CN116676328A - 一种提高尖孢镰刀菌基因敲除效率的方法及应用 - Google Patents
一种提高尖孢镰刀菌基因敲除效率的方法及应用 Download PDFInfo
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Abstract
本发明公开一种可用于提高尖孢镰刀菌基因敲除效率的方法及其应用，属于分子生物学技术领域。本发明是在尖孢镰刀菌ura3缺失突变体的基础上，利用ura3基因替换非同源末端连接（NHEJ）相关基因ku70和lig4，得到ku70和lig4缺失突变体，以此作为出发菌株,应用于CPS1基因的敲除，将敲除效率分别提高了38.06倍和40.07倍，该方法显著提高了尖孢镰刀菌中的基因敲除效率，为尖孢镰刀菌基因功能研究提供了高效的工具和方法，为其致病机理和遗传特性研究提供了技术支持。
Description
技术领域
本发明属于分子生物学技术领域，具体涉及一种提高尖孢镰刀菌基因敲除效率的方法及应用。
背景技术
乳清酸5′-磷酸脱羧酶（OMP脱羧酶）是参与嘧啶生物合成的关键酶，催化乳清酸单磷酸（OMP）脱羧形成尿苷单磷酸（UMP）。在尖孢镰刀菌（Fusarium oxysporum）中，合成OMP脱羧酶的基因为ura3。ura3基因缺失的突变体表现为尿嘧啶营养缺陷，需要依赖外源的尿苷、尿嘧啶才能正常生长。同样，将敲除了ura3基因的菌株中插入ura3基因后，在不添加尿苷、尿嘧啶的普通培养基上即可正常生长。
断裂DNA双链修复（Double strand break，DSB）在所有生物体中都极为重要，否则可能导致突变或染色体重排，产生致命的染色体畸变，最终导致细胞死亡。DSB有两种途径：同源重组（Homologous recombination，HR）和非同源末端连接（Non-homologous endjoining，NHEJ）。在高等真核生物如哺乳动物和植物以及丝状真菌中，NHEJ是主要的修复途径。但是在分子生物学领域的遗传转化过程中，NHEJ导致不精确的错配是基因敲除效率低的一大原因。在大多数高等真核生物中，lig4和ku70是NHEJ的关键因子。
尖孢镰刀菌（F. oxysporum）是引起植物枯萎病和根腐病的主要病原物，从寄主植物根部侵入，沿着维管束向上侵染，造成作物大规模减产，严重影响产量和品质。高效敲除体系的建立有利于提高遗传转化效率，为更好地开展尖孢镰刀菌致病机理的研究奠定基础，从而能更有效的控制枯萎病和根腐病的传播和发展，减少经济损失。精氨酸是生物体内最重要的氨基酸之一，影响生物的生化和生理功能。氨甲酰磷酸合成酶（CPS1）参与精氨酸生物合成，氨甲酰磷酸合成标志着精氨酸从头合成的开始。本发明运用高效基因敲除效率的方法获得尖孢镰刀菌中编码氨甲酰磷酸合成酶CPS1基因的缺失突变体。
发明内容
本发明通过PEG介导的真菌原生质体转化，利用转化片段与基因组中目的基因片段的同源置换，以尿嘧啶营养缺陷菌株为基础构建了ku70和lig4缺失突变体来提高尖孢镰刀菌基因敲除效率，为尖孢镰刀菌基因功能研究提供了高效的工具和方法。本发明的技术方案如下：
尖孢镰刀菌ku70和lig4缺失突变体的制备方法，步骤如下：
以含有尖孢镰刀菌的DNA作为模板，用引物对ura3-F/R扩增ura3基因（SEQ ID NO:7）；分别用引物对ku70-up-F/R、ku70-down-F/R、lig4-up-F/R和lig4-down-F/R扩增ku70的上游片段（SEQ ID NO:3）、ku70的下游片段（SEQ ID NO:4）、lig4的上游片段（SEQ ID NO:5）和lig4的下游片段（SEQ ID NO:6）；对上游片段、ura3片段和下游片段按摩尔质量比1：3：1进行融合分别得到lig4和ku70的同源置换臂；将融合片段通过PEG介导转化尿嘧啶营养缺陷菌株（∆ura3）的原生质体，并在普通T-TOP培养基上培养，筛选得到尖孢镰刀菌ku70和lig4的缺失突变体。
本发明提供了一种尖孢镰刀菌ku70和lig4缺失突变体在基因敲除中的应用，是将尖孢镰刀菌ku70和lig4缺失突变体作为出发菌株，实现高效率的基因敲除。本发明的有益效果为：本发明的方法在不引入其他抗性基因情况下，准确、有效的获得尖孢镰刀菌ku70和lig4缺失突变体，应用于其它目的基因的高效定点敲除。
附图说明
图1为尖孢镰刀菌ku70和lig4基因敲除策略。A图为ku70敲除策略，B图为lig4敲除策略；
图2为ku70和lig4缺失突变体PCR鉴定电泳图，所用引物在图中标出；
图3为尖孢镰刀菌CPS1基因的敲除策略图。
图4为野生菌株WT、ku70和lig4缺失突变体（∆ku70和∆lig4）为出发菌株敲除CPS1基因的敲除效率验证图。A图为以野生菌株为出发菌株的敲除效率验证图，B图为以ku70缺失突变体为出发菌株的敲除效率验证图，C图为以lig4缺失突变体为出发菌株的敲除效率验证图。1-48为转化子，WT作为对照。
具体实施方式
本发明所用培养基（1L），其组成成分如下所示：
PDA培养基：土豆200 g，琼脂20 g，葡萄糖20 g。PDB培养基：土豆200 g，葡萄糖20g。MM培养基：KH2PO4 1 g，MgSO4·7H2O 0.5 g，KCl 0.5 g，NaNO3 2 g，葡萄糖20 g，蔗糖200g，琼脂15 g。T-TOP培养基：KH2PO4 1 g，MgSO4·7H2O 0.5 g，KCl 0.5g，NaNO3 2 g，葡萄糖20g，蔗糖200 g，琼脂10 g。
本发明所用的主要试剂，如下所示：
Fastpfu酶、easytaq酶均购自北京全式金公司，UNIQ-10 柱式真菌基因组抽提试剂盒购自上海生工，崩溃酶购自Sigma公司。潮霉素购自索莱宝公司；TEC：1M PH=7.5的Tris-HCl，0.5M PH=8的EDTA，1M CaCl2·2H2O。STC（500mL）：山梨醇72.88 g，1M CaCl2·2H2O，1M PH=7.5的Tris-HCl。PEG：PEG4000 120g、MoPS（3-N-吗啉基）丙磺酸25.116g加蒸馏水水浴加热溶解至200mL，室温避光保存。试验所用菌株为尖孢镰刀菌粘团专化型2号生理小种（Fusarium oxysporum f. sp. conglutinans, FOC），由中国农业科学院蔬菜花卉研究所赠予。
在本发明中所使用的术语，除非有另外说明，一般具有本领域普通技术人员通常理解的含义。下面结合具体实施例，并参照数据进一步详细的描述本发明。以下实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。
实施例1 ku70和lig4缺失突变体（∆ku70和∆lig4）的获得
制备尖孢镰刀菌ku70和lig4缺失突变体，步骤如下：
（1）克隆尖孢镰刀菌ku70和lig4基因上下游片段以及ura3基因
使用UNIQ-10柱式真菌基因组抽提试剂盒提取尖孢镰刀菌的基因组DNA，并将其作为模板。分别用引物对ku70-up-F/R、ku70-down-F/R、lig4-up-F/R和lig4-down-F/R扩增ku70的上游片段、下游片段、lig4的上游片段和下游片段；用引物对ura3-F/R扩增ura3基因。所用的引物位置均在图1中标出，引物序列见表1。PCR体系：模板2 μL，dNTPs 4 μL，分别加引物up-F/R、down-F/R各1 μL，5×buffer 10 μL，ddH2O 31 μL，fastpfu酶1 μL。PCR程序：95 ℃预变性2 min，95 ℃变性20 s，57 ℃退火20 s，72℃延伸2 min，变性至延伸重复35个循环，最后72℃延伸5 min。将PCR产物进行琼脂糖凝胶电泳，纯化回收片段。
（2）融合片段的扩增
