CN116814671A - 组蛋白乙酰酶基因LeEaf6在调控香菇早熟性中的应用 - Google Patents
组蛋白乙酰酶基因LeEaf6在调控香菇早熟性中的应用 Download PDFInfo
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Abstract
本发明属于真菌基因工程技术领域,公开了NUA4组蛋白乙酰酶复合体Eaf6亚基编码基因LeEaf6在调控香菇早熟性中的应用。构建LeEaf6基因过表达和沉默载体,通过农杆菌介导的遗传转化方法研究该基因功能,结果表明:LeEaf6基因正向调控香菇的早熟性,过表达该基因,能够在不影响其他农艺性状(单菇重、单袋产量、单袋菇数、菌盖直径、菌盖厚度、菌柄长度和菌柄直径)的条件下,提早香菇原基的形成时间和子实体形成时间,显著缩短香菇出菇周期。
Description
技术领域
本发明属于真菌基因工程技术领域,具体涉及组蛋白乙酰酶基因LeEaf6在调控香菇早熟性中的应用。
背景技术
香菇是目前世界上产量最多的食用菌(Royse,D.J.et al.Current overview ofmushroom production in the world.In:Diego CZ,Pardo-Giménez A eds.,Edible andMedicinal Mushrooms:Technology and Applications.Hoboken:Wiley,2017.5-13.),在我国食用菌产业中发挥着极其重要的作用,中国是世界上最大的香菇生产、出口与消费国(李月梅.香菇产业具有良好的发展前景.食品科学.2005.07:261-266.)。香菇香气独特,味道鲜美,素有“菇中皇后”、“山珍”等美誉(Chang,S.T.Past and present trends in theproduction of Lentinula edodes in Asia.Mushroom Biology and MushroomProducts.2002.4:1-8.)。并且香菇富含蛋白质、氨基酸、多种维生素及矿质元素,具有防治肿瘤、增加免疫力、降血脂、抗血栓、健胃保肝等功效(边银丙.食用菌栽培学(第3版).北京:高等教育出版社.2017.138-140),因其具有的较高营养价值和药用价值,深受人们喜爱。
重要农艺性状的遗传改良是香菇产业可持续发展的基础(龚文兵等.基于F2群体的香菇遗传图谱构建及其在QTL定位中的应用.菌物学报.2014.33:297-311)。香菇重要经济性状多是数量性状,如产量、子实体数量(菇数)、单菇重、早熟性、菌丝生长速度等(肖扬等.关联分析及其在真菌遗传学研究中的应用.菌物学报.2016.35:782-790)。不同于质量性状,数量性状大多表现为连续变异,受多基因协同调控,遗传基础复杂,且易受环境因素影响,表型和基因型间没有明确对应关系,难以进行遗传改良(Santoyo,F.etal.Quantitative linkage mapping of lignin degrading enzymatic activities inPleurotus ostreatus.Enzyme and Microbial Technology.2008.43(2):137-143.)。自20世纪80年代以来,随着分子生物学和数量遗传学的发展,通过构建分子标记遗传连锁图谱和利用自然群体,进行数量性状位点(quantitative trait loci,QTL)的连锁分析和关联分析,进一步分析重要性状调控基因的功能及开发高效的分子育种工具,为解析数量性状遗传基础,进行遗传改良提供了有效途径(龚文兵等.食用菌QTL定位研究进展.园艺学报.2011.38:1800-1806.)。
