/L;所述PDA平板培养基在25℃下的pH为5.8-6.2。在30℃条件下培养3d,得到一个棕褐色单菌落(见图1(a))。挑选单菌落进行多次划线分离纯菌,最后将分离到的单菌用30%的灭菌甘油保存在-80℃冰箱中。
从新鲜固体培养基上挑取绿豆大小的上述菌株菌体到离心管内,用真菌基因组提取试剂盒提取菌株DNA,使用ITS通用引物ITS1和ITS4进行PCR扩增,其中,ITS1的引物序列如SEQ ID NO:1所示,具体为:5’-TCCGTAGGTGAACCTGCGG-3’,ITS4的引物序列如SEQ ID NO:2所示,具体为:5’-TCCTCCGCTTATTGATATGC-3’。PCR产物由生工生物工程(上海)股份有限公司进行测序,测序得到的ITS DNA序列并使用Bioedit软件拼接数据,然后上传到NCBI数据库(https://www.ncbi.nlm.nih.gov/),进行BLAST序列比对,并进行同源性分析,使用MEGAX软件按邻接法构建系统发育树,结果如图2所示。结果表明上述菌株与Aspergillusaculeatinus CBS 121060的序列有100%的同源性。
上述拟棘孢曲霉SF1于2023年7月7日送交中国典型培养物保藏中心保藏,保藏单位地址:武汉大学;分类命名:拟棘孢曲霉SF1(Aspergillus aculeatinus SF1),保藏编号:CCTCC NO.M 20231229。
实施例2
一种利用拟棘孢曲霉菌Aspergillus aculeatinus SF1制备黄酮醇,具体步骤如下:
发酵:本实验发酵所用培养基为PDB培养基,从保藏编号为CCTCC M 20231229的菌种保藏管中挑取适量菌液涂布于PDA平板培养基上,在30℃恒温培养箱中培养3-5d,得到单菌落。挑取单菌落,置于PDB液体培养基(马铃薯提取物20%,葡萄糖2%,pH自然值,115℃灭菌20min)中,在30℃、200rpm条件下培养4d得到对数生长期的菌液。再将菌液按1%的接种量接种到PDB培养基中,培养7d得到发酵液,培养温度为28℃,转速为180rpm。菌株Aspergillus aculeatinus SF1在PDB液体培养基中的形态如图1(b)所示,其在液体培养基中培养至生长稳定期时呈现出浅黄色。
发酵液萃取:将200mL发酵液置于细胞破碎仪上,设置总超声时间为2h,其中程序为超声2s间歇3s得到菌体完全破碎的发酵液,以释放可能存在于菌体细胞内的目标物质;将破碎处理后的发酵液与乙酸乙酯按1:1等比例混合,摇晃使其充分接触,静置放置后,分离得到下层有机相;将经过初提取后的发酵液与乙酸乙酯再次按2:1的比例充分混合,静置后取上层有机相。将两次提取收集到的有机相混合;将300mL乙酸乙酯萃取液进行旋蒸处理,用5mL甲醇分三次(1、2、2mL)清洗旋蒸馏瓶,收集5mL甲醇洗液,浓缩后的甲醇洗液过0.22μm滤膜后吸取适量置于棕色高效液相试样瓶中。
标准样品的制备:精确称取3,5,7,4’四羟基-8-甲氧基黄酮(sexangularetin)分别0.020g,用甲醇溶解并定容至50mL,配置成浓度为0.2mg/mL的母液。将母液分别稀释,配置成一系列浓度的黄酮标准品的标准溶液,浓度系列为0.100mg/mL、0.050mg/mL、0.025mg/mL、0.0125mg/mL、0.00625mg/mL。将标准溶液过0.22μm滤膜后置于棕色高效液相试样瓶中(-20℃保存)。
检测条件设置如下:自动进样器设置为10μL的进样体积,总流速1.0mL/min(A:B=1:1);色谱柱柱温为35℃;检测器设置检测波长为360nm;提取洗脱程序设置为0-10min停止。流动相A:乙腈,流动相B:双蒸水;色谱柱:YMC-TriartC18(250mm×4.6mm,5μm)。
清洗色谱柱:测定样品前使用A、B相清洗色谱柱半小时以上,直至无峰出现。
将上述甲醇洗液与标准品进行HPLC检测,检测结果如图3所示。
图3是拟棘孢曲霉菌Aspergillus aculeatinus SF1发酵液萃取后的甲醇洗液的色谱图,在图中可以看到在7.5分钟左右有明显的出峰,表示该菌发酵产3,5,7,4’四羟基-8-甲氧基黄酮(sexangularetin);经计算,发酵7d后,拟棘孢曲霉菌Aspergillusaculeatinus SF1实验组的3,5,7,4’四羟基-8-甲氧基黄酮(sexangularetin)产率为5.21mg/L,具有天然产3,5,7,4’四羟基-8-甲氧基黄酮(sexangularetin)的能力,用于3,5,7,4’四羟基-8-甲氧基黄酮(sexangularetin)的发酵具有可行性。
