CN106010989A - 一种灵芝酸高产工程菌株kmust-Crz - Google Patents
一种灵芝酸高产工程菌株kmust-Crz Download PDFInfo
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- CN106010989A CN106010989A CN201610342738.5A CN201610342738A CN106010989A CN 106010989 A CN106010989 A CN 106010989A CN 201610342738 A CN201610342738 A CN 201610342738A CN 106010989 A CN106010989 A CN 106010989A
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Abstract
本发明公开一种灵芝酸高产工程菌株kmust‑Crz，其在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC NO.12235本发明提供的灵芝工程菌，是通过选用灵芝强启动子P‑gpd表达钙离子依赖的锌指蛋白调控因子Crz基因，得到高产灵芝酸灵芝工程菌kmust‑Crz；通过摇瓶发酵实验表明，该菌在其细胞生长不受影响的情况下，Crz转化子菌株产单体灵芝酸GA‑Me，GA‑T，GA‑Mk，GA‑S分别是WT菌株的3.1，3.2，2.1，3.6倍，因此，本高产菌株可以作为生产灵芝酸的工程菌株，具有广泛的应用前景。
Description
技术领域
本发明属于基因工程和代谢工程领域的，具体涉及一种通过基因工程在灵芝中表达钙离子依赖的锌指蛋白转录因子 (Calcineurin-regulated zine-finger factor,Crz)基因的方法，构建了一个灵芝酸的高产工程菌株。
背景技术
灵芝 ( Ganodermalucidum ) 是担子菌纲，多孔菌科，灵芝属真菌。灵芝真菌在我国有20多种，包括赤芝，黄芝，紫芝，黑芝，薄盖灵芝，树舌等。我国应用灵芝作为药物已有两千多年的历史。灵芝对于增强人体免疫力，调节血糖，控制血压，辅助肿瘤放化疗，保肝护肝，促进睡眠等方面均具有显著疗效。由于其特殊的要用价值，灵芝的有效成分分析和药理学研究已经引起了国际上的广泛关注，尤其在日本，美国，韩国等国家。目前，灵芝的研究已经深入到了分子水平，一些专著相继出版，从不同的角度介绍了灵芝的生物学特性、栽培技术、药理学作用及临床应用等情况。
灵芝酸是一类分子结构中含有羧基的三萜类物质，从结构来看它属于高度氧化的羊毛甾烷衍生物。自1982年Kubota T等人首次分离得到灵芝酸以来，目前已有130多种灵芝酸被分离出来。它们多为四环三萜类化合物，含有30个碳原子，结构中一般都有羟基，在IR中有较强的羟基吸收峰。在紫外光谱中也呈现多个波长的特征吸收，多数是在250 nm、237nm、365 nm处有吸收峰。灵芝酸具有许多重要的药理活性如：抗癌，灵芝酸类物质能明显抑制小鼠肝肉瘤 ( HTC ) 细胞的增殖；护肝，灵芝中的灵芝酸提取物能加强其解毒作用；抗HIV；降血压；抗氧化；抑制血小板凝集，抑制组胺释放，镇痛，抑制真核细胞DNA多聚酶活性，抑制法尼基蛋白转移酶活性和促进体液免疫功能等作用。
随着人们生活水平的提高，各种疾病的发生率也逐年递增。于是，人们对健康的关注也越来越来广泛。灵芝酸作为灵芝中的重要活性成分，对提高人体免疫力，抗氧化等方面具有重要的地位，尤其是在治疗癌症方面表现尤为突出。
由于野生灵芝生长周期长，受环境因素影响大且野生灵芝资源有限，灵芝菌丝培养成为生产活性物质的重要方法。随着对灵芝活性物质需求的不断增加，如何提高灵芝的产量和增加灵芝的有效成分越来越引起人们的关注。液体深层发酵技术是进行快速工业化生产的重要手段。现在市售的灵芝酸主要是从灵芝子实体和液体深层发酵得到的菌丝体中获得。由于野生灵芝液体深层发酵时灵芝酸在灵芝细胞中的含量较低，分离纯化也较难，限制了灵芝酸的活性及作用机理研究及其广泛应用。近年来，基因工程与代谢工程迅速发展，成为了现代分子育种的重要手段。通过分子克隆和基因拼接等分子生物学方法来改造菌株的基因组，从而提高活性成分的含量越来越受学者们的青睐。