香菇早熟性是一个复杂的数量性状,包括原基期(FP)和出菇期(FB)。研究发现,早熟性性状与子实体形态特征负相关,与产量性状负相关,即出菇越早,子实体越大,子实体单菇重越大,产量越高(Gong,W.B.et al.Phenotypic evaluation and analysis ofimportant agronomic traits in the hybrid and natural populations of Lentinulaedodes.Scientia Horticulturae.2014,179:271-276)。此外,Gong等人也对香菇菇数、单菇重、早熟性、菌盖直径、菌盖厚度、菌盖重量、菌柄直径、菌柄长度、菌柄重量等一系列重要经济性状进行了系统的QTL定位研究(Gong,W.B.et al.Constructing a new integratedgenetic linkage map and mapping quantitative trait loci for vegetativemycelium growth rate in Lentinula edodes.Fungal Biology.2014b.118:295-308.Gong,W.B.et al.Genetic dissection of fruiting body-related traits usingquantitative trait loci mapping in Lentinula edodes.Applied Microbiology andBiotechnology.2016.100:5437-5452.Gong,W.B.et al.Detection of quantitativetrait loci underlying yield-related traits in Lentinula edodes(Agaricomycetes).International Journal of Medicinal Mushrooms.2018.20(5):451-458.)。
林范学在香菇中发现了8个与马铃薯葡萄糖培养基(Potato Dextrose Agar,PDA)上菌丝长速相关的QTL位点,5个木屑培养基上菌丝长速相关的QTL位点;并且发现菌丝长速和木霉抗性这两个性状之间存在显著的正相关(林范学.香菇分子遗传图谱构建和数量性状座位(QTL)分析[博士学位论文].武汉:华中农业大学图书馆.2008.)。基于171个F2双核体菌株,龚文兵等人构建了第一张双核体香菇遗传图谱,并定位到6个与麦芽浸膏培养基(Malt extract medium,MYG)上菌丝长速相关的QTL位点,对性状变异的贡献率为50.11%(龚文兵等.基于F2群体的香菇遗传图谱构建及其在QTL定位中的应用.菌物学报.2014.33:297-311.)。
总体而言,香菇重要数量性状遗传基础研究尚处于起步阶段,主要集中在QTL候选基因挖掘,基因功能验证鲜有报道。目前,还没有食用菌早熟性等生育期性状调控基因功能的研究报道。
本发明通过全基因组关联分析,最终定位到一个香菇早熟性调控的重要候选基因LeEaf6,功能研究表明LeEaf6基因正向调控香菇早熟性,过表达该基因,能够提早香菇原基和子实体形成时间,从而达到显著缩短香菇出菇周期的目的。
发明内容
本发明的目的在于提供了组蛋白乙酰酶基因LeEaf6在调控香菇早熟性中的应用,通过过表达LeEaf6基因显著缩短香菇出菇周期。
为了实现上述目的,本发明采用以下技术方案:
通过对香菇的全基因组关联分析发现,组蛋白乙酰酶基因LeEaf6为调控香菇早熟性的重要候选基因,该基因的核苷酸序列如SEQ ID NO.1所示,开放阅读框长336bp,其序列如SEQ ID NO.2所示,编码的氨基酸序列如SEQ ID NO.3所示。
本发明构建了LeEaf6基因过表达和沉默载体,通过农杆菌介导的遗传转化方法研究LeEaf6基因功能,结果表明,与对照空载菌株相比,过表达菌株的原基形成时间均提前了3-9天,子实体形成时间提前了6-12天,与对照相比过表达菌株的子实体菌柄长度、菌柄直径、菌盖厚度、菌盖直径、单袋菇数、单菇重等均无显著性差异,干扰菌株原基形成时间延迟了6-10天,子实体形成时间延迟了9-12天。因此,过表达LeEaf6基因可以显著缩短香菇的原基期和出菇期。
与现有技术相比,本发明具有以下优点及有益效果:
本发明是国内外首次公开LeEaf6基因在缩短香菇栽培周期中的作用。基于133个菌株的栽培实验数据进行全基因组关联分析,获得调控早熟性的重要候选基因LeEaf6。本发明实验结果表明,与空载对照菌株相比,LeEaf6基因过表达菌株的原基期提前3-9天,出菇期提前了6-12天。
附图说明
图1.早熟性性状的全基因组关联分析分析结果:原基期(图A)、出菇期(图B)。红色水平线以上为关联分析检测的显著关联位点,其±2kb范围内基因为性状关联候选基因。LeEaf6位于箭头所示关联位点±2kb范围内。
图2:LeEaf6基因结构(A)及保守结构域(B)图示。
