本发明属于生物工程
技术领域：
，具体地说，涉及一种分泌表达多种纤维素酶的酿酒酵母工业菌株及构建方法。
背景技术：
：石油、天然气和煤炭等化石能源是当前主要的能源形式，由于它们不可再生，大量消耗必然导致其快速枯竭。寻找能(部分)替代化石能源且污染较少的可再生能源是未来能源发展的一个方向。地球上每年光合作用产生的生物质高达1500-2000亿吨，5％的生物质所含的能量与人类对石油和天然气的需求量相当。生物质中的木质纤维素年产量约为1000亿吨，是自然界中最丰富的碳源，能以可持续的方式为生物燃料和化学品的生产提供潜在原料。因此，以木质纤维素为原料生产生物燃料已成为世界范围内生物燃料技术发展的趋势，其中木质纤维素燃料乙醇的发展力度最强。使用木质纤维素生物转化生产乙醇通常包括生物质预处理、纤维素酶生产、水解和发酵等多个步骤。纤维素酶生产需要单独进行，另外纤维素酶比活力比淀粉酶低100倍以上，木质纤维素乙醇用酶量是淀粉乙醇用酶量的30-50倍，这使得木质纤维素乙醇生产时所用纤维素酶的成本居高不下，成为仅次于原料的一大支出(20％-30％)。为降低成本，需要改进生物质技术，除需在瓶颈问题上(如预处理、酶制剂和发酵菌)有重大技术突破外，过程集成也很重要。将纤维素酶生产、水解和发酵组合在一起的统合生物工艺(consolidatedbioprocessing，cbp)因避免了纤维素酶生产过程中的设备投资、减少原料和其它材料的使用，从而降低水解和发酵成本而被认为是一种有应用前景的技术。该技术的关键是构建一种能有效地将纤维素类多聚糖降解成发酵糖并发酵产醇的微生物。在酿酒酵母中表达纤维素酶，使之具有利用纤维素发酵生产乙醇的能力即是其中的一种策略。经过二、三十年的研究，各种各样的纤维素酶在酿酒酵母中已成功得到表达，但研究多数集中在酿酒酵母实验室菌株，而在工业菌株中的研究还较少。与实验室菌株相比，酿酒酵母工业菌株具有更好的鲁棒性，是纤维素酶表达的更佳宿主。khramtsov等(khramtsovn,etal.industrialyeaststrainengineeredtofermentethanolfromligocellulosicbiomass.bioresourcetechnology,2011,102(17):8310-8313)采用δ整合策略和rdna同源重组在酿酒酵母工业菌株之衍生菌株k1-v1116染色体上整合了三种纤维素酶基因(trichodermareeseicbh2、egl2和aspergillusaculeatusbgl1)，筛选得到的最佳菌株发酵10％预处理过的玉米秸秆，96h后乙醇浓度达到2.6％(v/v)。重组菌株构建中采用了δ整合和rdna整合，纤维素酶基因整合的位置具有随机性，重组菌株构建难以重复；田沈和杨秀山(田沈,杨秀山.利用展示纤维素酶的酿酒酵母菌群发酵生产乙醇的方法.cn104673689a)在酿酒酵母工业菌株y5上分别单表面展示了内切葡聚糖酶ii、纤维二糖水解酶ii和β-葡萄糖苷酶i，得到三个重组菌株，将它们诱导培养后按2:1:1的比例混合后用于纤维素统合生物加工中。重组菌株是一混合菌群，各菌株之间的比例极易受发酵培养条件波动的影响。另外，在酵母表面展示表达纤维素酶，纤维素酶的表达量与细胞表面及直接相关，相反地，在分泌型表达中就没有这样的限制。细胞表面展示的另一个缺点是固定在细胞表面的纤维素酶比分泌型的酶扩散率低。技术实现要素：本发明的目的是克服现有技术的不足，提供一种分泌表达纤维素酶的酿酒酵母工业菌株。本发明的第二个目的是提供一种分泌表达纤维素酶的酿酒酵母工业菌株的构建方法。本发明的第三个目的是提供一种分泌表达纤维素酶的酿酒酵母工业菌株在纤维素统合生物工艺中的应用。本发明的技术方案概述如下：分泌表达纤维素酶的酿酒酵母工业菌株的构建方法，包括如下步骤：1)宿主菌kα的获得：安琪酿酒高活性干酵母进行生孢培养，筛选得到交配型为α型的单倍体菌株，用5-氟乳清酸平板进行反选得到宿主菌kα；2)纤维素酶基因表达盒定点整合质粒构建：将seqidno.1所示的dna片断h1经hindiii和psti双酶切后插入质粒puc18-ryur的hindiii-psti位点之间，得到质粒puc18-ryur-h1，再将seqidno.2所示的dna片断h2经smai和ecori双酶切后插入质粒puc18-ryur-h1的smai-ecori位点之间，构建得到定点整合质粒puc18-hru1；将seqidno.3所示的dna片断h3经hindiii和psti双酶切后插入质粒puc18-ryur的hindiii-psti位点之间，得到质粒puc18-ryur-h3，再将seqidno.4所示的dna片断h4经smai和ecori双酶切后插入质粒puc18-ryur-h3的smai-ecori位点之间，构建得到定点整合质粒puc18-hru2；将seqidno.5所示的dna片断h5经hindiii和psti双酶切后插入质粒puc18-ryur的hindiii-psti位点之间，得到质粒puc18-ryur-h5，再将seqidno.6所示的dna片断h6经smai和ecori双酶切后插入质粒puc18-ryur-h5的smai-ecori位点之间，构建得到定点整合质粒puc18-hru3；将seqidno.7所示的dna片断h7经hindiii和psti双酶切后插入质粒puc18-ryur的hindiii-psti位点之间，得到质粒puc18-ryur-h7，再将seqidno.8所示的dna片断h8经smai和ecori双酶切后插入质粒puc18-ryur-h7的smai-ecori位点之间，构建得到定点整合质粒puc18-hru4；将seqidno.9所示的dna片断h9经hindiii和psti双酶切后插入质粒puc18-ryur的hindiii-psti位点之间，得到质粒puc18-ryur-h9，再将seqidno.10所示的dna片断h10经smai和ecori双酶切后插入质粒puc18-ryur-h9的smai-ecori位点之间，构建得到定点整合质粒puc18-hru5；将seqidno.11所示的dna片断h11经hindiii和psti双酶切后插入质粒puc18-ryur的hindiii-psti位点之间，得到质粒puc18-ryur-h11，再将seqidno.12所示的dna片断h12经smai和ecori双酶切后插入质粒puc18-ryur-h11的smai-ecori位点之间，构建得到定点整合质粒puc18-hru6；将seqidno.13所示的dna片断h13经hindiii和psti双酶切后插入质粒puc18-ryur的hindiii-psti位点之间，得到质粒puc18-ryur-h13，再将seqidno.14所示的dna片断h14经smai和ecori双酶切后插入质粒puc18-ryur-h13的smai-ecori位点之间，构建得到定点整合质粒puc18-hru7；将seqidno.15所示的dna片断h15经hindiii和psti双酶切后插入质粒puc18-ryur的hindiii-psti位点之间，得到质粒puc18-ryur-h15，再将seqidno.16所示的dna片断h16经smai和ecori双酶切后插入质粒puc18-ryur-h15的smai-ecori位点之间，构建得到定点整合质粒puc18-hru8；将seqidno.17所示的dna片断h17经hindiii和psti双酶切后插入质粒puc18-ryur的hindiii-psti位点之间，得到质粒puc18-ryur-h17，再将seqidno.18所示的dna片断h18经smai和ecori双酶切后插入质粒puc18-ryur-h17的smai-ecori位点之间，构建得到定点整合质粒puc18-hru9；将seqidno.19所示的dna片断h19经hindiii和psti双酶切后插入质粒puc18-ryur的hindiii-psti位点之间，得到质粒puc18-ryur-h19，再将seqidno.20所示的dna片断h20经smai和ecori双酶切后插入质粒puc18-ryur-h19的smai-ecori位点之间，构建得到定点整合质粒puc18-hru10；将puc18-hru1、puc18-hru2、puc18-hru3、puc18-hru4、puc18-hru5、puc18-hru6、puc18-hru7、puc18-hru8、puc18-hru9和puc18-hru10分别用bamhi消化后，进行末端平滑化处理，分别得到载体片段v1、v2、v3、v4、v5、v6、v7、v8、v9和v10；将质粒pgem-teasy-ptpi-xyn2s-abgl1-tadhi经xhoi和scai双酶切，得到的3.6kbdna片断进行末端平滑化处理后与v1进行连接，得到纤维素酶基因表达盒定点整合质粒ip1；将质粒yeplac195-ptpi-xyn2s-tegii-tadhi经hindiii和ecori双酶切，得到的2.3kbdna片断进行末端平滑化处理后分别与v2和v5进行连接，得到纤维素酶基因表达盒定点整合质粒ip2和ip5；将质粒pgem-teasy-ptpi-xyn2s-aegi-tadhi经psti酶切，得到的1.8kbdna片断进行末端平滑化处理后与v3进行连接，得到纤维素酶基因表达盒定点整合质粒ip3；将质粒pgem-teasy-ptpi-xyn2s-tcbhi-tadhi经stui和scai双酶切，得到的2.6kbdna片断进行末端平滑化处理后分别与v4、v7和v10进行连接，得到纤维素酶基因表达盒定点整合质粒ip4、ip7和ip10；将质粒pgem-teasy-ptpi-xyn2s-acbhi-tadhi经mlui和scai双酶切，得到的2.8kbdna片断进行末端平滑化处理后分别与v6和v8进行连接，得到纤维素酶基因表达盒定点整合质粒ip6和ip8；将质粒pgem-teasy-ptpi-xyn2s-tcbhii-tadhi经stui和scai双酶切，得到的2.6kbdna片断进行末端平滑化处理后与v9进行连接，得到纤维素酶基因表达盒定点整合质粒ip9。