灵芝酸合成途径首先是由乙酰辅酶A ( Acetyl CoA ) 在硫解酶 ( AcetoacetylCoA thiolase ) 催化下缩合形成乙酰乙酰辅酶A ( Acetoacetyl CoA )，乙酰乙酰辅酶A经HMG-CoA合成酶 ( HMG-CoA synthase ) 催化与另一分子乙酰CoA缩合生成3-羟基-3甲基-戊二酸单酰辅酶A ( HMG-CoA )，然后HMG-CoA还原酶 ( HMG-CoA reductase, HMGR )催化HMG-CoA转化为甲羟戊酸。甲羟戊酸经甲羟戊酸激酶 ( MVK )、甲羟戊酸5-磷酸激酶( PMK ) 和MDD三步酶促反应转化为IPP。IPP进一步转化成法呢酯焦磷酸FPP。FPP 在鲨烯合酶squalene synthase ( SQS ) 的催化下合成鲨烯SQS后经过鲨烯单加氧酶的作用产生鲨烯2, 3-氧化物，再经过羊毛甾醇合酶 ( LS ) 的作用产生羊毛甾醇。羊毛甾醇又经过一系列氧化还原反应生成灵芝酸。
钙调磷脂酶信号是重要的钙离子信号之一。Crz 是钙调磷酸酶信号途径上的一个可以被激活的转录因子。转录因子Crz上有一个锌指序列，可以结合DNA,并且特异性地结合到钙调磷脂酶依赖的反应原件CDRZ上。钙调磷脂酶通过对细胞质内的转录因子Crz去磷酸化使Crz快速地从细胞质转移到细胞核，从而促进相关基因的表达，进而对次级代谢物的生物合成进行调控。
我们以前通过比较转录组的方法筛选了灵芝酸高产（液体静置培养）/低产（液体震荡培养）培养条件下差异表达的基因，发现Crz基因在灵芝酸高产条件下有显著地上调。早期的研究表明在灵芝细胞液体静置培养过程中外源添加钙离子能显著提高菌丝体中灵芝酸的含量，添加钙离子同时诱导了灵芝酸生物合成基因HMGR, SQS, LS的高表达，钙离子信号传导途径上Crz基因的高表达。以上结果表明灵芝酸的高产可能与Crz基因的过量表达相关。因此，对Crz基因进行过量表达有可能会提高灵芝细胞生产灵芝酸的能力。
在本研究中，我们率先在灵芝细胞中高表达了灵芝的Crz基因，获得了Crz基因过量表达的灵芝工程菌株，工程菌株生产灵芝酸的水平有显著提高。
发明内容
本发明的目的是解决野生型灵芝菌株自身产灵芝酸较低的问题，提供一种灵芝酸高产菌株，该菌株为高产灵芝酸的灵芝 ( Ganoderma lucidum ) 工程菌kmust-Crz，已于2016年3月21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，地址：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，保藏编号为CGMCC NO.12235。
本发明灵芝酸高产工程菌株的构建方法如下：
以PMD19-T为起始载体并使用灵芝本身的强启动子P-gpd、终止子T-sdhB、cbx基因，cbx基因 ( 具有carboxin抗性 ) 是来自灵芝本身的抗性基因，其特点是在蘑菇类担子菌的转化体系中同源标记基因的转化效率更高，能够稳定遗传，抗性强。
1、将灵芝启动子P-gpd与终止子T-sdhB与PMD19-T连接，将灵芝cbx抗性基因插入到Pst I酶切位点，从而构建成pJW-EXP载体；其具有灵芝的强启动子和终止子，并具有灵芝本身的抗性；
其中扩增灵芝强启动子P-gpd 的引物序列为：
P-gpd -F：5’-TCCAAAGCCGCTCTCATGGCATGGCAC- 3’，
P-gpd -R：5’-GCTAGCGTTGAGAGGGGATGAAGAGTGAGTAAGAAG- 3’；
扩增灵芝sdhB 基因终止子的引物序列为：
T-sdhB -F: 5’-ATGAGCGGGTCAGAGAGT- 3’，
T-sdhB -R : 5’-TGCTCTATGTCTTGCCTTGT- 3’；
扩增灵芝cbx 抗性基因的引物为：
cbx -F：5’-TCTGCTCTTCCCGATTGCTGCATTTGT-3’，
cbx -R：5’-CTATGTCTTGCCTTGTCTCGCGTCAACC-3’；
2、灵芝的Crz基因从灵芝基因组中克隆 ( 所用的引物为Crz-Nhe-F和Crz-Sma-R ) ，并插入到pJW-EXP载体中，得到pJW-EXP-Crz载体；引物序列为：
Crz-Nhe-F：5’-ATGGAAGACCGCAGTGCGT- 3’
Crz-Sma-R：5’-TCAGCTTGTCGCGCTCCGA- 3’；