图3:载体质粒pCAMBIA1300-g(A)、重组过表达载体LeEaf6-OE(B)和重组干扰载体LeEaf6-RNAi(C)的图谱。
图4:qRT-PCR检测LeEaf6-OE/RNAi转化子。
图5:A:香菇生长的不同发育阶段;B:工厂化栽培条件下,低温刺激之后不同菌株出菇情况。
图6:工厂化栽培条件下,不同菌株原基形成时间(低温刺激到原基形成时间)和出菇时间(低温刺激到子实体形成时间)统计分析结果(*p<0.05,**0.01<p<0.05,***p<0.01)。
图7:A:工厂化栽培条件下,不同菌株子实体形态相关性状统计分析结果;B:工厂化栽培条件下,不同菌株产量性状统计分析结果。
具体实施方式
下述实施例中所用方法如无特别说明均为常规生物实验方法,所用引物均由武汉天一辉远生物科技有限公司合成,测序由武汉天一辉远生物科技有限公司完成。pEASY-Basic Seamless Cloning and Assembly Kit购自TransGen公司;限制性内切酶购自NewEngland Biolabs公司;潮霉素B购自瑞士Roche公司,卡那霉素(Kan+)、利福平(Rif)、头孢噻肟霉素(Cef)和乙酰丁香酮(AS)购自清江生物科技有限公司;San Prep柱式DNA回收试剂盒购自上海惠凌生物技术有限公司;RNAiso Plus购自宝生物工程(大连)有限公司。实验中所用香菇单核体W1-26基因组数据、香菇子实体转录组数据、群体基因组数据为发明人所在课题组测序结果。香菇栽培菌株W1、大肠杆菌感受态Trans-T1、农杆菌感受态EHA105、载体质粒pCAMBIA1300-g(原35S启动子被香菇Legpd启动子替换,以启动潮霉素磷酸转移酶基因hph表达)等,均为发明人所在实验室保存。
实施例1:组蛋白乙酰基酶基因LeEaf6克隆
NUA4是一种组蛋白乙酰基酶,它是一个13亚基复合体,能够特异性乙酰化组蛋白H4和H2A N端尾部,NuA4组蛋白乙酰转移酶在真核生物中参与转录调节、细胞周期等进程(Hodges,A.J.et al.NuA4 acetyltransferase is required for efficient nucleotideexcision repair in yeast.DNA repair.2019.73:91-98.)。Eaf6是NUA4的一个亚基,酵母的EAF6亚基通过Yng2与另一种催化亚基蛋白Esa1相互作用(Mitchell,L.etal.Functional dissection of the NuA4 histone acetyltransferase reveals itsrole as a genetic hub and that Eaf1 is essential for complexintegrity.Molecular and cellular biology.2008.28(7):2244-56.)。发明人对133个香菇菌株开展两年的栽培试验,系统考察原基期、出菇期和单菇重等9个重要农艺性状,通过全基因组关联分析和QTL定位发现,LeEaf6(香菇的Eaf6基因)为调控香菇早熟性的重要候选基因(图1)。
基于香菇单核体W1-26基因组数据,设计引物LeEaf6-DNA-F:5’-ACGCGTACTCTCTACAAGGCCT-3’,LeEaf6-DNA-R:5’-GAACGACATTGTTGACCGTC AA-3’,以香菇栽培菌株W1 DNA为模板扩增并测序得到LeEaf6基因DNA序列。设计引物:LeEaf6-cDNA-F:5’-ATGGCCGAGGATGTCAAGACT-3’,LeEaf6-cDNA-R:5’-TCAGTTCCAAAGACTGTTTTCGTAA-3’,以W1菌株cDNA为模板扩增并测序得到LeEaf6基因cDNA序列。PCR扩增体系:2×Phanta Max Buffer25μL,引物(10μmol/L)各2μL,dNTP Mix(10mmol/L)1μL,Phanta Max Super-Fidelity DNAPolymerase(1U/μL)1μL,模板200ng,补ddH2O至50μL。反应参数:95℃预变性3min;95℃变性30s,60℃退火30s,72℃延伸1min,循环数35;72℃彻底延伸10min。