3)酵母转化与筛选：将所述质粒ip1经scai酶切、核酸纯化后，采用醋酸锂法转化宿主菌kα，使用缺省尿嘧啶的基本培养基平板筛选转化子，再用5-氟乳清酸平板进行反选得到去掉了ura3筛选标记的转化子kα1；将所述质粒ip2经scai酶切、核酸纯化后，采用醋酸锂法转化宿主菌kα1，使用缺省尿嘧啶的基本培养基平板筛选转化子，再用5-氟乳清酸平板进行反选得到去掉了ura3筛选标记的转化子kα2；将所述质粒ip3经pvuii酶切、核酸纯化后，采用醋酸锂法转化宿主菌kα2，使用缺省尿嘧啶的基本培养基平板筛选转化子，再用5-氟乳清酸平板进行反选得到去掉了ura3筛选标记的转化子kα3；将所述质粒ip5经scai酶切、核酸纯化后，采用醋酸锂法转化宿主菌kα3，使用缺省尿嘧啶的基本培养基平板筛选转化子，再用5-氟乳清酸平板进行反选得到去掉了ura3筛选标记的转化子kα4；将所述质粒ip4经scai酶切、核酸纯化后，采用醋酸锂法转化宿主菌kα4，使用缺省尿嘧啶的基本培养基平板筛选转化子，再用5-氟乳清酸平板进行反选得到去掉了ura3筛选标记的转化子kα5；将所述质粒ip7经scai酶切、核酸纯化后，采用醋酸锂法转化宿主菌kα5，使用缺省尿嘧啶的基本培养基平板筛选转化子，再用5-氟乳清酸平板进行反选得到去掉了ura3筛选标记的转化子kα6；将所述质粒ip10经scai酶切、核酸纯化后，采用醋酸锂法转化宿主菌kα6，使用缺省尿嘧啶的基本培养基平板筛选转化子，再用5-氟乳清酸平板进行反选得到去掉了ura3筛选标记的转化子kα7；将所述质粒ip9经scai酶切、核酸纯化后，采用醋酸锂法转化宿主菌kα7，使用缺省尿嘧啶的基本培养基平板筛选转化子，再用5-氟乳清酸平板进行反选得到去掉了ura3筛选标记的转化子kα8；将所述质粒ip6经scai酶切、核酸纯化后，采用醋酸锂法转化宿主菌kα8，使用缺省尿嘧啶的基本培养基平板筛选转化子，再用5-氟乳清酸平板进行反选得到去掉了ura3筛选标记的转化子kα9；将所述质粒ip8经scai酶切、核酸纯化后，采用醋酸锂法转化宿主菌kα9，使用缺省尿嘧啶的基本培养基平板筛选转化子，再用5-氟乳清酸平板进行反选得到去掉了ura3筛选标记的转化子kα10。上述方法构建的分泌表达纤维素酶的酿酒酵母工业菌株。上述分泌表达纤维素酶的酿酒酵母工业菌株在纤维素统合生物工艺中的应用。本发明的优点：本发明的方法能重复使用ura3筛选标记在酿酒酵母工业菌株之衍生菌株中实现多种纤维素酶基因的定点整合并分泌表达。本发明的重组菌株能有效提高商业化纤维素酶水解纤维素的效率，降低纤维素酶的生产成本，使木质纤维素资源高效转化生产乙醇。附图说明图1为纤维素酶表达盒示意图。图2为质粒puc18-ryur图谱。图3为定点整合质粒示意图。图4为纤维素酶基因定点整合质粒示意图。具体实施方式下面结合具体实施方式对本发明作进一步的说明。实施例1宿主菌kα的获得安琪酿酒高活性干酵母(市售)进行生孢培养，筛选得到交配型为α型的单倍体菌株，用5-氟乳清酸平板进行反选得到宿主菌kα。具体步骤如下：(a)取0.01g安琪酿酒高活性干酵母，接种于5mlypd液体培养基(1％酵母粉，2％蛋白胨，2％葡萄糖)中，30℃、150rpm培养20h。(b)用接种针蘸取部分菌液划线ypd固体(1％酵母粉，2％蛋白胨，2％葡萄糖，1.8％琼脂粉)平板，30℃培养至菌落2-3mm，挑取单菌落接种至3mlypd液体培养基中，30℃、150rpm条件下培养20h后，取50μl菌液接种至3mlypd液体培养基中，30℃、150rpm条件下培养6h。(c)离心收集菌体，用无菌水洗涤三次，将其接种到3ml预生孢培养基(醋酸钾1％，酵母提取物1％，蛋白胨2％)中，28℃、150rpm条件下培养12h。(d)离心收集菌体，用无菌水洗涤三次，将菌体重悬到3ml高效液体生孢培养基(醋酸钾1％，酵母提取物0.1％，葡萄糖0.05％)中，28℃、250rpm培养5d。(e)室温下500rpm离心5min收集酵母生孢培养物，用软化缓冲液(10mmol/l二硫苏糖醇，100mmol/ltris-so4，ph9.4)重悬细胞使之od600＝5，30℃下温育10min。室温下500rpm离心5min收集上述酵母细胞和孢子，用原生质体缓冲溶液(2.1mol/l山梨醇，10mmol/l磷酸钾，ph7.2)重悬使之od600＝25。加入200mg/ml蜗牛酶使之终浓度为0.5mg/od600，并在30℃下反应30min。上述反应液室温下500rpm离心5min收集沉淀，用等量的0.5％(v/v)的tritonx-100洗涤一次后，再用该溶液重悬使孢子反应液为总体积的1/4。超声波振荡使孢子充分分散。(f)上述得到的孢子悬液划线ypd平板，30℃培养至菌落2-3mm，随机挑选10个单菌落，分别接种至3mlypd液体培养基中，30℃、150rpm条件下培养20h后，离心收集菌体，提取染色体，用于菌株交配型的鉴定。(g)上述染色体为模板，引物mat-f(seqidno.21)、mat-a(seqidno.22)和mat-alpha(seqidno.23)为引物，进行pcr反应，反应体系如下：反应条件：94℃3min，(94℃30sec，54℃30sec，72℃1min)×30，72℃10min，25℃pause。pcr产物进行凝胶电泳，选择出现404bp条带的α交配型菌株(交配型为a型的菌株出现544bp条带，α型菌株出现404bp条带，而aα型菌株则出现544bp和404bp两条带)进行后续实验。(h)上述得到的α交配型菌株划线ypd固体平板，30℃培养至2-3mm，挑取适量菌体，用无菌枪头涂抹于5-foa(5-氟乳清酸)平板(ynb(bactoyeastnitrogenbasewithoutaminoacids(difco))0.67％，5-foa0.1％，葡萄糖2％，苏氨酸150mg/l，酪氨酸30mg/l，缬氨酸150mg/l，赖氨酸30mg/l，谷氨酸100mg/l，丝氨酸150mg/l，天冬氨酸100mg/l，甲硫氨酸20mg/l，苯丙氨酸50mg/l，异亮氨酸30mg/l，精氨酸20mg/l，腺嘌呤100mg/l，色氨酸200mg/l，组氨酸300mg/l，亮氨酸400mg/l，琼脂粉2％)，30℃培养2d。(i)上述得到的单菌落分别接种于2mlypd液体培养基中，30℃培养20h，各取50μl接种于5mlcmg培养基(0.67％ynb，2％葡萄糖，苏氨酸150mg/l，酪氨酸30mg/l，缬氨酸150mg/l，赖氨酸30mg/l，谷氨酸100mg/l，丝氨酸150mg/l，天冬氨酸100mg/l，甲硫氨酸20mg/l，苯丙氨酸50mg/l，异亮氨酸30mg/l，精氨酸20mg/l，腺嘌呤100mg/l，色氨酸200mg/l，组氨酸300mg/l，亮氨酸400mg/l，尿嘧啶50mg/l)和5mlcmg-u培养基(cmg培养基缺省尿嘧啶)中，30℃、150rpm条件下培养2d，在cmg-u培养基中不生长而在cmg培养基中生长最佳者(od660最大者)命名为kα，作为后续实验的宿主菌株。实施例2纤维素酶基因表达盒定点整合质粒构建纤维素酶基因表达盒示意图见图1，其中ptpi为(酿酒酵母磷酸丙糖异构酶启动子)启动子，xyn2s(瑞氏木霉木聚糖酶分泌肽)为分泌肽，cellulasegene为各纤维素酶基因外显子序列，tadhi(酿酒酵母乙醇脱氢酶终止子)为终止子。所述纤维素酶基因包括三种纤维二糖水解酶基因(瑞氏木霉trichodermareeseicbh1、cbh2和棘孢曲霉aspergillusaculeatuscbh1)、两种内切葡聚糖酶基因(a.aculeatusegl1和t.reeseiegl2)和一种β-葡萄糖苷酶基因(a.aculeatusbgl1)。以上六种纤维素酶基因表达盒构建见我们的工作(洪解放.表达纤维素酶的酿酒酵母工程菌株构建及其利用木质纤维素发酵产醇研究.2014年天津大学博士学位论文；张韦娜.表达纤维素酶基因的酿酒酵母菌株构建及其底物利用评价.2012年天津大学硕士学位论文；邹少兰.利用木质纤维素水解液产醇工程菌株构建及其产醇研究.2011年天津大学博士学位论文；jiefanghong,etal.developmentofacellulolyticsaccharomycescerevisiaestrainwithenhancedcellobiohydrolaseactivity.worldjournalofmicrobiology&biotechnology,2014,30(11):2985-2993；zhangweina,etal.comparisonoftheexpressioninsaccharomycescerevisiaeofendoglucanaseiifromtrichodermareeseiandendoglucanaseifromaspergillusaculeatus.bioresources,2012,7(3):4031-4045；邹少兰,等.编码β-葡萄糖苷酶的基因及重组表达载体及重组酿酒酵母表达菌株及应用.cn102154344a)，相应的质粒为pgem-teasy-ptpi-xyn2s-tcbhi-tadhi、pgem-teasy-ptpi-xyn2s-tcbhii-tadhi、pgem-teasy-ptpi-xyn2s-acbhi-tadhi、pgem-teasy-ptpi-xyn2s-aegi-tadhi、yeplac195-ptpi-xyn2s-tegii-tadhi和pgem-teasy-ptpi-xyn2s-abgl1-tadhi，它们的序列依次用seqidno.24～seqidno.29所示。质粒puc18-ryur(专利us2010/0291652a1)图谱见图2，序列见seqidno.30。在质粒puc18-ryur的hindiii-psti和smai-ecori限制性内切酶位点分别插入左、右同源臂，得到定点整合质粒puc18-hru1～puc18-hru10，如图3所示，构建过程简述如下：首先是同源臂的扩增。以酿酒酵母w303-1a染色体dna为模板，以不同的引物对为引物进行pcr扩增，反应体系如下：反应条件：94℃3min，(94℃30sec，54℃30sec，72℃1min)×30，72℃10min，25℃pause。pcr产物经核酸纯化后作为同源臂dna片断，用于后续构建。扩增得到的同源臂及大小与所用引物对名称见表1。