使用步骤（1）所得产物按up片段、ura3片段和down片段的摩尔质量比1:3:1添加作为模板，加dNTPs 4 μL，5×buffer 10 μL，fastpfu酶1 μL，用ddH2O补足至50 μL作为反应体系。PCR程序如下：95 ℃预变性2 min，95 ℃变性20 s，58 ℃退火10 min，72℃延伸2min，变性至延伸重复10个循环，最后72℃延伸5 min。使用上述所得产物2 μL作为模板，引物对ku70-up-F/ku70-down-R和lig4-up-F/lig4-down-R各1 μL，dNTPs 4 μL，5×buffer10μL，ddH2O 31 μL，fastpfu酶1 μL分别扩增ku70和lig4的同源置换臂。PCR程序如下：95 ℃预变性2 min，95 ℃变性20 s，57 ℃退火20 s，72 ℃延伸2 min，变性至延伸重复35个循环，最后72 ℃延伸5 min得到转化所需融合片段。
（3）制备尖孢镰刀菌原生质体
将崩溃酶液加到尖孢镰刀菌菌丝样品中，混匀，置于28 ℃、120 rpm的摇床崩溃4h。取6 mL酶裂解液，过滤，并不断用STC清洗，用灭菌的50mL的离心管收集滤液。3000rpm、4℃离心15 min，倒掉上清，用STC重悬原生质体，调整浓度为1×108 cfu/mL，备用。
（4）PEG介导原生质体转化
将2 μg融合片段加TEC至体积为60 μL，冰置20 min，12000 rpm、4 ℃离心2 min后吸取上清，每60 μL的转化片段对应100 μL原生质体。将上清样品加入到100 μL原生质体中轻轻混匀，冰置40 min。冰置结束后缓慢加入160 μL的PEG，用移液枪轻轻吹打充分混匀后，室温放置15 min，再加入1 mL的STC混匀，3000 rpm离心5 min，轻轻倒掉上清，沉淀用200 μL的STC重悬，置于冰上备用。用移液枪取50 μL溶液加入到10 mLMM培养中，然后倒入凝固好的MM培养基平板上，放入25 ℃培养箱中培养1 d。
（6）筛选尖孢镰刀菌的ku70和lig4缺失突变体
将培养1 d的MM培养基平板上覆盖T-Top培养基，继续培养5天。挑取转化子置于PDA培养基上培养3天，提取转化子的DNA作为模板，用引物ku70-check-F/R、ura3-F/R以及ku70-upcheck-F/R和ku70-downcheck-F/R进行PCR扩增鉴定ku70缺失突变体。用引物lig4-check-F/R、ura3-F/R以及lig4-upcheck-F/R和lig4-downcheck-F/R进行PCR扩增鉴定lig4缺失突变体。PCR扩增结果如图2所示，获得尖孢镰刀菌ku70和lig4缺失突变体（∆ku70和∆lig4），分离单孢纯化培养。
分别使用尖孢镰刀菌野生型WT、∆ku70和∆lig4作为出发菌株敲除CPS1基因，所用引物位置如图3所示，引物序列见表1。
以尖孢镰刀菌的DNA作为模板，用引物对CPS1-up-F/R扩增CPS1的上游片段（SEQID NO:9），用引物对CPS1-down-F/R扩增CPS1的下游片段（SEQ ID NO:10）。以含有hygR基因的质粒作为模板，用引物对hygR-F/R扩增hygR基因（SEQ ID NO:11）。再对上游片段、hygR片段和下游片段进行融合得到CPS1的同源置换臂。分别制备WT、∆ku70和∆lig4的原生质体，转化过程同上。T-TOP培养基添加80 μg/mL的潮霉素。
各挑取48个转化子计算敲除效率。提取转化子的DNA作为模板，用引物CPS1-check-F/R、hygR-F/R以及CPS1-upcheck-F/R和CPS1-downcheck-F/R进行PCR扩增鉴定CPS1缺失突变体。结果如图4所示，以野生型菌株为出发菌株，获得的CPS1缺失突变体的个数为1，敲除效率为2.08%，以∆ku70为出发菌株，获得的CPS1缺失突变体的个数为39，CPS1基因敲除效率为81.25%，相比于野生型提高了38.06倍；以∆lig4为出发菌株，获得的CPS1缺失突变体的个数为41，CPS1基因敲除效率为85.42%，相比于野生型提高了40.07倍，表明本发明所提供的方法及菌株可实现对目的基因的高效敲除。
上述实施例中所涉及的基因序列，如下所示：
SEQ ID NO:1 ku70基因：
ATGGCTGATAAACAGAATTGGCGAAAAGAAGACGAAGATGAGGAGGAGCAAGAACTTGATGAGAACGTGAGTTGATCGAACATTGGTACTCACCCCAACTCCATATTGATCCACCCATAGAACTATAAGGCACAGAAAGATGCCATCTTGCTTGCAATCGATGTTAGAAAATCTATGTTAGAGCCACCGCCGCCTTCGGACTCCAAAAAGGCAGATCGTGACAGCCCTGTCCAGGCGGCCTTAAAATGCGCCTATCATCTCATGGAACAACGCATCATCTCTAATCCCAAGGACATGATGGGTATTCTTCTCTTTGGAACGAAGAAATCAAAATTCCAGGACTCTGTAGATGGTCGAAGTGGTCTAGGTTACCCTCATTGTTACCTATTCACTGATCTCGACGTGCCCGCGGCAGAAGATGTGAAGGTCCTCAAAGCCCTGGTCGAAGATGGCGAGGACGAAGATGAGGTACTGACACCTTCGGATGAGCCTGTCAGCATGTCGAATGTGCTATTCTGCGCGAACCAGATCTTCACAACAAAGGCTGCGAACTTTGGCAGTCGTCGTTTGTTTATAGTAACTGACAACGACAATCCCCACGCATCTGACAAACAGGCCAAGTCCGCTGCAGCGGTACGAGCCAAGGATCTGTACGACCTTGGGATCCTGATAGATCTATTCCCTATCACCCGTGGAGACGAGAAATTCGACCTCAACAAGTTTTATGATGTAAGAATCGGTTTGAAGATAACAGGGAACCAGCTAACACGCGAAGGATATTATCTATCGTGACCCTGTTGGAGAGGCGAACATGACAGAAGTCCGAACATCGAAATCTGGAGACGGACTGACATTACTCAACTCGCTCATCTCAAATGTCAATTCCAAACAAACTGCAAAGAGAGCCCTGTTCTCAAATTTGCCATTCGAGATTGCTCCTGGGCTACGCATCTCAGTGAAGGGGTATAACATTGTTCATCGTCAAACACCAGCACGCACTTGTTACATTTGGCTGGACGGTGAAAAGCCGCAAATTGCTACAGGTGAAACTACACGAATCGCGGAGGATAGCACCAGGACCGTTGAAAAAGCAGAGATAAAGAAGGCGTACAAGTTTGGCGGCGAATACGTTTACTTCTCCCCAGACGAACAGAAGTCATTGAAGGATTTCGGATCACCCATCATTCGTATCATCGGCTTCAAACCGCGCAGTCTGCTCCCCGTTTGGGCAAGCACCAAGAAGTCTACATTCATCTTCCCCAGCGAAGAAGATTATGTTGGTTCGACCAGGGTTTTCACAGCGCTTTGGCAGAAACTTCTCAAGGATGACAAAATGGGTGTCGCTTGGTGTATCACTCGTGCTAATGCGCAGCCAATGTTGGCCGCCATCATACCGTCTAGAGAGCGGTCTGATGCTGACTCGGGAACGCCGTATCTGCCTGCTGGACTTTGGATCTATCCTCTACCATTCCAGGATGACCTGCGAAATATCAACCCACCTAGTGAAGTGCTGCGAAGTTCTGGAGAACTTACGACGCAGATGCGAACTATCATTCAGCAACTCCAGCTGCCAAAGGCTATGTACGATCCATTGAAATACCCCAATCCCGCGTTGCAGTGGCATTACAGAATCCTTCAAGCTCTTGCTTTGGAGGAAGAGGTACCCGAGAAGGCAGACGATGCAACGGAGCCAAAATACAAAGCGATCAGCAAACGAGCAGGCGGCTACTTGGAAGAATGGTCTGAATCTCTAGAAGAAGAGTCTGGTAAAGTTGCGAACAAGAAATCGACAAAGCGTGAAACGGACGATGAAGACATGGATAGACCGGTGAAGAAATCGAGAGGTTCATCAGAGAAAGCAACTGGTTCTTCCTTCAGTATGGCTCAATTGAAGGCCGCAATCGAAGGTGGGACTATTCAAAAAATGACGGTAGTTCAGCTCAAAGATATACTGGCTACTAAGGGTATGAGTACTGCTGGACGAAAGGTTGAGTTGATCGAGAGAATCGAGCAATGGATAGAGGAAAGTTCATGAGACTGAGGTCGAAGGGCACAATAGAGTGCATTGTCAGTGCTTTATATGAAGAATGGGTGGCCGGCGGAACAGGCGTTTTTGCTACTTTCTAAGGACATTGTTTATCAAGCTGCGAGCACTTGATCGTAATGAATCAAAATACTGAGGAGAATGGCCCCGCTCATTTGTTCCTTATTGTGATTGTGATTAAATGCTTTTTTTCATGGAAGCTTTCGAAGCCGTCGAAGCTTTCGCAACCACTTCCTTTTCATCACATTCACCAAAGCTTCTTTTACTCGACAGCAACATCACAAGTTGAACATCCTGACAAGCGAATCATAAAAACTGCCATAGTTGATTTCTATTCACTTTGATCTGCGTATCTCCTTGAAATATTCCAATATGAAATATGGCGACTGCAGGCGGGAGGATAGATTCATCCTCTATACTCACACCCCCTTCAGCACCCGTTAGTGGCTCTCAGTGGCGAGGCGCTCAACTCACACCCACTAAACTTCAGCTAAACACTCCCCGGCCCCTTACCTCATCTATCAAATCCGTAGCTCCACCATCACCTACCTGCGCGTTCGTGAGCCCGCAAACGCCAGGACAGCGTCACCCAAACATCCCATGGGCAACACTTGTCGCCTCATCACCTCAGAAGGAGCCGGGCGCTGACGAGTATAACTCAAGGCTATTGCGCCTGGCGCGGCGCCGGCCAACTTGCGAGCAACGAAAGACCATGGTCGATTTCCTCGCAAGCCTTGGTCTTGGCGGCGCGCAGACACCCGCAGCGTCAAGTAACTCGCAATGCGATTCGGTCGAGGAGCTCCAGCTTAAACTACGAAGCATTCTAGATGCCAAGGTGACACCGACACGGGCCGAAAATGTGGCTTTGACAATCGAATTGAGAGCCACGGTACGATTTCAGTTGCCTGTGTCCGAATCGGAGAATGGGACACTCGAGAGCCTAAACAACATCGATCCGTCACTGGGAGGAGCGCAATCTGTAGGAATGAATGGCAATCAGGATGAAGGACAGCTATCGCGCGTGGTATTTGCCAATGACACGCTCATGAATCAGCCACAAGACGACCCGACGCTCCAACGGTCTGTCGCAAAACATATCATTTCAATAATCAGCTCAACCGATGGGAGTACCTGGACCGTCCGAGAGGTGTCGCGCGTTACACAGGGTTGGACTTTTACGTATTTGTGCAAGGACTCGTATCAGCAATGGACTCGACAGAATGCGAAGAATCCAACGAAAACAATAGTAGGAGAGTTCAGTCAGAGAGAGCCTGACCCAGTTCTGCATGGTAAGTTCAGAGCGCCAGCCAAAATTGCTTCACTGGCTGACAATGAGATAGCTCGGCCTGCTTTTGACTGTCGAGGATCTATCACGATTGCCTTTAACCGTGGCAGTCGCTCCATCAACGTAAAATACAACCATACTCCTTTGCACAAAACAGTTGCAGAGCTAGCCGCCCACTTCAAGTCTGCTCCTCGTCAACTGGGACCAGGCGCGCAGAGACTCCTGGAACAGCAGCAGAAGGCTAAGGAGAAGACGCCGAGGAAATCTAGAGGTGAGAAGAAAGACAAGAAGGAGCGAAAGAAGCGGGATTCTATTATAAAGGCGCAAGATGAGAATGGTAATCCCCGGAAACGAAAGAAGAAAAACAACGGAGCATCTCAACCAGCGGGCTCAGTAGACGGACCGATAATCCCCCCAGATTGTCCTGGGGCACCAGCAATCGATGGGCAAAATCAAGGGGGTCCATCGTATATGAACGGAGGAGAAGCGTCGGGCAACCCTGGGCAGCAAGGATCCAACGATTATCCTCAAGGGTTGATGGGGGACGCATCGGCAACGACGAATGGAACTTCGTCTCAGCAGGCGAACGGCCAGCTGGCTTCAGTTGCTTTCCCAGTTAATGTCTCAGCAGCTGAGGCAGCACGCCGAAAAGAGGCAGCCACGACAATGCTCAGCAACGCTGGTGTCGATCCCACCACATTGTCTCCTGAGCAGTTTGGTATCTTCGCCAACCAAGCACCAGAGTTGCAGCGCGAGTCACTGAACATGCTTGTCAAATATGGTGCAGAACGGCTCCGCATTGTCCATCCCGGTAACAAGGAAGGCTCAACACAGGCAAGCTCTTCAACACAGAGCAGCCAGACAACTTCAACTGGACCTAGAACTACCAAAGAGCTAGTGCCACAGTCAAGTGCTCAGAGCAACGCGAACACAGATACTGAGACAGCAGTAGCTCAGGCTGCTGGCGACACTTCCGCTCCTAAGCGTAAGAGGAAGAAGATGGGCAAGTCGAGAACAGCTTGTTTCCCCTGTAAGGCACGGAAGGCAAAGGTAAATGCCTCTCAAACTAATCAAATGCTGCTGAACTAATATAAAATAGTGCCCAAGGGAGAGACCAATCTGCACCGAATGTGCAGATCATGGGACTGCATGTGAATATGCGCCACAGAAACCAAGAAATAAGGCAACGAAGCCCAAGAAGTCTGAGGCTATCGTTGTTGTTGATGATGACGAAGACGATGGCGATGAGGAAGATGAAGAGGTTGAACCGCAGGAAGATGCTGAGGCAGAAGGTTCCGAACAGGAAGATGACGAACAACAAGACACTTCACAGGACTACTCATACCCTCAGATGAATATCGGAAACATGGTCACCAACACTGATGCAACAACTCAAGATACCCAGTCCACACAAAACAATTACTATCAGCCTGCTTCCGGCCTAGTTCTTCCTCAGCCAGACCCTAACTCGACCAGCTCTCAAGCAATGGGCACATCAGTATCGCAGAGAGGCAACTACTCTCATAACCTGCCAGAAGTGACACAACCCATGGTCCACCCAGCCCCTCCTATCCCAGCTCCTGCTGGTACGATCGCTCCCTCTGAAACAAGGCGATGGACCGCCTTTGGTAGCGGTAGTGTTGGTGTTGCTTCCAGCAACACCACCAGCAAGACACGAAGAAGTCTTCCGTCCGAGCCCAGCCATACGAATGCGCAAAACATGCCTGCAAACCCACAGCCATCAGACTGGGCTCAAACTTCCAACGCGACTATGCCTGCAACAACAATGGCATCGGTCTCTCCGCAGATGACATACGACACACGCTCAAGCGGGAGTACTCGACAGAAGAACGAGAGCCTAACGAATGATGCTTCCCAACAAGCTGCAGCAGTGTCCAACACGGTCATGCAGCAGGCTCAGGCACGTCAATCGCCCGTCGCTGCAGCAGCCATGATGGCACATACCCGCAAATCCCCTTATCAACAAGCTGCGGTTCCTCACACGACTTCTAGAACAAGCCAGCGAAACCAGTCTCGCACACCTGTAACTGACCAGGCACGAGGTTACCAGCCTCCTTCCGACATGGCTCAGCAGCAAAATCCACTAGGCAGCACTCATTATGATACATCACACATATCCAGTGGTTCTGGCTATAATGATAATGGGCGTTACAGCAGCACTAGCACCAGCGGAAGCCACCAACCACCGACCACCATGAGCACATCTTATCAGACTACTTCTTCCACAGCAAACCAGTGGTCTAATTCTTCGAGTCGAAACGATCGCTCATATAGCACCAGCCCATCATATCAAACACCTAATACTCACGCACAGACAACAGCGTCCAAGCCAACGGCGGCATCCCGCCAGAACTTTAATATGCGTTCAAACACACAACAGCATATTCGTGCTAGCAGTGGCTCATACAACCAGCAGCAGCCACAACAGCAGCAGCAGGGTTATGCATCATACTCTGGGTCTACACACCAGAGCCAACAGGGTCAGACATCAATCCAGCAACAGCCGTCTAGCTGGTACTTCCAAAACTCGCACAACCCTTCTATTCATCCTGGGGGCCAGTCATCTGGCTATAACTATGAGCCATGGTCAGGCGTTTAG
SEQ ID NO:2 lig4基因：
ATGTCGCAAACTGATGAGGCACAGCATGAAGACGAGATCCAGTATGGCATCGTGGGCATGACCCGCGAAGAGCTGGAGGAAAAGTGAGCAATTCATAATCCGTTAAGAATTCGATCTTCATACTGAACTTGCGCAAGTTTTCCGAATCGCCCAAAGAATCACTCAAAGACGCTCATCTTCTCGGAACTGTTTCGAGAAGTCTTCAACCCCCTCAATGAGAATAAGAAGCAGAATGCCATCGGTGCAGGCCCTAGAAAGGGATTCCGTGGTGTGAACAAGCTTTCTCCTCATGAACAGCGTAGGCACATCATTGAGCGCTTCATCACTCGATGGCGCAAAGAAGTTGGTCCTGATTTCTATCCCGCTTTGCGTCTGATCCTGCCAGACAAAGACCGTGACCGTGGAGTTTACGGACTCAAAGAAAACACAATTGGAAAGTTGCTCGTTAAGCTTATGAAAATCGACAAAAACTCCGAAGATGGCTACAACCTGTTGCATTGGAAGCTACCAGGTCAGACGACAGCGTCGCGCCTGGCTGGTGACTTTGCAGGACGATGTTTTGAAGTCATCTCAAAGCGGCCAATGCGCACCGATGTTGGGAATATGACCATTGCAGAAGTCAATGAGCAGCTTGACAAACTCGCTGCTTCAACAGGAGAGTCAGAGAATCTGCGCGTCTTTGAGACTTTCTACAATCGCATGAATTCAGAAGAGCTCATGTGGCTCATTCGGATCGTCCTCAAGCAGATGAAAGTCGGGGCCACGGAGAAGACTTTCCTTGACTTGTGGCATCCAGACGGAGATGCTCTATTTAGTGTGTCCTCAAGTTTAAGACGTGTTTGCTGGGAGCTGCATGACCCAGAAACCCGCCTTGAGCAGGAGGAAACTGGAGTTGCTTTGATGCAATGTTTCCAACCACAGCTCGCCCAATTTCAGATGCCTGCATCATTTCAAAAAATGATCGAGCTTCTTCGACCAACCGAGGAAGACCCCGAATTCTGGATCGAGGAAAAGCTGGACGGTGAGCGTATGCAAGTCCATATGGTCCAAGACAAGAGCCACCCGGGCGGCAAAAGGTTCTGTTTTTGGTCACGAAAAGCCAAAGATTACACCTATCTCTACGGCGATGGTCTTCAAGACGAGAACTCAAGTCTGACCAGACATTTGAAAAACGCATTTGCATCTGGAGTAAAAAACTTGATCCTGGACGGCGAAATGATCACCTGGGATATGGGTGTCGACAAGATAGTGCCTTTTGGCACCCTCAAAACAGCTGCGCTTTCGGAACAGAAGAATAAGTCTGATACAGACTCAGCTGGCCACAGGCCATTGTTCCGCGTTTTTGATATCCTTTACCTGAACAACAAGCCCCTAACTCAATATACCCTGCGGGATCGACATCGAGCTTTGGAAAAGGCTATCAAGCCTGTTCATCGCCGACTTGAAATTCACAGTTTTGACAAGGCAACGAGTGCCGACGCAATCGAGCCTCTCCTCCGTGAGGTCGTCGCTAATGCTTCTGAAGGCCTTGTGCTGAAGAACCCGCGATCTATGTACCGTCTCAACAGTCGAAATGATGACTGGCTCAAGGTCAAACCTGAATACATGTCCGAGTTTGGCGAGTCCCTCGACTGTGTCATTATTGGTGGCTACTATGGTTCAGGGAAACGAGGTGGCATGCTATCAAGCTTTCTGTGTGGACTGCGTGTTACGCAGAATCATATCCAAGCTGGCGCCAACCCCGAGAAATGTTTCAGTTTCTTCAAAGTCGGTGGCGGTTTCCGTGCAGAAGACTATGCGGAGATTCGGCATCGAACAGAAGGCAAGTGGATTGAGTGGGATCAGAAGAATCCGCCATCAAAGTACATCGAACTTGGAGGAGGAGAGCTTAGACAGTATGAGAGACCGGATGTCTGGATCCGACCTAAAGACTCTGTTGTTGTTTCTGTGAAAGCAGCCAGTGTGGGACCTTCAGATCAATTCGCCCGAGGCTTCACACTGAGATTCCCACGATTCAGACGGCTTCGACTGGACCGAACCTGGGATACAGCCCTCTCCCTTGAAGAGTTCCAGGAGTTGAAGGAGCGAGTTGATGAAGAGTCAAAGGAGAAGGCGATGACGGTTGAGGACAGGAAGCGGCGCAACCCCAAGCGAGTGAAGAGAGAGCTGACCATTGCTGGAGAAGACGCGACTGTAGCTGAGTTCAAGGGAGAGATGACAAACCTTTTCGAGGGACTGGACTTCTGCGTGCTTTCAGAAGCGCTCCGGCCTTATAAAAAGACCAAGGCCCAGCTTGAGGCTATCATCAAGGAGAATGGCGGCACAGTCTTACAACGAGCCGTACCAGGGACCAACATGGTTCTGATAGCCGACAAGAAGGTGGTAAAGGTGGCGAGCCTCATTAAAGGAGGGGCCGTGGACATTATCCGCCCCAAATGGATAAGAGATTGTCTTGAACAGGAATTGGGAGCGTTTCTTTTGCCATTTGAGCCGTCGCATCTGTTTCACGCCACTGATGCATTACACCGAGCGGCAGAAGATAACACAGATCAGTTTGGGGACAGCTACGCGCGGGATCTTTCCATCGAAGAACTCAGAGAGCTAATGCAAGACATCCCTAAGATTGAGGACGGCACGCAATTTGACAAGAATGAGTTTCTCCAACAGCTTGAGGAACATGGGAAAGACTTAGGACGATTAAGGAGCTTTACCTTTCGACGTTGTGTGGTGCTGTTTCATGCCGTGGAAGCTGGCGACAACAAGATTTCGAGGCTAAAGCACCTTGTCAAGTACGGCGGGGGTAATGTTACAGATGACACCGATGATCTTTCTGTGACACATGTGGTGATCGAAGGCGACGACCCGATGCAAGTGGGCGAGGCCGCTGATATGGTACGAAAAGAGCTGAGCTCAAGGCGCGTGCAACCAAGATTAGTGACAGGGAAATGGATTGACGAGTGCTGGAACGAGAGCACATTACTTGACGAGGAGCAGTTTGTAATTCCTTGA
SEQ ID NO:3 ku70基因上游片段：
ATCTTAAGCGAGGCCAGCCTGCTGGCTCGCAAAGAATCGTTTCGATTCAGCACATCTCTCGCTCTTACACCCAGGGCATCGAAGACCCATCTGCTTAGAGCAAGCCTGACCTGCTGCCAGGTGCGCCTTGGCAATGTCGGCGAGAGCTTGGATAAAGACAGGACTTCCGTTGAGAGACTCAACTCGTTTGACAGTTTCGGGGTGACCACTGTCGCCGATGACTTCCTGATCCAACTCATATAGAGTTTCGATATGGTCGGATGTGAAAGCGATAGGGATGAGCACGAGATCCTTCTGACCCTTGGATATGTAGTGCTCCACGCTGTCCGAGGTCTGAGGCCCCAACCAGGCCGACGGGCCGACCTGAGACTGCCAGCAAAGTCGGTAAGGGTTGGAGAAACCGAGGCGCTGCATGACAGCGTAGACAGTGGCCGCAACTTCAGCAGGGTAAGGATCACCTTTATCATGTTAGTATCTGTTACGAATATACGTGACGAAGCAACAAACCTCGGTTAACGACAGACATGGGCAGACTATGCGCAGAGAAGAGCAGCACAGCATCCTTTCTTCGCTCCTCAGGGTATTCCGCAAGCTTGGCCTCGATGTTTTGGGCAAAAGCCTCGACCAATCCACTGTGGTTAGGCCAACGGTCGATTACACTCCAGGTAATGGTACCGTCACCAGACTCCTTGTTGGTCTTGCCTTCAAGTCGGTGCCTCCACTTCCATAGCTCGTTCAAGCTGCTACCTGTTGTTGAGCAAGAGTACTGAGGATATTGTGTGAATGCGACGGCGCGGCCACCTTTTCCATTTCCAAAGCCGTCCTTCAACAGCTGCTCGTACATTTCCTCGGTTAATGGGTCCGCGTATCGGAAAGCGACGTAGGGCTTGTGAGGCGCAGTCTCGGGAGAGATCTTGTCCAAGATCTTACACATCTCGGCGTTTTGGTATTCAGACCACTTTCGAATTGGTGATCCTCCACCGATGACCGAGTATTGCTTGATGATCTTGGGTGTTCGCCGCTTCGAAATCAATGGACCGAGATAGTTTTGTAGAGGGCCAAGAGGGATTAGATCGCCATCGGCCTGTGGTGGTTGTTAATATACATGATTCGGGGGTCTCGGTGTTTCGTTAATGACTTACAAAAAGGCGGCTCAAAAAGTCACCAACCTCATCGGTAGTCGATGGTCCCCCCATGTTGAGGAAGACCATAGCCGTAGGGCCCTTTGATCCAGTAGCATCTTGAGTGACAGGATGGACGGGAGTGGCCAGATATCGCTTCTGAGTAGCGATTTGGGGTAGCGCAAGTCGATGCGTACTGGCCTGTGAAATGCTCTTGCAGAGCTGGCCTCCGGATCTTCGCAGAGCCATTGTCGATGAACTGTCACTGACTTCAAGAGACAAGGCGTGGAAGGGGCAAGAAAATCCAATTTATTGATCAATTGGAAGTGGTGCGAGGTTTGGAATCATAAAACAGCTGAGGGGTGTAAAGAAATCTTATGTTACGTACATGCAACTAGTGCCGACGGCGATGATGCACGCATCTCCATGGGCTCCGGGATCGCCTACAACGCATC
SEQ ID NO:4 ku70基因下游片段：
GCTGAGAGATCGCAGCTTGCCACCGTAAAAGTACAGCAGCCAGATACCAATGATACCGATAACACTCAGACCACCAAGAAGACTGGTACCCTTAGCAACACCGAGATTGTCGAATAAGGGCTGGGCAAAGAGAACACTGCCACAAGCAAGAGCACTTCGGAAGAAGTCATTCGCCGCAAAGAGACTGGCAGCATACATTGGGTAGCTGAGAGGAATGTACACAAAGATACACTGCATGACAATGAAGACTGTAGCTCCGTAGATGGTGATGCCAATGGTGGGCACGATCCAGTGGATAGAGGCCCGAGCTGTCCAGGCGAAGAGGAACAAGCCGATAGTAGGACCAAGGGCGGCAGGAAGAGCTGGAACGAGACGACTTTCTTGAATGGGCCAACCACGCTTAGCGATGCGGGGGTCCATGTAGAAGTAGAGGTAGGAGACGTAGAGGCCTACGCCAATCATACAAGAGACGAGAACGCAGAGGAAGACGAGACCGATTTGACCAAGGTTCATGTTGTAGTAGACTGGGTAGACACGGGGGAAAACCTCGAAGACTAAGAAGAGTAAGTTATCTGAGTTACTACTGATATGGTGGAGCAAACTTACATGAATAGTAGATACCATAGATAATGGCAGTGTAGACCTGCACGAACAGCACGGCAGGATCCTTGATGGTGATCTCCATGGGCTTAATGAGGGCATCAACTGCAATAGCAGAAACCTTCATATGACGCTGGTCGATCTCACTCTGCGACATGAAACGTTGGTTACCGGTGAGCTTTCGAAGACGCTCTGCGCGGCGGAGGAGAATGTTGGCACCGCTGGTCTCGGGGAGCAGAAGGAACATGAGGATGAAGATAGGGGCCGACGCCCAGATACTCTCGTAAAGAGACCATCGCCAGTTCTTTGCGGGGACGGCGAAACCGCTGATCAGAGGACCAAGAGCAGCTAGATATGTTAGTTGAAAGCAAAGAGACAGTGAGAGGAAAAGAGACTTACGACCACAGTAAGCAGCCGAGACCCAGGCCATCATAGCATAGGGGAGAGCCATGAGACTGTAAATATCTCCCAGTGAAGCGCCACCAGAAGCCAAACAAGGCGAGCCGAAGAAGCCTTGAAGGAATCGAAGAACCATGAGACCGGGATAGTTGTTCACTAGTGCCGTAGGAATAGAGATGATAACAAATAGAAACATCGTGACAATGTAAACGGGGTTTCGGCCAATGCGAGGAATCTCACTCAGGGGTGAAAAAATCAGAGGACCAATACCATAGCCCAAAACATATAGTGAAAGACCAAGGGTAGCTTTGAGTTGACTAACTCCAAAAGCGCGCATGACACCTTCAGTTGAGGAAGTGTAGATCGCAGAGGATGTATAAACGACGAAGGTGTACAGACAGATGATGAGAGAGACACCTAGGCGGCGGTTGTTGGTCCAGTTATGAGGATTCTCGTTATCATCACTGAAGTACCAATCGACGAGGATAGAACCATCCTTTGTCCGCTTAGGAACGACAGGGATAGACTTGACCTTCTCAACATCGTGCTCTTCATCGGCTTCGAGTCTGTCG