序列分析结果显示:LeEaf6基因全长为898bp,包含2个外显子和1个内含子(图2A),开放阅读框全长为336bp,编码NUA4组蛋白乙酰基酶的亚基Eaf6。LeEaf6基因序列如SEQ ID NO.1所示,开放阅读框序列如SEQ ID NO.2所示,编码蛋白质氨基酸序列如SEQ IDNO.3所示。对基因编码蛋白的理化性质进行分析,结果表明其包含111个氨基酸,分子式为C562H898N164O175S2,分子量为12816.43,理论等电点为9.22,不稳定系数31.68,脂肪族指数为78.29,LeEaf6是一个稳定的亲水性非分泌蛋白(图2B)。
实施例2:LeEaf6过表达(OE)和基因沉默(RNAi)载体构建
过表达载体构建:用Eco RⅠ和KpnⅠ对质粒pCAMBIA1300-g(图3A)进行双酶切,用1%琼脂糖凝胶电泳检测回收。根据酶切位点两端核苷酸序列,设计重组引物OE-F:5’-attgactgcttgaatggtaccACGCGTACTCTCTACAAGGCCT-3’,OE-R:5’-gggaaattcgagctcgaattcGAACGACATTGTTGACCGTCAA-3’扩增LeEaf6基因片段。引物Legpd-F:5’-tgccactggcTGAAAAGACATGGATTGAGC CA-3’,Legpd-R:5’-tctagaggatccccgggTACCGTACATCCCTTGCTCTGC-3’扩增香菇Legpd启动子片段。纯化回收PCR扩增产物。将线性化片段pCAMBIA1300-g、Legpd启动子片段、LeEaf6基因片段按一步克隆法进行同源重组(酶切线性化片段pCAMBIA1300-g 160ng,Legpd启动子20ng,LeEaf6基因片段30ng,2×BasicAssembly Mix 5μL,补ddH2O至10μL),PCR仪中50℃反应15min后转化至大肠杆菌感受态细胞Trans-T1,以引物CX-POE-NployA-F:5’-CATGCTTAACGTAATTCAACAG-3’,OE-F:5’-attgactgcttgaatggtaccACGC GTACTCTCTACAAGGCCT-3’进行阳性克隆测序检测,并送至武汉天一辉远生物科技有限公司进行测序。测序结果显示阳性克隆基因片段与参考序列有100%相似性。提取阳性克隆的载体质粒,即为重组过表达载体LeEaf6-OE(图3B)。
RNAi载体构建:用Eco RⅠ和KpnⅠ对质粒pCAMBIA1300-g进行双酶切,1%琼脂糖凝胶电泳检测回收。根据酶切位点两端核苷酸序列,设计重组引物RNAi-F:5’-attgactgcttgaatggtaccTGGCTCAAATTCCGAAGAAGA-3’,RNAi-R:5’-ctcgcagctcttcacgaattcATATGTAAGACTGCTATTCGAGAATATACG-3’以扩增263bp LeEaf6基因保守区片段。重组引物LeActin-F:5’-ccacctcaaacttcggaattcGCAGTATTTATACCTACGGAGCG-3’G,LeActin-R:5’-tcttccgagCGTGAAGAGCTGCGAGTGTTG-3’扩增香菇Leactin启动子片段。纯化回收PCR扩增产物。将线性化片段pCAMBIA1300-g、Legpd启动子片段、Leactin启动子片段、LeEaf6基因保守区片段按一步克隆法进行同源重组(酶切线性化片段pCAMBIA1300-g 100ng,Leactin启动子50ng,Legpd启动子40ng,LeEaf6 antisense片段20ng,2×Basic Assembly Mix 5μL,补ddH2O至10μL)。PCR仪中50℃反应15min后转化至大肠杆菌感受态细胞Trans-T1,以引物LeEaf6-RNAi-F:5’-attgactgcttgaatggtaccTGGCTCAAATTCCGAAGAAGA-3’,Actin-jy:5’-GATATGTGCGGAACTTGCTTG-3’进行阳性克隆测序检测,并送至武汉天一辉远生物科技有限公司进行测序。测序结果显示阳性克隆基因片段与参考序列有100%相似性。提取阳性克隆的载体质粒,即为重组干扰载体LeEaf6-RNAi(图3C)。
实施例3:农杆菌介导遗传转化筛选转化子