3、通过PEG介导原生质体融合的方法，将pJW-EXP-Crz转化到野生型灵芝细胞中，以carboxin作为抗性来筛选灵芝转化子，在含有carboxin抗性的CYM平板上筛选出转化子；
4、将转化子在carboxin抗性的CYM平板中进行传代培养；
5、灵芝细胞的液体培养：
PDA培养基 ( g/L )：葡萄糖10，琼脂20 ，硫酸镁 1.5 ，磷酸二氢钾 3，维生素B1 0.05和制备好的土豆汁；
土豆汁的制备方法：将200 g去皮新鲜土豆切成小块，加入去离子水1.0 L煮沸30 min，用八层纱布过滤，取滤液，用于PDA培养基的配制；
种子培养基( g/L )：葡萄糖35，蛋白胨5，酵母膏2.5，磷酸二氢钾1，硫酸镁0.5和维生素B1 0.05。
发酵培养基( g/L )：蛋白胨5、酵母膏5、磷酸二氢钾1.0、硫酸镁0.5、维生素B10.05，乳糖35，起始pH 5.5。
CYM培养基( g/L )：葡萄糖 20，麦芽糖10，酵母粉 2，蛋白胨 2，MgSO4 0.5，KH2PO44.6，琼脂 10。
斜面培养：接种菌丝于土豆汁-葡萄糖-琼脂 ( PDA ) 斜面中28℃培养5-7天。
一级种子培养：在250 mL摇瓶中添加40 mL培养基和10 mL菌丝体悬液 ( 从一支斜面中获得 ) ，并在30 ℃，120 rpm下培养5天。
二级种子培养：在250 mL摇瓶中加入45 mL培养基和5 mL一级种子培养液 ( 大约500 mg DW/L，一级培养物用玻璃珠打碎后接种 ) ，在30℃，120 rpm下培养2天。
发酵培养：在250 mL摇瓶中加入45 mL发酵培养基和5 mL二级种子发酵液 ( 大约500~600 mg DW/L，二级培养物用玻璃珠打碎后接种 ) ，在30℃，120 rpm下培养。
6、单体灵芝酸的分离和提取
准确称取干细胞粉末100 mg，加入3 mL 70%（v/v）乙醇浸泡过夜，超声处理3次，每次30min。10000 rpm离心5 min后获得上清液。在50℃烘箱中烘干。用200μL色谱级甲醇彻底溶解，用0.22μm滤膜过滤。用HPLC检测灵芝酸单体。HPLC检测条件为：HPLC色谱柱为C18柱（Agilent 1200 series，5 μm Agilent Zorbax SB-C18 column，250×4.6 mm）；进样量 20μL；流速 1 mL/min；流动相A为甲醇/乙酸（100:0.5（v/v）），流动相B为超纯水， 0-20 min：A相为80 %-100 %等梯度洗脱；20 min-30 min：A相为100 %洗脱；30 min-35 min：A相为80 %洗脱；紫外检测波长为245 nm；洗脱时间35 min。记录相应的峰面积和出峰时间。按照标准曲线计算出各个灵芝酸单体的浓度和含量。
本发明的优点和技术效果：通过摇瓶发酵实验表明，该灵芝酸高产工程菌株在其发酵性能不受影响的情况下（培养温度、培养基组成、接种量和培养方式相同的条件下），Crz转化子菌株产单体灵芝酸GA-Me，GA-T，GA-Mk，GA-S分别是WT菌株的3.1，3.2，2.1，3.6倍，在同等情况下，使用本发明构建的工程菌株可以节约劳动力，缩短生产周期，降低成产成本。因此，本高产菌株可以作为生产灵芝酸的工程菌株，适用于工业化生产，具有广泛的应用前景。
附图说明
图1为本发明中灵芝基因组；图中：G为灵芝基因组，M为DNA Marker；
图2为本发明中pJW-EXP载体结构示意图；
图3为本发明中扩增Crz基因的电泳图；图中：M为DL10000的核酸标准品 ( Takara ) ；C为Crz基因；
图4为本发明中pJW-EXP-Crz载体结构示意图；
图5为本发明中验证Crz阳性转化子的电泳图；图中：M为DL2000的核酸标准品 (Takara ) ，C为转Crz基因的阳性转化子，WT为野生型菌株，N为阴性对照，P为pJW-EXP-Crz质粒。
具体实施方式
下面通过实施例对本发明作进一步详细说明，但本发明的内容并不局限于此，本实施例中方法如无特殊说明的均按常规方法操作，所用试剂如无特殊说明的采用常规试剂或按常规方法配置的试剂。
实施例1：pJW-EXP载体的构建
1、灵芝基因组DNA的提取