表1同源臂及扩增同源臂所用引物同源臂名称长度，bp同源臂序列所用引物引物序列h1318seqidno.1p1/p2seqidno.31/seqidno.32h2278seqidno.2p3/p4seqidno.33/seqidno.34h3428seqidno.3p5/p6seqidno.35/seqidno.36h4488seqidno.4p7/p8seqidno.37/seqidno.38h5296seqidno.5p9/p10seqidno.39/seqidno.40h6318seqidno.6p11/p12seqidno.41/seqidno.42h7282seqidno.7p13/p14seqidno.43/seqidno.44h8305seqidno.8p15/p16seqidno.45/seqidno.46h9468seqidno.9p17/p18seqidno.47/seqidno.48h10453seqidno.10p19/p20seqidno.49/seqidno.50h11418seqidno.11p21/p22seqidno.51/seqidno.52h12418seqidno.12p23/p24seqidno.53/seqidno.54h13418seqidno.13p25/p26seqidno.55/seqidno.56h14418seqidno.14p27/p28seqidno.57/seqidno.58h15473seqidno.15p29/p30seqidno.59/seqidno.60h16454seqidno.16p31/p32seqidno.61/seqidno.62h17306seqidno.17p33/p34seqidno.63/seqidno.64h18308seqidno.18p35/p36seqidno.65/seqidno.66h19318seqidno.19p37/p38seqidno.67/seqidno.68h20318seqidno.20p39/p40seqidno.69/seqidno.70puc18-ryur经hindiii和psti双酶切，琼脂糖电泳回收4.8kb条带，得到载体片断vp。dna片断h1、h3、h5、h7、h9、h11、h13、h15、h17、h19分别用hindiii和psti双酶切，核酸纯化后作为插入片断分别与上述载体片断vp连接，得到10个含有单同源臂的质粒puc18-ryur-h1、puc18-ryur-h3、puc18-ryur-h5、puc18-ryur-h7、puc18-ryur-h9、puc18-ryur-h11、puc18-ryur-h13、puc18-ryur-h15、puc18-ryur-h17和puc18-ryur-h19，然后这10个质粒分别经smai和ecori双酶切，核酸纯化后作为载体片断vp1、vp3、vp5、vp7、vp9、vp11、vp13、vp15、vp17和vp19。dna片断h2、h4、h6、h8、h10、h12、h14、h16、h18和h20分别用smai和ecori双酶切，核酸纯化后分别与vp1、vp3、vp5、vp7、vp9、vp11、vp13、vp15、vp17和vp19连接，得到10个定点整合质粒puc18-ryur-h1-h2、puc18-ryur-h3-h4、puc18-ryur-h5-h6、puc18-ryur-h7-h8、puc18-ryur-h9-h10、puc18-ryur-h11-h12、puc18-ryur-h13-h14、puc18-ryur-h15-h16、puc18-ryur-h17-h18和puc18-ryur-h19-h20，分别简称为puc18-hru1～puc18-hru10。上述质粒puc18-hru1、puc18-hru2、puc18-hru3、puc18-hru4、puc18-hru5、puc18-hru6、puc18-hru7、puc18-hru8、puc18-hru9和puc18-hru10分别用bamhi消化后，进行末端平滑化处理，得到的dna片断记为v1、v2、v3、v4、v5、v6、v7、v8、v9和v10，作为载体片断。将质粒pgem-teasy-ptpi-xyn2s-abgl1-tadhi经xhoi和scai双酶切后，琼脂糖电泳回收3.6kbdna片断，然后末端平滑化处理后与v1进行连接，得到纤维素酶基因表达盒定点整合质粒ip1。质粒yeplac195-ptpi-xyn2s-tegii-tadhi经hindiii和ecori双酶切后，琼脂糖电泳回收2.3kbdna片断，然后末端平滑化处理后分别与v2和v5进行连接，得到纤维素酶基因表达盒定点整合质粒ip2和ip5。质粒pgem-teasy-ptpi-xyn2s-aegi-tadhi经psti酶切后，琼脂糖电泳回收1.8kbdna片断，然后末端平滑化处理后与v3进行连接，得到纤维素酶基因表达盒定点整合质粒ip3。质粒pgem-teasy-ptpi-xyn2s-tcbhi-tadhi经stui和scai双酶切后，琼脂糖电泳回收2.6kbdna片断，然后末端平滑化处理后分别与v4、v7和v10进行连接，得到纤维素酶基因表达盒定点整合质粒ip4、ip7和ip10。质粒pgem-teasy-ptpi-xyn2s-acbhi-tadhi经mlui和scai双酶切后，琼脂糖电泳回收2.8kbdna片断，然后末端平滑化处理后分别与v6和v8进行连接，得到纤维素酶基因表达盒定点整合质粒ip6和ip8。质粒pgem-teasy-ptpi-xyn2s-tcbhii-tadhi经stui和scai双酶切后双酶切后，琼脂糖电泳回收2.6kbdna片断，然后末端平滑化处理后与v9进行连接，得到纤维素酶基因表达盒定点整合质粒ip9。上述构建得到的纤维素酶基因定点整合质粒示意图如图4所示。实施例3酵母转化及筛选将质粒ip1经scai酶切、核酸纯化后，采用醋酸锂法转化宿主菌kα，使用缺省尿嘧啶的基本培养基平板筛选转化子，再用5-氟乳清酸平板进行反选得到去掉了ura3筛选标记的转化子kα1。将质粒ip2经scai酶切、核酸纯化后，采用醋酸锂法转化宿主菌kα1，使用缺省尿嘧啶的基本培养基平板筛选转化子，再用5-氟乳清酸平板进行反选得到去掉了ura3筛选标记的转化子kα2。将质粒ip3经pvuii酶切、核酸纯化后，采用醋酸锂法转化宿主菌kα2，使用缺省尿嘧啶的基本培养基平板筛选转化子，再用5-氟乳清酸平板进行反选得到去掉了ura3筛选标记的转化子kα3。将质粒ip5经scai酶切、核酸纯化后，采用醋酸锂法转化宿主菌kα3，使用缺省尿嘧啶的基本培养基平板筛选转化子，再用5-氟乳清酸平板进行反选得到去掉了ura3筛选标记的转化子kα4。将质粒ip4经scai酶切、核酸纯化后，采用醋酸锂法转化宿主菌kα4，使用缺省尿嘧啶的基本培养基平板筛选转化子，再用5-氟乳清酸平板进行反选得到去掉了ura3筛选标记的转化子kα5。将质粒ip7经scai酶切、核酸纯化后，采用醋酸锂法转化宿主菌kα5，使用缺省尿嘧啶的基本培养基平板筛选转化子，再用5-氟乳清酸平板进行反选得到去掉了ura3筛选标记的转化子kα6。将质粒ip10经scai酶切、核酸纯化后，采用醋酸锂法转化宿主菌kα6，使用缺省尿嘧啶的基本培养基平板筛选转化子，再用5-氟乳清酸平板进行反选得到去掉了ura3筛选标记的转化子kα7。将质粒ip9经scai酶切、核酸纯化后，采用醋酸锂法转化宿主菌kα7，使用缺省尿嘧啶的基本培养基平板筛选转化子，再用5-氟乳清酸平板进行反选得到去掉了ura3筛选标记的转化子kα8。将质粒ip6经scai酶切、核酸纯化后，采用醋酸锂法转化宿主菌kα8，使用缺省尿嘧啶的基本培养基平板筛选转化子，再用5-氟乳清酸平板进行反选得到去掉了ura3筛选标记的转化子kα9。将质粒ip8经scai酶切、核酸纯化后，采用醋酸锂法转化宿主菌kα9，使用缺省尿嘧啶的基本培养基平板筛选转化子，再用5-氟乳清酸平板进行反选得到去掉了ura3筛选标记的转化子kα10。重组菌株纤维素酶活性测定参见文献(洪解放.表达纤维素酶的酿酒酵母工程菌株构建及其利用木质纤维素发酵产醇研究.2014年天津大学博士学位论文)，经检测，菌株kα1具有bgl(β-葡萄糖苷酶)活性，菌株kα2～kα10均具有bgl(β-葡萄糖苷酶)、eg(内切葡聚糖酶)、cmcase(羧甲基纤维素酶)和fpase(滤纸酶活)活性。重组菌株kα1～kα10经ypd液体培养基培养后(30℃、150rpm、20h)，取5ml菌液加入50ml带螺旋口的摇瓶中(3g酸碱预处理的玉米芯，12ml稀硫酸溶液，ph4.0)，然后加入0、10fpu/g纤维素料和氏壁纤维素酶(和氏壁生物技术有限公司)，30℃、150rpm发酵10天。菌株kα为对照菌株。发酵液用hplc法测定乙醇浓度，结果表明：加入10fpu/g纤维素料和氏壁纤维素酶(和氏壁生物技术有限公司)，菌株kα1～kα10乙醇浓度(g/l)分别为35.61、38.87、42.20、47.73、49.23、49.99、51.34、53.28、54.72和55.03，对照菌株kα乙醇浓度为29.66g/l；未加纤维素酶时，菌株kα1～kα10乙醇浓度(g/l)分别为0、3.41、5.13、9.73、11.26、14.74、16.51、19.16、21.38和22.45，对照菌株kα不产乙醇。以上质粒构建过程中包括pcr、核酸纯化、dna片断和质粒限制性内切酶消化、dna片断末端平滑化、琼脂糖凝胶电泳回收dna片断、连接、转化等在内的分子操作，以及酵母生孢、孢子筛选、酵母转化、转化子筛选、酵母菌株酶活测定和发酵纤维素产醇性能评价等操作，均可参考文献(洪解放.表达纤维素酶的酿酒酵母工程菌株构建及其利用木质纤维素发酵产醇研究.2014年天津大学博士学位论文)，为本专业人士所熟悉。