SEQ ID NO:5 lig4基因上游片段
CCTCCATCGGGAAGCTTTGGGTCATAACCAGGCTCGGAGCCAGGCTGCCAACCAGGGCGATCGCCAAAGGCAGTGTCGAACTCATCATAATCCTCCACCGTCTTGAACGTTGGGGCGCGAGTCATTCGGCGTCGCAATATCGAGGGCGAAAATGTCATTTCGCGATGGACCTCTGTATCGGAACGCACTGGCCGCATAGGCTCAGCCTTGGGCGGGGAGAGGTTCTGTGTGTTGAGGGTGAGGTTGTAGCGGACATGCTGATCCTTCTCGTCAGATACGTTGGTGTTGGAAGAATGGCTTTGGCGACTGCTCTGCTCTATATCGTCCTGGGGCTGCATCGTATTATCGTGCTCCGCTGGGACAGCGAAGTCTCGTGATGTTGACATGGATGGGAAGATGCCCTGCTGAAAGGCAGAGGCGAGAAATAGAGTATATATTGCAGCCAGTCGAACGAGCGTGTAGAATAAGAGTAACCTTTAAAGCTGCAGAAAGCCCTATCAGCGCAGCTGAGAGAGATGGAATGAAAAGGATCAGGTCCAGTTCGAGTTACCTAAGACTCGCATAAAAATAAATTCAAGGGTGCTCGATTCCTCATGGATGGGTCGCGGCGTCACGATGCAGGGGAAGTTCAGCCCGTCAGCACCAATTTCAGTGGTCGGTTTAGGGGAGTAGGGGGTTGTTTCTCAATGGAGCTTTCAACCATTCAACTCGTATATCGCAGCCGCTGTTGAATAACCCGACCTTTAGTGTGTTCGAAAGGTCCAGACTTTGATGGGCAGAAGCCGATTACCCGTCCCTGGTCCATCGACGAATGCCATCGTCTATTATGACGTCCTGACGATGTCTTTCACGATGAATCTCCACGCCAATCGCTGATGAGCAGTCTCATTCTCAAAGGCCAAGCTTTCACAACAAGCTTCTTATCCACATTTTTCCGCCTCGGCATAGAACAACACAGAATAAATCAAGGTCGAATACGACCACACTTTATCCATTTGTGTACGATAGAAATGATTGATCATGAGTCTCTCAAAGACCCGAAACAATAGTCTACAGATATAACTGTTTCAGAGACCGCAATGACCACCGGATGATTTCTTAACACAATTTATAAAAATGCAAGGTTCGGGAAAAGCGGCCAGTAGCACTGCACCATTAGGTCACGATCTTGGCTTTAATAGACAAAAATTCGATGCTGAAGGCTAACAATAGAACACTCCTTAAGAAGTCAACTAAAGAAAGATCGGTAAACTTATGAATTTTAAGTCAGACGCTTCCAAACTTATAAATATAATAAGAGATTTACATTCTTTTTATTAAACTTTTTCTAATATTATACAAGCTTATTAATTACCTTATTGGACTCCTCTGAATAGCTCCCTCCAGGAATTATCATCAGACAATGGATATCATTTGTTAGTTTAACGTCATGTTTCTTGACATTGCATACTTAGGTAGTACGAGGGCCAACGTCGTATTTCATGACCACAACATTCCCCGTTCCACTTCTAGCATCATACCCCTCGACTATCATGAAGCAGCTTTCAGAAGATCTTCTCCTTTCCAATTCGGTTATTGAAGGCACTCGGAGTTACCAGCGGATCACACTTCAATGTTTCCATCTCATCTGTTATGATCATGTTGAGACACGCTTCAGGTGCTG
SEQ ID NO:6 lig4基因下游片段：
GGCAGCGACTCTCTGAACACCCTCGTCAGCTCCAATAACGGCGAGAACAATGATTCTCTTTGCGCCCCATTCACGAAGCGTCTGGATGGCGGCGGCGCAGGTCCCACCGGTTGCAATAACAGGATCGACCAAGATAGCAAGGCTACTAGCGTTGCTGTTGGAGGAGTCACCTGGAATTTGCTGGGGAAGATTGTTGTAGTACTCGACTGGGTCGAGAGTAGACGGCTCACGGTACATGCCCAGATGATGAACTGGAACTGGGATAGGGAGCATGGTTTGCACAGCTGGAGTGGTCAGTTTAGGCAATATGAGTGAAGTGGCAGCTGGACTAACCGTCAACCATGCCAAGACCTGATCGCAGAATGGGAACCAGACACATGGTACTCGGTTGCACTGTTGTGGAAGTGAACTGGAAGCCCAAAGGCGTCTCATCCTGTTCATGGTTAGTGAAGCTTCGAAAGTACCTATTATGCGTCATACCTGAGTCCCTTCCGTAGCTTCCAAAGACGAAGCAAGAGCTTCACAAGAGACAATCAGAGAGATCTCATGAATCAGTGATTTGACCTCTCTAGGGCTGTTGGCCTTTGAGCGAAGCTGGCTCAGCTTTGCCCGTAGACACGGGTGCTGAGAGACATGGACGTTTGAAGGCAACGAAGACATTATGAGTCAAGTACGAAAGCACAGATTATTTCACAAAAGTAAAGAATCTACGAGGAGACGATGAAGCATCACAAGATGAGATGCTGGGACGAAGCCAAGGAGAGAGAAGAAGGAGAAGAAAGAAAGAATCAGAAAGAAAAAGGAGACGGAGGGGGTTTTGTATGTGAGCGAGAGCAAGCAGTGTAAGGTATTTACTTGGAGACACGTTGAGCACTGTACCTGTACCTCTGCCTAGCTGTACAGTAGCTGTTTATCAGTTTGTTGGTACTACGTGAGATTGGATAATCCAATCGTCGTTCCAAGAGAGATCACAAGGACAATGAATGATAGTCGAGGCGGGCGGTAGCGTATGTCGGCGGGGTTAGAGCTTAAGCTACAGGGGAATGCTCGGAGGAGGGCGGGGTGCGATAAGATAAGACGGAGCTCACCGCTGTCAGTGATAGGAGCTACATTGAAACGCGGTGAGGACTCAACAGTCAGATATTCATGTTTGTTGTTAGTAAATACTTTCTTGGGCATCAATTGAATCTTATTGGACAAGATACAAACTATCATCCTGTTTTCTCTATCACCCCGTGTCTTGTTATCTCTTTCTTATATATACCCTTGCTCTCCTCTCAACTCAAAAGACTCCACAGCAAACGCCTCCACAATATGAAATCAAAACGCCAGTCATCCTTTGTGGCAGAGCTCGGGATGTTTCTTTCCTTTAATGCCGTCATATCCGACGAATCTAGCCACAAACTATGCCATCAAACCTTAGACATCGTAATTTAACAACAGCAAGAGCTTCTTCCATTCAAATAAAATGCCTTGTTACCACAAACTGCGTCGGCATAGCACGTTGCGATC