称取约0.2 g冻干野生型灵芝 ( CCGMC 5.0616 ) 的菌丝体在液氮中研磨成粉末，将粉末转入1.5 mL经65℃预热的CTAB ( 十六烷基三甲基溴化铵 ) 抽取缓冲液中，65℃保温30 min，然后在 4℃下，10000 g离心20min，取上清液加等体积的氯仿：异戊醇 ( 24：1 )的混合物，轻轻摇匀30 min以上，4℃下、10000 g离心20min；将上清液移入1.5 mL离心管后，加入2/3体积经-20℃预冷的异丙醇，轻轻摇动5min，用玻璃棒捞出DNA后，用75%的乙醇洗涤2-3次，室温凉干后，溶于适量含20g/mL RNase的TE，37℃消化RNA 30 min后即可到灵芝基因组DNA。琼脂糖凝胶电泳结果显示：灵芝的基因组为单一条带，且大小在10000bp以上( 见图1 ) 。
2、灵芝强启动子的克隆
以灵芝基因组DNA为模板，用P-gpd -F、P-gpd -R作为引物进行PCR扩增，扩增得到灵芝的启动子P-gpd，引物序列如下：
P-gpd -F：5’-TCCAAAGCCGCTCTCATGGCATGGCAC- 3’，
P-gpd -R：5’-GCTAGCGTTGAGAGGGGGATGAAGAGTGAGTAAGAAG-3’；
PCR条件如下：95℃ 10min， 95℃ 30s，55℃ 30s，72℃ 90s，72℃ 10min。
3、启动子P-gpd 与PMD19-T连接
将克隆后的启动子P-gpd 进行胶回收，将回收产物与PMD19-T用T4连接酶在16℃进行连接，得到PMD19-T-P中间载体。
4、灵芝终止子的克隆
以灵芝基因组DNA为模板，用引物
T-sdhB -F：5’-ATGAGCGGGTCAGAGAGT- 3’
T-sdhB -R：5’-TGCTCTATGTCTTGCCTTGT- 3’
进行扩增，得到2480bp的序列；PCR条件为：95℃ 10min， 95℃ 30s，55℃ 30s，72℃150s，72℃ 10min。
5、终止子T-sdhB 与PMD19-T-P连接
将克隆后的终止子T-sdhB 胶回收，把PMD19-T-P中间载体用SacI单酶切，PCR回收酶切产物，再用T4聚合酶补平酶切位点，然后再用T4连接酶在16℃进行平末端连接，得到PMD19-T-P-T中间载体。
6、cbx 基因的克隆
以灵芝基因组DNA为模板，用引物
cbx -F：5’-TCTGCTCTTCCCGATTGCTGCATTTGT-3’
cbx -R：5’-CTATGTCTTGCCTTGTCTCGCGTCAACC-3’ 进行PCR得到灵芝的sdhB基因；PCR条件为：95℃ 10min， 95℃ 30s，55℃ 30s，72℃ 90s，72℃ 10min；再设计一对定点突变引物：
sdhB -MR：5'-GAAGATCGTGAGGCAGCGGTATAGGC-3' ( 其中A为突变位点 )
sdhB -MF：5'-GCCTATACCGCTGCCTCACGATCTTC-3' ( 其中T为突变位点 )。
第一轮PCR用引物cbx -F和sdhB -MR进行扩增，PCR条件为：95℃ 10min， 95℃30s，58℃ 30s，72℃ 2min20s，72℃ 10min，得到片段sdhB1；第二轮PCR用引物cbx-R和sdhB-MF进行扩增，PCR条件为：95℃ 10min， 95℃ 30s，55℃ 30s，72℃ 1min20s，72℃10min，得到片段sdhB2；获得相应片段后，采用重叠PCR的方法把两个片段连接。第三轮重叠PCR的引物为cbx -F 和cbx -R，扩增条件为：94 ℃变性10 min，66 ℃退火30s，72 ℃延伸4min，共35个循环，最后72 ℃延伸10 min。最终获得了3171 bp突变的灵芝sdhB基因序列 (定点突变后对carboxin抗生素产生抗性，我们把定点突变后的sdhB基因定义为cbx ) 。经序列测定后确认相应位点已经突变。
7、将突变的cbx基因插入到PMD19-T-P-T的Pst I酶切位点
将PMD19-T-P-T载体用Pst I酶在37℃下，单酶切2h，回收酶切后的片段，用T4聚合酶补平酶切位点，再用T4连接酶在16℃下把cbx基因与回收后的片段进行连接，即得到pJW-EXP载体 ( 见图2 ) 。
实施例2：pJW-EXP-Crz载体的构建
1、Crz基因的克隆
以灵芝基因组DNA为模板，用引物
Crz-Nhe-F：5’-ATGGAAGACCGCAGTGCGT- 3’
Crz-Sma-R：5’-TCAGCTTGTCGCGCTCCGA- 3’
进行PCR得到Crz基因，其核苷酸序列如SEQ ID NO:1所示，编码如SEQ ID NO:2所示的钙离子依赖的锌指蛋白调控因子；PCR条件为：95℃ 10min， 95℃ 30s，60℃ 30s， 72℃150s，72℃ 10min ( 见图3 ) 。
2、将Crz基因插入到pJW-EXP载体
将pJW-EXP载体用SmaI和NheI双酶切，回收酶切后的片段，用T4连接酶在16℃下，将Crz基因插入到pJW-EXP载体的NheI和SmaI之间即得到pJW-EXP-Crz载体 ( 见图4 ) 。
实施例3：通过PEG介导原生质体融合的方法，将pJW-EXP-Crz转化到野生型灵芝细胞中
1、灵芝原生质体的制备及转化
先用溶壁酶将野生型灵芝菌丝制备成原生质体，然后将灵芝原生质体悬浮在100μL的STC ( 0.55 M的山梨醇，10 mM的CaCl2，10 mM的Tris-HCl缓冲液，pH为7.5 ) 中，再加入1μg的质粒DNA和PTC缓冲液 ( 60%的PEG4000 ( W/V ) ，10 mM的Tris-HCl缓冲液，pH为7.5，50 mM的CaCl2 ) ；在冰上培养10min，再加入1mL的PTC缓冲液混合均匀并在室温培养20min；用10mL融化的CYM固体培养基与转化后的原生质体混合倒平板并加入carboxin使carboxin的终浓度为2 mg/L；在30℃下培养10天后可长出若干单菌落。
2、在含有carboxin抗性的平板上传代培养
把单菌落转移到含有2 mg/L的carboxin的CYM平板中，在30℃下传代培养约7天；经过3次在抗性平板上传代可获得稳定的转化子，用CTAB法提取转化后的灵芝基因组，具体操作步骤见实施例1步骤1。分别以转化后的灵芝基因组、野生型 ( WT ) 灵芝基因组、水 ( 阴性对照 ) 为模板，用
Crz-F：5’-GAGTGACGCAGGTGGTGA- 3’
Crz-R：5’-TAGGAATGGGGAGAAGGA- 3’
作为验证引物进行PCR，PCR条件为：95℃ 10min，95℃ 30s，60℃ 30s，72℃90s，72℃10min ( 见图5 )。以Crz-F 和Crz-R 为引物和基因组DNA为模板进行PCR，能够扩增出约1393bp条带的菌株即可认为是导入了Crz基因的转基因灵芝。如图5所示，从转基因菌株上能够扩增出约1393bp的条带，而野生型中没有出现此条带，只出现了一些非特异性条带。结果表明：Crz基因确实整合到了灵芝基因组上。
3、斜面培养
将阳性转化子转移到固体PDA培养基中 ( 含有2 mg/L的carboxin ) ，在28℃下培养5-7天。
4、一级种子培养
在250 mL摇瓶中添加40 mL种子培养基和10 mL菌丝体悬液 ( 从一支斜面中获得 ) ，并在28℃，120 rpm下培养5天。
5、二级种子培养
在250 mL摇瓶中加入45 mL种子培养基和5 mL一级种子培养液 ( 大约500 mg DW/L，一级培养物用玻璃珠打碎后接种 ) ，在28℃，120 rpm下培养2天。
6、发酵培养
在250 mL摇瓶中加入45 mL发酵培养基和5 mL二级种子发酵液 ( 大约500~600 mgDW/L，二级培养物用玻璃珠打碎后接种 ) ，在28℃，120 rpm下培养。
7、单体灵芝酸的分离和提取
准确称取干细胞粉末100 mg，加入3 mL 70%（v/v）乙醇浸泡过夜，超声处理3次，每次30min。10000 rpm离心5 min后获得上清液。在50℃烘箱中烘干。用200μL色谱级甲醇彻底溶解，用0.22μm滤膜过滤。用HPLC检测灵芝酸单体。HPLC检测条件为：HPLC色谱柱为C18柱（Agilent 1200 series，5 μm Agilent Zorbax SB-C18 column，250×4.6 mm）；进样量 20μL；流速 1 mL/min；流动相A为甲醇/乙酸（100:0.5（v/v）），流动相B为超纯水， 0-20 min：A相为80 %-100 %等梯度洗脱；20 min-30 min：A相为100 %洗脱；30 min-35 min：A相为80 %洗脱；紫外检测波长为245 nm；洗脱时间35 min。记录相应的峰面积和出峰时间。