实施例3
一种拟棘孢曲霉菌SF1菌株基因编辑体系的构建方法
(1)准备菌株与培养基:
菌株:拟棘孢曲霉菌SF1;
培养基:
PDB液体培养基:马铃薯提取物20%,葡萄糖2%,pH自然值,115℃灭菌20min;
上层等渗筛选MM培养基:1.2M山梨醇,20×Salts溶液2%,1000×trace elements溶液0.1%,葡萄糖1%,琼脂1%,补充100ug/ml潮霉素,pH自然值,115℃灭菌20min;
下层等渗筛选MM培养基:1.2M山梨醇,20×Salts溶液2%,1000×trace elements溶液0.1%,葡萄糖1%,琼脂1.5%,补充100ug/ml潮霉素,pH自然值,115℃灭菌20min;
STC溶液:1.2M山梨醇,50mM氯化钙,50mM Tris-HCl。
(2)质粒与DNA
所用质粒p19ACas9g由pUC19质粒与真菌质粒克隆片段AMA1加Cas9基因与sgRNA片段构建而成,以潮霉素基因为同源插入片段。
(3)拟棘孢曲霉菌原生质体的制备
1)将新鲜的活化菌株SF1接种到100mL PDB液体培养基中,30℃培养5-7天;
2)使用0.1%吐温80溶液冲洗菌体表面,收集孢子,然后离心,用无菌水洗涤2次;
3)接种孢子至100mlPDB液体培养基,控制孢子浓度为10的8次方,37℃,180rpm,6.5h;
4)待孢子发芽至体积2至3倍长度,8000rpm,4℃,15min,离心收集置于无菌三角锥瓶中,悬浮于经过滤除菌的10mL 3%溶壁酶+2%yatalase酶溶液+1%纤维素酶酶解液中,进行酶解;
5)将上述溶液放于恒温震荡培养箱中,设置30℃培养,转速为90rpm,培养2.5-4h后,每隔半小时观察一次;
6)在显微镜下观察到原生质体酶解好后,使用1.2M山梨醇溶液冲洗;
7)将上述溶液用falcon细胞滤网过滤,除去残余的菌丝体,收集原生质体液;
8)将上述原生质体液置于离心机里,5000rpm,室温,10min;
9)离心后,去上清,将原生质体悬浮于STC溶液调整浓度为107/ml,将得到的原生质体在电镜下进行观察,其电镜图如图4所示,由图4可知,拟棘孢曲霉菌SF1菌株可成功制备出原生质体。
(4)拟棘孢曲霉菌原生质体的转化
1)向100uL原生质体中加入10ug质粒,吹打混匀,冰上静置50min;
2)加入1.5mLPEG缓冲液,轻轻摇晃混匀后室温静置20min;
3)加入4mL STC溶液,再将体系与上层等渗筛选MM培养基混匀,铺在含有100ug/ml潮霉素的下层等渗筛选MM培养基平板上,待上层培养基凝固后,用封口膜封口,37℃恒温静置培养2天。
(5)转化子的PCR鉴定和测序验证
1)将上述去除了细胞壁的拟棘孢曲霉SF1的原生质体在等渗筛选MM培养基(上层为等渗筛选MM培养基,下层为下层等渗筛选MM培养基)中培养2天,恢复成一个小菌落,用无菌竹签蘸取培养2天的上述菌体,在筛选培养基(等渗筛选MM培养基)上划线,再次培养2天。
2)刮取少量生长的菌丝体,置于0.5mLEP管中,加入20ul无菌水,混匀后,90℃水浴5min,冷却后作为模板。
以上述菌丝体为模板,靶点基因两端序列设计引物进行PCR扩增,扩增片段约5000bp。
hph-F:5’-CATCGCCGAGTGAGTAGGTC-3’SEQ ID NO:3
hph-R:5’-TGCCCCCTGATCCCAATTTC-3’SEQ ID NO:4
PCR体系:
PCR反应程序:
循环32次
1%琼脂糖凝胶电泳检测PCR产物,并使用紫外凝胶成像系统观察并拍照保存。其凝胶成像结果如图5所示,由图5可知,可以得到菌落PCR产物,说明拟棘孢曲霉菌SF1菌株基因编辑体系的构建成功。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本领域技术的技术人员在本发明公开的技术范围内,可轻易想到的变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以所述权利要求的保护范围为准。