利用冻融法,将上述构建完毕的重组载体LeEaf6-OE、LeEaf6-RNAi和pCAMBIA1300-g(CK)转入农杆菌感受态细胞EHA105中。步骤如下:1)将保存的EHA105感受态细胞从-80℃冰箱中拿出,处于冰水混合物时插入冰上;2)100μL感受态细胞加入约1μg构建好的载体质粒,轻柔混匀;3)分别在冰上静置5min,液氮5min,37℃水浴5min,冰浴5min;4)加入700μL无抗生素的LB液体培养基,28℃,200r/min,培养2-3h;5)6000r/min离心1min,留取200μL左右上清轻轻吹打重悬菌块并涂布到含50μg/mL Kan+和20μg/mL Rif的LB培养基平板上;6)28℃,培养60h;7)以引物CX-POE-NployA-F:5’-CATGCTTAACGTAATTCAACAG-3’,OE-F:5’-attgac tgcttgaatggtaccACGCGTACTCTCTACAAGGCCT-3’进行OE阳性克隆检测;以引物CK-MCS-F 5’-AAGCTTGCATGCCTGCAGG-3’,CK-HYG-R 5’-ATGTTGG CGACCTCGTATTG-3’进行CK阳性克隆检测;以引物LeEaf6-RNAi-F:5’-attg actgcttgaatggtaccTGGCTCAAATTCCGAAGAAGA-3’,Actin-jy:5’-GATATGTGCG GAACTTGCTTG-3’进行RNAi阳性克隆检测;7)经PCR检测为阳性的单克隆摇菌至OD600=1.8-2.0(紫外分光光度计检测),用等体积的50%甘油保菌于-80℃超低温冰箱,以备后续研究使用。
以香菇W1菌株为受体,进行农杆菌介导的遗传转化,方法如下:分别将含有线性载体质粒pCAMBIA1300-g(CK)和重组载体LeEaf6-OE、LeEaf6-RNAi的农杆菌EHA105在LB抗性平板(50μg/mL Kan+,20μg/mL Rif)上划线培养2d,挑取单菌落于1mL液体LB(50μg/mL Kan+,20μg/mL Rif)中培养48h,28℃,200r/min。随后将培养好的农杆菌菌液加入到100mL基本培养基MM(50μg/mL Kan+)中,28℃、200r/min培养24h后,4℃、5000r/min离心10min,弃上清,收集菌液。用适量诱导培养基IM重悬菌体,使得初始OD600约为0.4,并添加乙酰丁香酮(AS)至终浓度为200μmol/L,28℃,200r/min摇培3-5h,使OD600约为0.8左右。香菇菌株W1的菌丝在MYG培养基上活化7d后,用灭菌枪头打取直径5mm的菌丝块,并用上述OD600为0.8左右的农杆菌菌液浸没25min,每5min摇匀一次。之后用灭菌滤纸吸干菌液,将菌丝块接种于共培养培养基(200μmol/L AS)中,25℃培养2d。共培养2d后,将菌丝块从培养基上挑取下来,用灭菌的ddH2O清洗3次,并用含400μg/ml头孢噻肟霉素(Cef)的无菌水浸泡15min,每隔5min摇匀一次,抑制农杆菌的生长。用灭菌滤纸吸干菌丝块表面水分,转移至含有4μg/mL潮霉素和400μg/mL Cef的MYG培养基上,25℃培养15d进行第一次筛选。将第一次筛选后萌发的菌丝,用灭菌枪头打孔,转至加入4μg/mL潮霉素的MYG培养基上,25℃培养7-10d,进行第二次筛选。经过五次筛选后仍然能够生长的菌株做为假定阳性转化子进行下一步实验。
基本培养基(Minimal Media,MM):K-buffer 10mL,M-N buffer 20mL,20%葡萄糖(w/v)10mL,0.01% FeSO4(w/v)10mL,20%(NH4)2SO4(w/v)2.5mL,1%CaCl2·2H2O(w/v)1mL,ddH2O 1L,用H3PO4或NaOH调节pH为6.7-7.0。
诱导培养基(Induction Media,IM):K-buffer 10mL,M-N buffer 20mL,20%葡萄糖(w/v)5mL,0.01% FeSO4(w/v)10mL,20%(NH4)2SO4(w/v)2.5mL,1%CaCl2·2H2O(w/v)1mL,50%甘油(w/v)10mL,7.808g MES(40mmol/L,MW 195.2),ddH2O 1L,用H3PO4或NaOH调节pH为5.6。
共培养培养基(Co-induction Media,Co-IM):K-buffer 10mL,M-N buffer 20mL,20%葡萄糖(w/v)2.5mL,0.01% FeSO4(w/v)10mL,20%(NH4)2SO4(w/v)2.5mL,1% CaCl2·2H2O(w/v)1mL,50%甘油(w/v)10mL,7.808g MES(40mmol/L,MW 195.2),琼脂20g,ddH2O 1L,用H3PO4或NaOH调节pH为5.6。