通过野生型和Crz菌株灵芝酸的含量的测定，结果表明：通过摇瓶发酵实验表明，该菌在其发酵性能不受影响的情况下，Crz转化子菌株产单体灵芝酸GA-Me，GA-T，GA-Mk，GA-S分别是WT菌株的3.1，3.7，2.9，3.6倍，因此，本高产菌株可以作为生产灵芝酸的工程菌株，具有广泛的应用前景。
序列表
<110>昆明理工大学
<120>一种灵芝酸高产工程菌株kmust-Crz
<160>14
<170>PatentIn version 3.5
<210>1
<211>2480
<212>DNA
<213> 灵芝菌株CCGMC 5.0616
<400> 1
atggaagacc gcagtgcgtg tgcggtcacc ggtggctcag ctgtcgcgtg cgtgcaaagg 60
gcgatcggcc tgtcgatgtc gtaagtgcca tgcggtgccg cgggttggtg cattatccct 120
tcacgatgct gtgccgcgac gtcgaccggg acgcgttgcg tgcccggtgt tgctgggggg 180
gaagcttgga atgttctgtg ctcgctactg tacttggagt gaggagagcg tgagaagcgg 240
ggtgccggag gaagaagatt tccgacattg acgcgacggg ggacggacgg tgcccggcga 300
gaagggtggg ccggagaccg aggtggacct tgaagcggga tcgtagcggc gcgtcggagt 360
ggctgatgca ggaatgctgt agtgctatat agcttggcgg gctatcgaat cgagaacctg 420
caagtaaaca gtcgactcca agtatgcgct gaccgctagg tctgagggta tgaaacgaga 480
gccaagcaag aaaaaaaaga atcaggatta gtccgtatgt agttgctaga aaataggaga 540
agatcgacgt ggaggaggat gtggccatga gggcgggttt gatgggcttg gctgggaagg 600
gcccaggatt cagatctcag agggcgggaa gagctttggt ggagagggcg gaggaccccg 660
gggagcgtga ttggcgcggg gagggaaatg gatgcgaaac agagtggtgg gtggcggtac 720
ggtgagggat caaaggatgt ctgtctatcg agattggcca tgacacatcg acatcgcgcc 780
tgcccccgtc gctcctatcg ttatatgcta taaagcctcc ctttttgccc ttttcccccg 830
ccgcccgcct cgtcctccac aacgtcccga cgctcgcccg tctcgctctc ctcgacgtcg 900
ccccttgctt ccctccacca tcctccaccg atcgagccct tccgcccacc gatcccggct 960
cccctgacct atcgcacctc agcatcagca ccagtgatta ccagcgctac aagactgaca 1020
tggaagtaga ttcctctttc cgtcttccaa caatgccccc accttcagga gctcctttcc 1080
gtgctctcca cccctaccag ttcgcccctg ccgaatacaa tcccctcatg ccctcgccag 1140
accatccccc aaatgctgcg ccccatcccc caccagcacg ccccgaacac agtggcctct 1200
ctgaacccca cgatcggaat ccttctcccc attcctacta ctcccatcct cctccacctc 1260
ctgcacatgc ctcctccagc cgtacccatt accaatcccc tccaagcccc cagttccgca 1320
cccatgctgt caactggggt ttacctccca tcggctcctc tcagcagcag cagcagcagc 1380
aactcccacc cccatctcaa cttgccggcc cttcgatgcc ccctgcccag tttccccctc 1440
caacaacaac atctcgtcgt tcaaatgacg atgcccagcg cgcgcctcaa gcagcgatgc 1500
tctacgccca atcccaacag ctacctccaa tccaaacaaa ccaagcaggt atcgcgcccg 1560
ccacccgcgg acgctcatcc tccgtgatcg ccgccgcgtc ctcgttcaca caaggcgcaa 1620
atgttccgcg gctcccccca atcatgcaag tggagaagca gcaagtgacc actagcgcga 1680
cccaagcagc gagcgcgagt cggcggcgca acgaggccaa cttcgtttgc ccggtccccg 1740
gctgcgggag cacgtttact cggcgtttca acctgagagg tacgtcccct actctccgcg 1800
tcaccttcga cgcccgtgtc cgcatcggtc attcaacctg tctcctttct cgcctcgtgc 1860
acctgccgcg acatttgcgt cctctcctcc gtctccgccg acatccaggc cacctccgct 1920
cccacaccgc cgaacgcccc ttcctatgcg agtggcccgg atgcaacaaa ggattcgcgc 1980
gccagcatga ttgcaagtga gtggcctttc ctgagaccct gacatcgctt tcagttagct 2040
aatcgttccc ttgcttgttg cgcatttgct gcgcgcttcg tttatctcag gcgacatcag 2120
gcgctacata catcgcggtc ccaatcaaac gtatgccagg gatgtgggaa gacattcagt 2160
cgtctagatg cgcttaacgt aagtcccatt ggctcatccg cctagctgtg cgcagctact 2220
gacgccgttt cctcccacag aggcatcgta cgttcacaaa cgaagtttga gaaccatgat 2280
ccccggctga cttttgaact cctcctccca acacagtgcg ttcggacgga ggcgctgact 2340
gccgccaggc ggcggaggcc gcgaatgtcg cgaaccgact gagcccaccg gacgacgact 2400
gggagaacgc gagcaattcc acagagcttt cagaacgcgg gggctcgcgc cggtctccgc 2460
atcggagcgc gacaagctga 2480
<210>2
<211>299
<212>PRT
<213>灵芝菌株CCGMC 5.0616
<400> 2
MET Pro Ser Pro Asp His Pro Pro Asn Ala Ala Pro His Pro Pro Pro Ala Arg Pro Glu
1 10 20
His Ser Gly Leu Ser Glu Pro His Asp Arg Asn Pro Ser Pro His Ser Tyr Tyr Ser His
21 30 40
Pro Pro Pro Pro Pro Ala His Ala Ser Ser Ser Arg Thr His Tyr Gln Ser Pro Pro Ser
41 50 60
Pro Gln Phe Arg Thr His Ala Val Asn Trp Gly Leu Pro Pro Ile Gly Ser Ser Gln Gln
61 70 80
Gln Gln Gln Gln Leu Pro Pro Pro Ser Gln Leu Ala Gly Pro Ser MET Pro Pro Ala Gln
81 90 100
Phe Pro Pro Pro Pro Thr Thr Ser Arg Arg Ser Asn Asp Asp Ala Gln Arg Ala Pro Gln