Claims (10)
1.一种拟棘孢曲霉(Aspergillusaculeatinus)SF1菌株,其特征在于,该菌株的保藏编号是CCTCCNO.M20231229。
2.一种如权利要求1所述拟棘孢曲霉的应用,其特征在于,所述应用为利用权利要求1所述的拟棘孢曲霉SF1菌株制备黄酮醇。
3.根据权利要求2所述的应用,其特征在于,所述应用包括以下步骤:
步骤1:将拟棘孢曲霉SF1菌液加入到液体培养基中培养,获得发酵液;
步骤2:将发酵液置于细胞破碎仪上破碎,得到菌体完全破碎开的发酵液;
步骤3:将破碎处理后的发酵液采用有机溶剂提取、浓缩,获得黄酮醇浓缩液。
4.根据权利要求3所述的应用,其特征在于,在步骤1中,拟棘孢曲霉SF1菌液的加入量为液体培养基的1-2wt%,液体培养基为PDB液体培养基,培养时间为7-10d,培养温度为28-30℃,培养的转速为180-200rpm。
5.根据权利要求3所述的应用,其特征在于,在步骤2中,破碎的温度为28-30℃,破碎采用的变幅杆的末端直径为6mm,破碎总时长为20min,其中,每超声2s,间隔3s。
6.根据权利要求3所述的应用,其特征在于,所述步骤3具体为:
步骤31:将破碎处理后的发酵液与乙酸乙酯混合,静置放置后,分离得到上层有机相,备用;
步骤32:将初提取后分离了上层有机相的发酵液再次与乙酸乙酯混合,静置后取上层有机相,将两次提取收集到的有机相混合;
步骤33:将提取收集到的有机相浓缩,得到黄酮醇浓缩液。
7.一种如权利要求1所述的拟棘孢曲霉SF1菌株基因编辑体系的构建方法,其特征在于,包括以下步骤:
步骤a:将拟棘孢曲霉SF1采用组合酶酶解液酶解,制备成原生质体;其中,组合酶酶解液为3%溶壁酶+2%yatalase酶溶液+1%纤维素酶酶解液;
步骤b:向原生质体中加入质粒p19ACas9g,经混匀、静置、加PEG缓冲液、混匀、静置;其中,质粒p19ACas9g由pUC19质粒与真菌质粒克隆片段AMA1加Cas9基因与sgRNA片段构建而成;
步骤c:加入STC溶液后,再将体系与上层等渗筛选MM培养基混匀,铺在含有潮霉素的下层等渗筛选MM培养基平板上,待上层培养基凝固后,用封口膜封口,37℃恒温静置培养2天。
8.根据权利要求7所述的拟棘孢曲霉SF1菌株基因编辑体系的构建方法,其特征在于,步骤a具体为:
步骤a1:将拟棘孢曲霉SF1接种到100mLPDB液体培养基中,30℃培养5-7天;
步骤a2:使用0.1%吐温80溶液冲洗菌体表面,收集孢子,然后离心,用无菌水洗涤2次;接种孢子至100mlPDB液体培养基,控制孢子浓度为108,37℃,180rpm,6.5h;
步骤a3:待孢子发芽至体积2至3倍长度,8000rpm,4℃,15min,离心收集置于无菌三角锥瓶中,悬浮于经过滤除菌的10mL3%溶壁酶+2%yatalase酶溶液+1%纤维素酶酶解液中,进行酶解;
步骤a4:将上述溶液放于恒温震荡培养箱中,设置30℃培养,转速为90rpm,培养2.5-4h后,每隔半小时观察一次;当在显微镜下观察到原生质体酶解好后,使用1.2M山梨醇溶液冲洗,并将上述溶液用falcon细胞滤网过滤,除去残余的菌丝体,收集原生质体液;
步骤a5:将上述原生质体液置于离心机里,5000rpm,室温,10min;离心后,去上清,获得原生质体。
9.根据权利要求7所述的拟棘孢曲霉SF1菌株基因编辑体系的构建方法,其特征在于,所述STC溶液:1.2M山梨醇,50mM氯化钙,50mMTris-HCl;所述上层等渗筛选MM培养基:1.2M山梨醇,20×Salts溶液2%,1000×traceelements溶液0.1%,葡萄糖1%,琼脂1%,补充100ug/ml潮霉素,pH自然值,115℃灭菌20min;所述下层等渗筛选MM培养基:1.2M山梨醇,20×Salts溶液2%,1000×traceelements溶液0.1%,葡萄糖1%,琼脂1.5%,补充100ug/ml潮霉素,pH自然值,115℃灭菌20min。
10.根据权利要求8所述的拟棘孢曲霉SF1菌株基因编辑体系的构建方法,其特征在于,所述PDB液体培养基:马铃薯提取物20%,葡萄糖2%,pH自然值,115℃灭菌20min。
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