实施例4实时定量PCR鉴定阳性转化子
经过五次筛选后仍然能够生长的菌株作为假定阳性转化子,在贴有玻璃纸的MYG培养基上25℃培养7d后收集菌丝,用RNAiso Plus试剂盒(TaKaRa)提取总RNA,用HiScriptII One Step RT-PCR Kit(Vazyme)进行反转录。用AceQTM qPCR SYBR Green Master Mix(Takara)试剂盒在CFX Connect Real-Time PCR System(BIO-RAD)中进行实时定量PCR(qRT-PCR)。反转录后的cDNA稀释10倍后作为模板。
定量引物为LeEaf6-Qpcr-F:5’-GACTCGCTATGAGGCGATAAAG-3’、LeEaf6-Qpcr-R:5’-GGGTACCGGTCTCTGATAGAT-3’,内参基因为Leactin,内参引物为Actin-F:5’-GCATCCTGTCCTTCTTACCGAG-3’、Actin-R:5’-AAGAGCGAAACCCTCGTAGATG-3’,对照为转入空载体的W1菌株转化子。基因相对表达量计算方法采用2-ΔΔCT(Luo et al.Selection andvalidation of references for qRT-PCR in Lentinula edodes under differentexperiment conditions.2019.Genes.10(9),647.)。
qRT-PCR反应体系为:AceQTM qPCR SYBR Green Master Mix 5μL,引物(10μM)各0.5μL,cDNA3μL,ddH2O 1μL。反应参数:95℃预变性5min,95℃变性10s,60℃退火30s,72℃延伸30s,循环数40;溶解曲线的温度设置为65℃到95℃,每0.5℃读取一次数据。
根据定量结果,最终选取W1野生型菌株、1个CK转化子(CK-W1)、4个OE转化子(O-1、O-2、O-3、O-4)、4个RNAi转化子(R-1、R-2、R-3、R-4)进行后续表型实验(图4)。
实施例5LeEaf6-OE/RNAi转化子早熟性、产量、子实体形态性状测定
对LeEaf6-OE/RNAi阳性转化子、对照菌株CK-W1和野生型W1菌株进行栽培实验。用MYG培养基活化菌种,25℃避光培养10d后用于接种。用木屑培养基制作栽培袋,每个菌株制作16个栽培袋。栽培袋规格为15cm×30cm的聚乙烯袋,每袋装料湿重为1kg,接种量约为8%(w/w)。接种后将栽培袋放于室内25℃避光养菌,45d-50d菌袋长满后再放置7d-10d进行菌丝后熟。对满袋菌丝刺孔增氧,并给予适当光照,刺激转色。20d-25d后将转色完成的栽培袋在工厂化栽培模式下进行出菇管理(王卓仁等.应用灰色关联度分析筛选香菇优良杂交子.食用菌学报,2010:26-31.)。开伞程度6-7分时采收和测量农艺性状。
在香菇的不同发育阶段(图5A),统计分析栽培试验两批香菇表型数据,主要包括:原基期、出菇期、菌盖直径、菌盖厚度、菌柄直径、菌柄长度、单袋总菇数、单菇鲜重和鲜菇总产量(表1)。结果表明,与对照空载菌株相比,过表达LeEaf6基因可以明显缩短转化子的原基期和出菇期(图5B),所有超表达菌株的原基形成时间均提前了3-9天,子实体形成时间提前了6-12天,干扰菌株原基形成时间延迟了6-10天,子实体形成时间延迟了9-12天(图6)。并且和对照相比,过表达菌株的形态性状(菌柄长度、菌柄直径、菌盖厚度和菌盖直径)(图7A)和产量性状(菇数、单菇重和产量)等性状表型均无显著性差异(图7B)。
表1 9个香菇农艺性状测定标准
Claims (2)
1.LeEaf6基因在调控香菇早熟性中的应用,其特征在于,所述LeEaf6基因序列如SEQID NO.1所示,早熟性包括原基期和出菇期,所述的原基期为香菇接种后到第一个原基形成时间,出菇期为香菇接种后到第一个子实体收获的时间。
2.一种缩短香菇出菇周期的方法,其特征在于,在香菇中过表达LeEaf6基因可以缩短香菇出菇周期。
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