序列表<110>天津大学<120>分泌表达纤维素酶的酿酒酵母工业菌株及构建方法<160>70<170>siposequencelisting1.0<210>1<211>318<212>dna<213>酵酒酵母(saccharomycescerevisiae)<400>1cgcaagctttgagtaaatttggcaattgtgttcaagtcaacgcctggaatcattcccagc60gttttattatggactgggactcttgcaactgttctactggcacagtggatttatagtgaa120ctggatttttcaaaaatggcttcactttgagaaacactatttactatgtttgttaacttc180ttaacagtgtgtattacttttttgccatattgatattaaaaaagtcatttcgactgctga240cagagaatacaactagtaactggagagatagctggtgcagtgagtgattccacataatgc300cagacttctctgcagaca318<210>2<211>318<212>dna<213>酵酒酵母(saccharomycescerevisiae)<400>2cgcaagctttgagtaaatttggcaattgtgttcaagtcaacgcctggaatcattcccagc60gttttattatggactgggactcttgcaactgttctactggcacagtggatttatagtgaa120ctggatttttcaaaaatggcttcactttgagaaacactatttactatgtttgttaacttc180ttaacagtgtgtattacttttttgccatattgatattaaaaaagtcatttcgactgctga240cagagaatacaactagtaactggagagatagctggtgcagtgagtgattccacataatgc300cagacttctctgcagaca318<210>3<211>428<212>dna<213>酵酒酵母(saccharomycescerevisiae)<400>3ctgaagcttatcataaagaaattaaccaactattgagaatgaacacttgggatacaaaca60aatattatgatagaaatgacatagatcctaagaaagtaataaactcaatgtttatattta120acaaaaaacgtgatggtacacacaaagccagatttgttgcaagaggcgacattcaacacc180ccgatacatatgattctgatatgcaatccaataccgtacatcactatgcactgatgacgt240cattgtcaatcgcattagacaatgactattatatcacacagctggacatatcctctgctt300acttatatgctgatatcaaagaagaattatacataagacctccaccacatttaggtttga360atgataagttactacgtttgagaaaatcactctatggtttgaaacaaagtggtgcaaacc420tgcagtca428<210>4<211>488<212>dna<213>酵酒酵母(saccharomycescerevisiae)<400>4tatcccgggtggtatgaaaccattaaatcatatttaataaattgttgcgacatgcaagaa60gttcgcggatggtcatgcgtatttaagaatagtcaagtaacaatttgcttattcgttgat120gatatgatattgttcagcaaagacttaaatgcaaatgagaaaatcataacaacactcaag180aaacaatacgatacaaagataataaatctgggtgaaagtgataacgaaattcagtacgac240atacttggattagagatcaaatatcaaagaagcaagtacatgaaattaggtatggaaaaa300tccttgacagaaaaattacccaaactaaacgtacctttgaacccaaaaggaaagaaactt360agcgctccaggtcaaccaggtcattatatagaccaggatgaactagaaatagatgaagat420gaatacaaagagaaagtacatgaaatgcaaaagttgattggtctagcttcttatgttggg480aattcagt488<210>5<211>296<212>dna<213>酵酒酵母(saccharomycescerevisiae)<400>5cgtaagcttttctcgtgccctgagtggtgcacaaaagaagaaaagggaaaaaaaaagcga60acgaggagtgacgtggacgcgatgaaggtgactcagccaatccgggtgagccgcaccctt120cgtcctcagtgacctctcacgtgccctgtatcagccagccactgacagttttgtttttgc180cgcccagctcaaaactcagatcggatgtgctttagtttttatcttcgttatgtggatgtt240tcattagcttaaagcataggataccgagaaaccgtaacggtttcatgctgcagtgt296<210>6<211>318<212>dna<213>酵酒酵母(saccharomycescerevisiae)<400>6atacccgggttgaccggtttttgagtttatggttttaaagttcgtcagttgttccgacgc60ttagattttaaggagcttgtcaaaatgcctaaaccacaatccttacagcactcaaaatca120acaaaaagtgagaaggtttgaaagtagtacatcccctcttccctttgatttttagccgcc180aaaactaccaacccacacagcacaaaatgtgttactcctcgaaaaaatctgttacccttt240tcccgccatggttgccagggaaacagtggggacttctgcgttgcttctatttttcccccc300tccttcctcgaattctgc318<210>7<211>282<212>dna<213>酵酒酵母(saccharomycescerevisiae)<400>7cactaagctttcagaaatcaaataaggcagaggaagtggttttgtcgtattcaatgacta60attgtaagactcgagtataacttatgcagtttccaagaagtaagcaaacagtttctctca120acctttggtgacacactatgcgcatgcaagtcgtaatgcttctctatttatgtgtgggtt180tattaaaatagttagaattatctaggtttgtgggaatttggccatgaatattgaatagta240acaagaacaaagcgtgaaaatgttgaatataatctgcagtac282<210>8<211>305<212>dna<213>酵酒酵母(saccharomycescerevisiae)<400>8atacccggggaaagctgaagacatcaaatcatgttctccaaatctttctgtctcgtatat60tttctttgatatttgttttatgcggaaattgcagtggaaacaatcgcgaaacattcgaga120aagaaactaggtagctacaaaagtatggaaaaaagggtaataagcattccttttttttta180gccgcaaagcctgcttaaccatacatgaagcactttaacatatagatgctgctcttcact240gttacattatatacagaaaaaagtgctcgcaataagaaacaaaccttcagaaaaaggaat300tctgc305<210>9<211>468<212>dna<213>酵酒酵母(saccharomycescerevisiae)<400>9tcgaagctttatcgaacaaggagcgttgaagtaagaaaaacaaacgaagaactgaataaa60cacagcctgcttcaaattatggaaggagctgttacacatggtaattttcttatttttttt120gatagatggtggaaagtggtagcgtggaagttgcggattatcacgacgttttatgggctc180taaatcggtataagctcaacaaatggttgagggccgaataaacgaaaaattattagacca240taattgctaccagctgaaagattatctcattataatacgcttgaaaccctctaaaatgtc300attcgtagttaagtacacttttttgatctctccaaggttagccgcccatataaaaaactc360taggaaccaaatttggtatactgcgtgcgcttgagattcagtttttgtgtaaatcggttg420ctgcaaactatttatattaatgttacatcgatgcaagatctgcagtac468<210>10<211>468<212>dna<213>酵酒酵母(saccharomycescerevisiae)<400>10tcgaagctttatcgaacaaggagcgttgaagtaagaaaaacaaacgaagaactgaataaa60cacagcctgcttcaaattatggaaggagctgttacacatggtaattttcttatttttttt120gatagatggtggaaagtggtagcgtggaagttgcggattatcacgacgttttatgggctc180taaatcggtataagctcaacaaatggttgagggccgaataaacgaaaaattattagacca240taattgctaccagctgaaagattatctcattataatacgcttgaaaccctctaaaatgtc300attcgtagttaagtacacttttttgatctctccaaggttagccgcccatataaaaaactc360taggaaccaaatttggtatactgcgtgcgcttgagattcagtttttgtgtaaatcggttg420ctgcaaactatttatattaatgttacatcgatgcaagatctgcagtac468<210>11<211>418<212>dna<213>酵酒酵母(saccharomycescerevisiae)<400>11cgtaagcttaattgtagccgctgaaggcggatggttttgagagaagttgatgttcaaaga60atcgtgtatgataaaaaaaaaaaattgtaaccaattcgtaaatactgaaactctttgcag120taagaaatctttcattcccagagagaacgcggaaatattcagcctaagtctctccaaatt180gatttctttcattttggtagcgaaagacttagtatcaacctcaaaaagctgaaatttttg240tagatatatgtgcagatgacatgaagtatccagctcttttgtgaatatggaagcaagttt300ctttcaaattattatcgacttttcctctgccccttttaccttataactttaggaaacact360accctattcatactcaactaaaaaagtagtacgttttcttctttgagagctgcagctg418<210>12<211>418<212>dna<213>酵酒酵母(saccharomycescerevisiae)<400>12tatcccggggtatggtgcaaaccagtgcaatctgattccagattgccctcacgtgaggct60tcagtaaatctcgatccctctacatattatattatctcctacttggcaaaatagatgtgg120ctttagttgatgaaaatttaccacgtcaactatttatgtcctttctacaataaaccccga180cctactatcctctatgttgtttttaacataccatacgagcgtcatatgtcttttggttca240ctggggaagaccatcactgttgaacgtttgttgaaaagttcgagatgatgaagattgcat300aacaaaatgtaaagttatatttagaaactataacggttcaagacattcaacactataaca360tagcttggctggcttgaacaaaaagaggaagaagcaaaagcaagagtaagaattcgac418<210>13<211>418<212>dna<213>酵酒酵母(saccharomycescerevisiae)<400>13tatcccggggtatggtgcaaaccagtgcaatctgattccagattgccctcacgtgaggct60tcagtaaatctcgatccctctacatattatattatctcctacttggcaaaatagatgtgg120ctttagttgatgaaaatttaccacgtcaactatttatgtcctttctacaataaaccccga180cctactatcctctatgttgtttttaacataccatacgagcgtcatatgtcttttggttca240ctggggaagaccatcactgttgaacgtttgttgaaaagttcgagatgatgaagattgcat300aacaaaatgtaaagttatatttagaaactataacggttcaagacattcaacactataaca360tagcttggctggcttgaacaaaaagaggaagaagcaaaagcaagagtaagaattcgac418<210>14<211>418<212>dna<213>酵酒酵母(saccharomycescerevisiae)<400>14tgacccgggaaatgtaaagaaattgcagaatgttctgattacttttcatcacccaaagag60tccatactaccactaccaccaaccattgccggtgagcggtttcttgctcattaacatgac120gtacaagggaacggaatgtcccttgtacgttgtaatgtcttttgctctcgtaaagaagcc180tatacacgccccttccttccgcccttcggcatccgctgccaactgggtgggggctcaatg240gatctctttactctggcccttagaagccttctgaaattctcggcttaggcatcgaacgat300gaagaaactaactaaaaatatacgccatttttttggggagggggggccgcactgaaacgc360gaaatgccaaaaaaacgcgaaacgccgcagtaaaaggtcagtaccgcgcgaattctga418<210>15<211>473<212>dna<213>酵酒酵母(saccharomycescerevisiae)<400>15ctgaagcttggggaaaaacttcaaaaactaacaaaaagaagtgttaaagatgaacttttt60tcttttgtatcgtcggcttgccacattcgataagttaataaaacacaaacgggcaacaca120tctacgtactttttatgtgaagacaagagaaagaatacatcactgatacgtgcgtctatt180gcgcagcactgtactattgcgaagaactgagtgaaggcaaagaatgagatgatcgatgct240tagccgactgttaaacttactgagcggtagcctctactagttccttagtcagatcgcact300caacttggaatgttccgtcaggcccacggaagaaaaagcaacaaaagaaagaaaaaccgg360acaacatgcgggaaaaatgcaaactgtgtgagggaaaaattaactttaaagattacatca420acggcgggaaaagatccttccgcagtccttctactgcggtttccctgcaggca473<210>16<211>454<212>dna<213>酵酒酵母(saccharomycescerevisiae)<400>16tatcccgggcagctaaccggagggaagacaagtcaagcggacatgcagttcccgctcagg60cttctttgcagagaaatcggaatatgaattttttgtgttttttgttaacttctttaccat120tcgcatcacgggaagagtgtgttggcataagaggcgcaagaaaaaggtgacccacggtgg180tgggaaagagaagaaaaagccgtttgcggaaagcaatgttttgcccattcagaaacaata240tctttgctgtatccctacatgtattatgccgcgttgcttgtgcaggtacggagtccctgc300acgatttttcaacaaggtctagatttgctaagacgatgaggcggagcccgaaacaaacaa360gccaaaaaccggaatttcgcgcttggtgtgctttcgcatcttttcgtgtggtgaatgcac420ctggtgcacgctgcgaaggtgacgtgaattcgta454<210>17<211>454<212>dna<213>酵酒酵母(saccharomycescerevisiae)<400>17tatcccgggcagctaaccggagggaagacaagtcaagcggacatgcagttcccgctcagg60cttctttgcagagaaatcggaatatgaattttttgtgttttttgttaacttctttaccat120tcgcatcacgggaagagtgtgttggcataagaggcgcaagaaaaaggtgacccacggtgg180tgggaaagagaagaaaaagccgtttgcggaaagcaatgttttgcccattcagaaacaata240tctttgctgtatccctacatgtattatgccgcgttgcttgtgcaggtacggagtccctgc300acgatttttcaacaaggtctagatttgctaagacgatgaggcggagcccgaaacaaacaa360gccaaaaaccggaatttcgcgcttggtgtgctttcgcatcttttcgtgtggtgaatgcac420ctggtgcacgctgcgaaggtgacgtgaattcgta454<210>18<211>454<212>dna<213>酵酒酵母(saccharomycescerevisiae)<400>18tatcccgggcagctaaccggagggaagacaagtcaagcggacatgcagttcccgctcagg60cttctttgcagagaaatcggaatatgaattttttgtgttttttgttaacttctttaccat120tcgcatcacgggaagagtgtgttggcataagaggcgcaagaaaaaggtgacccacggtgg180tgggaaagagaagaaaaagccgtttgcggaaagcaatgttttgcccattcagaaacaata240tctttgctgtatccctacatgtattatgccgcgttgcttgtgcaggtacggagtccctgc300acgatttttcaacaaggtctagatttgctaagacgatgaggcggagcccgaaacaaacaa360gccaaaaaccggaatttcgcgcttggtgtgctttcgcatcttttcgtgtggtgaatgcac420ctggtgcacgctgcgaaggtgacgtgaattcgta454<210>19<211>318<212>dna<213>酵酒酵母(saccharomycescerevisiae)<400>19ctgaagctttgactctgtgaaaataatgtataagagtgggcaacggtgttctcgtagata60tagcgctgtgctgaaccaagtttagttttttttactttccaccaaaggcaaagaaactga120ttaatttattgctgtgacgaagggatggagtatgattgcggttgtgactgaatctctttg180tagactttcggacaaccacaagatccactttctggtaaagttaatttcaagatcgtgttt240ttttctgtcttttctttctcttctcatagccaaaatcgtaccacaaggaaaaaaagcacc300gggttaccactgcagtcg318<210>20<211>318<212>dna<213>酵酒酵母(saccharomycescerevisiae)<400>20ctgaagctttgactctgtgaaaataatgtataagagtgggcaacggtgttctcgtagata60tagcgctgtgctgaaccaagtttagttttttttactttccaccaaaggcaaagaaactga120ttaatttattgctgtgacgaagggatggagtatgattgcggttgtgactgaatctctttg180tagactttcggacaaccacaagatccactttctggtaaagttaatttcaagatcgtgttt240ttttctgtcttttctttctcttctcatagccaaaatcgtaccacaaggaaaaaaagcacc300gggttaccactgcagtcg318<210>21<211>23<212>dna<213>人工序列(artificialsequence)<400>21agtcacatcaagatcgtttatgg23<210>22<211>23<212>dna<213>人工序列(artificialsequence)<400>22actccacttcaagtaagagtttg23<210>23<211>23<212>dna<213>人工序列(artificialsequence)<400>23gcacggaatatgggactacttcg23<210>24<211>5701<212>dna<213>人工序列(artificialsequence)<400>24gggcgaattgggcccgacgtcgcatgctcccggccgccatggcggccgcgggaattcgat60tgaccacatatggccgcctaggcctcgagctgcagtaacgggagcgtaatggtgatggaa120ctggacgaatccatcaatagatacgtcctgaggaccgtgctacccaaatggactgattgt180gagggagacctaactacatagtgtttaaagattacggatatttaacttacttagaataat240gccatttttttgagttataataatcctacgttagtgtgagcgggatttaaactgtgagga300ccttaatacattcagacacttctgcggtatcaccctacttattcccttcgagattatatc360taggaacccatcaggttggtggaagattacccgttctaagacttttcagcttcctctatt420gatgttacacctggacaccccttttctggcatccagtttttaatcttcagtggcatgtga480gattctccgaaattaattaaagcaatcacacaattctctcggataccacctcggttgaaa540ctgacaggtggtttgttacgcatgctaatgcaaaggagcctatatacctttggctcggct600gctgtaacagggaatataaagggcagcataatttaggagtttagtgaacttgcaacattt660actattttcccttcttacgtaaatatttttctttttaattctaaatcaatctttttcaat720tttttgtttgtattcttttcttgcttaaatctataactacaaaaaacacatacataaact780aaaaatggtctccttcacctccctcctcgccggcgtcgccgccatctcgggcgtcttggc840cgctcccgccgccgaggtcgaatccgtggctgtggagaagcgccagtcggcctgcactct900ccaatcggagactcacccgcctctgacatggcagaaatgctcgtctggtggcacgtgcac960tcaacagacaggctccgtggtcatcgacgccaactggcgctggactcacgctacgaacag1020cagcacgaactgctacgatggcaacacttggagctcgaccctatgtcctgacaacgagac1080ctgcgcgaagaactgctgtctggacggtgccgcctacgcgtccacgtacggagttaccac1140gagcggtaacagcctctccattggctttgtcacccagtctgcgcagaagaacgttggcgc1200tcgcctttaccttatggcgagcgacacgacctaccaggaattcaccctgcttggcaacga1260gttctctttcgatgtcgatgtttcgcagctgccgtgcggcttgaacggagctctctactt1320cgtgtccatggacgcggatggtggcgtgagcaagtatcccaccaacaccgctggcgccaa1380gtacggcacggggtactgtgacagccagtgtccccgcgatctgaagttcatcaatggcca1440ggccaacgttgagggctgggagccgtcatccaacaacgcgaacacgggcattggaggaca1500cggaagctgctgctctgagatggatatctgggaggccaactccatctccgaggctcttac1560cccccacccttgcacgactgtcggccaggagatctgcgagggtgatgggtgcggcggaac1620ttactccgataacagatatggcggcacttgcgatcccgatggctgcgactggaacccata1680ccgcctgggcaacaccagcttctacggccctggctcaagctttacccacgataccaccaa1740gaaattgaccgttgtcacccagttcgagacgtcgggtgccatcaaccgatactatgtcca1800gaatggcgtcactttccagcagcccaacgccgagcttggtagttactctggcaacgagct1860caacgatgattactgcacagctgaggaggcagaattcggcggatcctctttctcagacaa1920gggcggcctgactcagttcaagaaggctacctctggcggcatggttctggtcatgagtct1980gtgggatgattactacgccaacatgctgtggctggactccacctacccgacaaacgagac2040ctcctccacacccggtgccgtgcgcggaagctgctccaccagctccggtgtccctgctca2100ggtcgaatctcagtctcccaacgccaaggtcaccttctccaacatcaagttcggacccat2160tggcagcaccggcaaccctagcggcggcaaccctcccggcggaaacccgcctggcaccac2220caccacccgccgcccagccactaccactggaagctctcccggacctacccagtctcacta2280cggccagtgcggcggtattggctacagcggccccacggtctgcgccagcggcacaacttg2340ccaggtcctgaacccttactactctcagtgcctgtaagcgaatttcttatgatttatgat2400ttttattattaaataagttataaaaaaaataagtgtatacaaattttaaagtgactctta2460ggttttaaaacgaaaattcttattcttgagtaactctttcctgtaggtcaggttgctttc2520tcaggtatagcatgaggtcgctcttattgaccacacctctaccggcatgccgagcaaatg2580cctgcaaatcgctccccatttcacccaattgtagatatgctaactccagcaatgagttga2640tgaatctcggtgtgtattttatgtcctcagaggacaacacctgttgtaatcgttcttcca2700cacggatagtactgactatcgtcggccgcctaggccatggtacaacatcactagtgaatt2760cgcggccgcctgcaggtcgaccatatgggagagctcccaacgcgttggatgcatagcttg2820agtattctatagtgtcacctaaatagcttggcgtaatcatggtcatagctgtttcctgtg2880tgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaa2940gcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgct3000ttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggaga3060ggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtc3120gttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaa3180tcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgt3240aaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaa3300aatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgttt3360ccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctg3420tccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctc3480agttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagccc3540gaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgactta3600tcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgct3660acagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatc3720tgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaa3780caaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaa3840aaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaa3900aactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatcctt3960ttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgac4020agttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatcc4080atagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggc4140cccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaata4200aaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatc4260cagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgc4320aacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttca4380ttcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaa4440gcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatca4500ctcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttt4560tctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagt4620tgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtg4680ctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgaga4740tccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcacc4800agcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcg4860acacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcag4920ggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggg4980gttccgcgcacatttccccgaaaagtgccacctgatgcggtgtgaaataccgcacagatg5040cgtaaggagaaaataccgcatcaggaaattgtaagcgttaatattttgttaaaattcgcg5100ttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatccct5160tataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagt5220ccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgat5280ggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagca5340ctaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaac5400gtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgta5460gcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcg5520tccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgc5580tattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccag5640ggttttcccagtcacgacgttgtaaaacgacggccagtgaattgtaatacgactcactat5700a5701<210>25<211>5551<212>dna<213>人工序列(artificialsequence)<400>25gggcgaattgggcccgacgtcgcatgctcccggccgccatggcggccgcgggaattcgat60tgaccacatatggccgcctaggcctcgagctgcagtaacgggagcgtaatggtgatggaa120ctggacgaatccatcaatagatacgtcctgaggaccgtgctacccaaatggactgattgt180gagggagacctaactacatagtgtttaaagattacggatatttaacttacttagaataat240gccatttttttgagttataataatcctacgttagtgtgagcgggatttaaactgtgagga300ccttaatacattcagacacttctgcggtatcaccctacttattcccttcgagattatatc360taggaacccatcaggttggtggaagattacccgttctaagacttttcagcttcctctatt420gatgttacacctggacaccccttttctggcatccagtttttaatcttcagtggcatgtga480gattctccgaaattaattaaagcaatcacacaattctctcggataccacctcggttgaaa540ctgacaggtggtttgttacgcatgctaatgcaaaggagcctatatacctttggctcggct600gctgtaacagggaatataaagggcagcataatttaggagtttagtgaacttgcaacattt660actattttcccttcttacgtaaatatttttctttttaattctaaatcaatctttttcaat720tttttgtttgtattcttttcttgcttaaatctataactacaaaaaacacatacataaact780aaaaatggtctccttcacctccctcctcgccggcgtcgccgccatctcgggcgtcttggc840cgctcccgccgccgaggtcgaatccgtggctgtggagaagcgccaagcttgctcaagcgt900ctggggccaatgtggtggccagaattggtcgggtccgacttgctgtgcttccggaagcac960atgcgtctactccaacgactattactcccagtgtcttcccggcgctgcaagctcaagctc1020gtccacgcgcgccgcgtcgacgacttctcgagtatcccccacaacatcccggtcgagctc10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aagacacgacttatcgc3360cactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacag3420agttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcg3480ctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaa3540ccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaag3600gatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaact3660cacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaa3720attaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagtt3780accaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatag3840ttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggcccca3900gtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaacc3960agccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagt4020ctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacg4080ttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattca4140gctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcgg4200ttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactca4260tggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctg4320tgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgct4380cttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctca4440tcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatcca4500gttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcg4560tttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacac4620ggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggtt4680attgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttc4740cgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacat4800taacctataaaaataggcgtatcacgaggccctttcgtc4839<210>31<211>29<212>dna<213>人工序列(artificialsequence)<400>31cgcaagctttgagtaaatttggcaattgt29<210>32<211>29<212>dna<213>人工序列(artificialsequence)<400>32tgtctgcagagaagtctggcattatgtgg29<210>33<211>29<212>dna<213>人工序列(artificialsequence)<400>33atacccgggccactcgtcagccagttcga29<210>34<211>29<212>dna<213>人工序列(artificialsequence)<400>34gtcgaattcattcttgagaacaggtaaat29<210>35<211>29<212>dna<213>人工序列(artificialsequence)<400>35ctgaagcttatcataaagaaattaaccaa29<210>36<211>29<212>dna<213>人工序列(artificialsequence)<400>36tgactgcaggtttgcaccactttgtttca29<210>37<211>29<212>dna<213>人工序列(artificialsequence)<400>37tatcccgggtggtatgaaaccattaaatc29<210>38<211>29<212>dna<213>人工序列(artificialsequence)<400>38actgaattcccaacataagaagctagacc29<210>39<211>29<212>dna<213>人工序列(artificialsequence)<400>39cgtaagcttttctcgtgccctgagtggtg29<210>40<211>29<212>dna<213>人工序列(artificialsequence)<400>40acactgcagcatgaaaccgttacggtttc29<210>41<211>29<212>dna<213>人工序列(artificialsequence)<400>41atacccgggttgaccggtttttgagttta29<210>42<211>29<212>dna<213>人工序列(artificialsequence)<400>42gcagaattcgaggaaggaggggggaaaaa29<210>43<211>29<212>dna<213>人工序列(artificialsequence)<400>43cactaagctttcagaaatcaaataaggca29<210>44<211>29<212>dna<213>人工序列(artificialsequence)<400>44gtactgcagattatattcaacattttcac29<210>45<211>29<212>dna<213>人工序列(artificialsequence)<400>45atacccggggaaagctgaagacatcaaat29<210>46<211>29<212>dna<213>人工序列(artificialsequence)<400>46gcagaattcctttttctgaaggtttgttt29<210>47<211>29<212>dna<213>人工序列(artificialsequence)<400>47tcgaagctttatcgaacaaggagcgttga29<210>48<211>29<212>dna<213>人工序列(artificialsequence)<400>48gtactgcagatcttgcatcgatgtaacat29<210>49<211>29<212>dna<213>人工序列(artificialsequence)<400>49tatcccgggtttagccatgttcctagttt29<210>50<211>29<212>dna<213>人工序列(artificialsequence)<400>50acggaattctttttctcaacctagcaaag29<210>51<211>29<212>dna<213>人工序列(artificialsequence)<400>51cgtaagcttaattgtagccgctgaaggcg29<210>52<211>29<212>dna<213>人工序列(artificialsequence)<400>52cagctgcagctctcaaagaagaaaacgta29<210>53<211>29<212>dna<213>人工序列(artificialsequence)<400>53tatcccggggtatggtgcaaaccagtgca29<210>54<211>29<212>dna<213>人工序列(artificialsequence)<400>54gtcgaattcttactcttgcttttgcttct29<210>55<211>29<212>dna<213>人工序列(artificialsequence)<400>55tcgaagcttgatctttaaaaggtccggtg29<210>56<211>29<212>dna<213>人工序列(artificialsequence)<400>56gtactgcagccaatacttgctccatttaa29<210>57<211>29<212>dna<213>人工序列(artificialsequence)<400>57tgacccgggaaatgtaaagaaattgcaga29<210>58<211>29<212>dna<213>人工序列(artificialsequence)<400>58tcagaattcgcgcggtactgaccttttac29<210>59<211>29<212>dna<213>人工序列(artificialsequence)<400>59ctgaagcttggggaaaaacttcaaaaact29<210>60<211>29<212>dna<213>人工序列(artificialsequence)<400>60tgcctgcagggaaaccgcagtagaaggac29<210>61<211>29<212>dna<213>人工序列(artificialsequence)<400>61tatcccgggcagctaaccggagggaagac29<210>62<211>29<212>dna<213>人工序列(artificialsequence)<400>62tacgaattcacgtcaccttcgcagcgtgc29<210>63<211>29<212>dna<213>人工序列(artificialsequence)<400>63cggaagctttttaagattagtgcggaggc29<210>64<211>29<212>dna<213>人工序列(artificialsequence)<400>64tcactgcaggttaaaacgggctatccata29<210>65<211>29<212>dna<213>人工序列(artificialsequence)<400>65gtacccgggttcagaatctcatctgtaaa29<210>66<211>29<212>dna<213>人工序列(artificialsequence)<400>66gatgaattcacaacaattttacttaataa29<210>67<211>29<212>dna<213>人工序列(artificialsequence)<400>67ctgaagctttgactctgtgaaaataatgt29<210>68<211>29<212>dna<213>人工序列(artificialsequence)<400>68cgactgcagtggtaacccggtgctttttt29<210>69<211>29<212>dna<213>人工序列(artificialsequence)<400>69gtacccgggtgatggtggaatcccactgt29<210>70<211>29<212>dna<213>人工序列(artificialsequence)<400>70gacgaattcgtaaataactaatacataga29当前第1页12