101 110 120
Ala Ala MET Leu Tyr Ala Gln Ser Gln Gln Leu Pro Pro Ile Gln Thr Asn Gln Ala Gly
121 130 140
Ile Ala Pro Ala Thr Arg Gly Arg Ser Ser Ser Val Ile Ala Ala Ala Ser Ser Phe Thr
141 150 160
Gln Gly Ala Asn Val Pro Arg Leu Pro Pro Ile MET Gln Val Glu Lys Gln Gln Val Thr
161 170 180
Thr Ser Ala Thr Gln Ala Ala Ser Ala Ser Arg Arg Arg Asn Glu Ala Asn Phe Val Cys
181 190 200
Pro Val Pro Gly Cys Gly Ser Thr Phe Thr Arg Arg Phe Asn Leu Arg Gly His Leu Arg
201 210 220
Ser His Thr Ala Glu Arg Pro Phe Leu Cys Glu Trp Pro Gly Cys Asn Lys Gly Phe Ala
221 230 240
Arg Gln His Asp Cys Lys Arg His Gln Ala Leu His Thr Ser Arg Ser Gln Ser Asn Val
241 250 260
Cys Gln Gly Cys Gly Lys Thr Phe Ser Arg Leu Asp Ala Leu Asn Leu Leu Thr Pro Phe
261 270 280
Pro Pro Thr Glu Ala Ser Tyr Val His Lys Arg Ser Leu Arg Thr MET Ile Pro Gly
281 290 299
<210>3
<211>27
<212>DNA
<213>人工序列
<400>3
tccaaagccg ctctcatggc atggcac 27
<210>4
<211>37
<212>DNA
<213>人工序列
<400>4
gctagcgttg agagggggat gaagagtgag taagaag 37
<210>5
<211>18
<212>DNA
<213>人工序列
<400>5
atgagcgggt cagagagt 18
<210>6
<211>20
<212>DNA
<213>人工序列
<400>6
tgctctatgt cttgccttgt 20
<210>7
<211>27
<212>DNA
<213>人工序列
<400>7
tctgctcttc ccgattgctg catttgt 27
<210>8
<211>28
<212>DNA
<213>人工序列
<400>8
ctatgtcttg ccttgtctcg cgtcaacc 28
<210>9
<211>26
<212>DNA
<213>人工序列
<400>9
gaagatcgtg aggcagcggt ataggc 26
<210>10
<211>26
<212>DNA
<213>人工序列
<400>10
gcctataccg ctgcctcacg atcttc 26
<210>11
<211> 19
<212>DNA
<213>人工序列
<400>11
atggaagacc gcagtgcgt 19
<210>12
<211>19
<212>DNA
<213>人工序列
<400>12
tcagcttgtc gcgctccga 19
<210> 13
<211>18
<212>DNA
<213>人工序列
<400> 13
gagtgacgca ggtggtga 18
<210> 14
<211>18
<212>DNA
<213>人工序列
<400> 14
taggaatggg gagaagga 18
Claims (1)
1.一种灵芝酸高产工程菌株kmust-Crz，其在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC NO.12235。
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