SEQ ID NO:7 ura3基因
ATGTCGCCGCATCCTACCCTCAAGGCGACTTTCGCCAGTCGAGCTGAGACAGCTACCCATCCTCTCAGTCGACATCTCTACAAGCTCATGGACCTCAAGGCCTCGAATCTTTGCCTCAGCGCCGATGTCGCAACCGCTCGCGAACTGCTCTACTTCGCCGACAAGATTGGTCCCTCCATCGTCGTCCTCAAGACTCATTATGACATGGTGGCCGGCTGGGACTTTGATCCTAGGACAGGTACTGGTGCCAAGCTCGCCTCACTAGCACGCAAGCATGGTTTCCTCATCTTTGAGGATCGCAAGTTTGGTGATATTGGCAATACGGTCGAGTTGCAGTATACTAGCGGTGCTGCGCGCATCATCGAGTGGGCACATATTACCAATGTAAATATGGTTCCAGGCAAGGCCTCGGTTACATCTCTGGCCCACGCTGCCAACCGATGGCTCGAGCGATACCACTACGAGGTCAAGACATCTATCAGCATTGGAACTCCCACCGCCAGTCAACTGGATGAGGATAGCGAGCGTTCAGACAACGAGAACCAGAAGGACACGCCGGAACCTGCTCGCGCTGACAGCGGCCGAAAAGGTAGCATTGTTTCCACCACCACCGTCACCCAACAGTACGAGTCGGCCGATTCACCACGCCTCGTCAAGACGATCCCTGAGGGCGATGAGACAGTATTCCCCGGCATCGATGAAGCGCCGATCGAGAGGGGCCTGCTTATCCTGGCACAGATGTCGAGCCAGGGCAACTTCATGAACAAGGAATATACGCAAGCATGTGTAGAGGCCGCGCGGGAACACAAGAACTTCGTCATGGGCTTTGTCTCACAGGAGTGTCTCAACACAGAACCCGAGGATGACTTCATCCACATGACCCCCGGTTGCCAATTGCCCCCTGAGGGCGCGGATGAGAATGAAGCCATCAAGGGTGATGGCAAGGGTCAACAGTACAACACGCCACAGAAGATCATTGGTATTGCCGGTGCTGACATTGCTATTGTTGGACGCGGTATCATCAAGGCAAGCGATCCCGAGGAGGAAGCTGAGCGATATCGGTCCGCGGCATGGAAGGCTTACACGGAGCGGGTTCGTTAA
SEQ ID NO:8 CPS1基因：
ATGTTCTCCCGACTGGCTTCCTTTGCCAAACCCGCGGCCAAGTCCGCGTCGGCTTCTGGCCGAACCATGCTTCAGGTCCGATACCTGTCTGAGAAGGCTGTTGCTGGCAGCAAGGGCAGGACTATGCCCTTCTCTGCTGCTCGAGCTACTGCTCCTGCTGCTTCTCTCCCCGCCACTCTCACCATTCGAGTATGTGCTCCTGCAATGGCATCTCACTTGTCGAACCAATCTAATATGCCTATAGGATGGTCCTGTTTTCCAGGGCAAGGCTTTCGGCGCCAACGCCAACATCTCTGGTGAGGCTGTCTTCACCACCTCTCTTGTTGGCTATCCCGAGTCCATGACGGACCCCTCCTACCGAGGCCAGATCCTCGTCTTCACCCAGCCCCTGATTGGCAACTATGGCGTTCCCTCCGCTGTCCGAGACGAGTATAATCTTCTCAAGTACTTCGAGTCCCCCCACGTTCAGTGTGCTGGTATCGTTGTCTCCGATGTCGCTGTCAACTACAGCCATTGGACTGCCGTCGAGAGTCTCGGCGAGTGGTGCGCCCGTGAGGGAGTTCCCGCCATTTCTGGCGTTGATACCCGCGCCATTGTCACCCACCTCCGAGAGCAGGGCTCTTCCCTGGCTAGGATCTCCATTGGAGAAGAGTACGATGCCGACGAGGATGAGGGTTTCGTTGACCCCGGCCAGATCAACCTGGTCAAGCGTGTCACCACCAAGGCTCCCTTCGTTGTTGAGTCCCCCGGTGCCTCTCTCCACGTTGCTCTGATCGACTGCGGTGTCAAGGAGAACATCCTTCGTAGCCTGGTCAGCCGAGGTGCTTCCGTCACAGTCTTCCCCTATGACTACCCCATCCACAAGGTCTCGCAGCACTTCGATGGTGTCTTCATCTCCAACGGTCCCGGTGACCCTACTCATTGCCAGGCTACTGTTCACAATCTCGCCCGTCTGATGGAAACCTCCAACATTCCTATCATGGGTATTTGTCTCGGCCACCAGCTTCTGGCTCTTGCTGTCGGTGCTCGCACCATCAAGCTCAAGTACGGTAACCGAGCTCACAACATTCCCGCTCTCGACTTGACCACTGGCTTGTGCCACATCACCAGCCAGAACCACGGATACGCCATCGATGCCAGCACCCTTCCCAGCGACTTCAAGGAGTACTTCGTCAACCTCAACGACGGATCCAACGAGGGTATGCTCCACAAGACCCGCCCCATCTTCTCGACCCAGTTCCACCCCGAGGCGAAGGGTGGCCCTATGGACAGCTCTTACCTCTTCGACAAGTACCTCCAGAACGTCCAGCTTGCCAAGAGTAACCAGGTGGTTTTCAAGGATAACCGACCTACCCCTCTGATGCTCGATATCATGAGCCGAGAGCGCGTCGGTGTTGAGCCTACCCCTCTTGCCGCCACTGCTTGA
SEQ ID NO:9 CPS1基因上游片段：
GGATGTCACACAAGGAGCCTGAGATGTGCCAGGGCTCTATTTCCGTAAACAAGCACGCATGTGAGCAGTTTTCTGTCAGCGACATGATTTTCTGTTAGTAAGTTGAGGCTCAAGAGACTCTATAAAGCATTCATCATATTGTGACACCACGACCAGGACTTGCAAGGTTGTCTCATGAATGGATTCCGACTGCCGAACGCCGCGTGCCTGGCAGGGCCGTCGAGGGCTCCACAGACTCTTATCAGAGCGATCGACAGTGCTACGCTAGAAATACTTCTAGCTTGGCTTCCATGCTTTGACCGCCAACAGAGGAGAAAACTCAACAAGGGCGAATCAAATGACGTCTCAACATTTCCGGAGTTTGTCTGGGGACGGTGGATCTTGTATGGATTCATTGACATGATAAAGGGCATGGCTGTGCTGGAGTGCTTGACTCAGTCGTTGAATGAATACTACCTACGGCATGGAGTGCAAGAGTGATGCTGAGTTGTAGCATCTGAGACAAGATATTCAATGGTCGCGATATGCCTTCTTCTGGCGGTATATGTGGTATGAAGAAAAGATAGGCTCAATGATCGGTTCAGGTTGTGTTGTGACGTGTAATTGGTGTTGGTCAAGGACTCACACAGCGACAAGAGTCAAAAACGATATCGGAGGTTGAGAGATGTTGTCAAAGGGTTCAGTTCCTGGCAGCGGGGCTCTAATAGTGCCAATAGGCGGAGCGCTCAGGCCGCCACCACTGTTTTCTCGGCGGAAGAGCGGGTTTTTGGTTCCGCCAGGAGGAAATTTGCAGCCAGGAACCCACTCAGCTCGAAAAAGCTTGCCAATGACCAAGGCCGTGTTGTCAGGTTTCCCTATCGGGGCGGCACCTGCGGAGCGGGATCGCACCAACCTATCCTATCGTCCATCCTGTCCATGGTTGGATCCTAACTAAAGGTATCCTATCTTTTCTTCCCAGCTCCCACCCTCTTGACTCTTTTCTTCTTTTTCTTCTCCTCCAACAGAAAGAAATCCTCGCGACAAATTCACACAATCTTGACCTTCAAGCTCTGACTCAACGTTCCTGCAACACGATTGCATCTTCTCTTATAACTCTGAACTGAGCTCGTCTCTTTAATCCGCCCACATGCACGGGCGCCCATCCGTCTTCACCTCGCAGGTACGTTCACTTTTTTTTTCTTATTACGTCGCGTGTGAGATATATTCGATGTATCAACCCCGCCATCACTGAACACATTTTTATCGGTGGCTCTGCAACGTTCGTTGTAGGAGCTATAGATCGATGTGTATCCACCTGATGGCTCCTCTCCTCATCACCAAGACTTCCATTATGTATCAATTGGCACAATGCTAACTCATCTTATAGGATTATCTCTCTGGTTCGTACTCTTAATTGGTTTTTGACTCACCGCTCGAAACCAT
SEQ ID NO:10 CPS1基因下游片段：
TTGGCGTCTGGGATAGGTCGTTGGTAACGTCTGTTAGTTTCTCGGGAGCAAGGATAATGGCTGCTATTTTAATGTTATATTCAGAAACATCGGTCTTATAAGAAAGATTGTGATACCACTACTACACCTCAAGCCGCCATGCCATGCCTCCTGTGCTGAAATGTACTGACATCAATGACCCAAGCAAGTGCTGCATCCAGCTTGTAGCGTCTCATTTAGTGATGTTAATACTCATACACAAAATATGACTAGGATAAAAGCAGTCTTCAGAATTAAAACGATGATTAAAATTAAAGTTACTCAATTCACTGATGTCTAAACACCTATTGTTTATTTCCTTGCCTTTTACTGTCTCAACGCCGCCCTCCATGTGATATAGCTCCTAATACTGTGCACACAATGCAAAGTTATTAATTTAAATTTACTGTCTTTTCTTCGCACAGCATAGCACATAATGCGCACCTAGCCCTCAGTCCCTCATTCGAACATCGCCGTCGTCAGACAAGGAGCCACGACGAGGACGGTGGTCGTCTCTTGGTGGAGGAAGATACCGATCACTACGCGGTCGACCGCCACCGCCGCCGCCGCGTCCACGACGTCTCCCACCACGGCGCTCACGGCCCATGGCTCGCTCGCGGTGTGGAGGCCGGATGCCTTCAAAGACCTCCTTGCCCCGGTCAACGAATGAAGTACCGTTCTCAAAGGAGTAGCGGTTAATCTCGCGGCCGACATCGCGGTTGCGATCGTTTGTGTAATAAGAGCGCATATTGTCGATGCGGCGGCGCGCAAGGTCTTCTGATACAGGTTGGTAGTCGATAACTGGGCGAGTGTTGGTTCGCTTGAATGCACCGCCTGGGTTGATAGGTGGAGGTCTTGGTCCAGGTCCAGTGCCGGGGCGAACAGGGCGTGGTCCAGTAGGAATGGCAGGGTCGCGGAGTGCTCTGGGTCCAAGAGGGATAGAATTAGTCGCCATCGTCGAGCTCGGGGGTGGTCTTGCGCTCAGTTGAGGCATCATGCGAGATGTTGATGCTGATGCTTCCTCGAGATGTGCTTCAACATCGCCGTTAGGGCCATCCTCATAAACGATTTCCCATCGAGCACCAGAGCGGCCGCGTGGACCACCGCTGGCTTGTTCTGTATCAATCCAGTCCCGGACTTTCTCAGGAGGGACAAAGTCGACCCAGAGCGCTTTGCGATTGCTCTCGTTAGGCCAGACACTGTCGTGAAGTGCAGTGCGAACGCGGGAAGCAGCAGCGGTGTTTTTGAAGATAACAAAACCATGAGTGCGGATCTGGTCCAGAAAGAATTCGGTCACGATCCCGTCGTCATCGTCCGCTGTGTCCTGAGAACGTCTCGCAAGCGTGACGAGATGGGCCTTGACATCTTGAGGTCGGAGAGGGCGCATGAAGTTCTTGATGTAAAGTGCAGAAGTGGCCGGGTGCACCGCCGGTGCAACGTCTCGCTCGTAGTT
SEQ ID NO:11 hygR基因：
CTTGGCTGGAGCTAGTGGAGGTCAACAATGAATGCCTATTTTGGTTTAGTCGTCCAGGCGGTGAGCACAAAATTTGTGTCGTTTGACAAGATGGTTCATTTAGGCAACTGGTCAGATCAGCCCCACTTGTAGCAGTAGCGGCGGCGCTCGAAGTGTGACTCTTATTAGCAGACAGGAACGAGGACATTATTATCATCTGCTGCTTGGTGCACGATAACTTGGTGCGTTTGTCAAGCAAGGTAAGTGGACGACCCGGTCATACCTTCTTAAGTTCGCCCTTCCTCCCTTTATTTCAGATTCAATCTGACTTACCTATTCTACCCAAGCATCCAAATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAGTTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTCCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCATGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCCTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGCCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGAGTAGATGCCGACCGGG
表1 实施例中涉及到的引物名称及序列
以上所述，仅是本发明的较佳实施例而已，并非是对本发明作其它形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本发明技术方案内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本发明技术方案的保护范围。
Claims (5)
1.一种提高尖孢镰刀菌基因敲除效率的方法，其特征在于，以尖孢镰刀菌ura3缺失突变体为出发菌株，通过敲除ku70和lig4来提高尖孢镰刀菌基因敲除效率。
2.权利要求1所述方法，其特征在于，提取尖孢镰刀菌基因组DNA作为模板，分别用引物对ku70-up-F/R、ku70-down-F/R、lig4-up-F/R和lig4-down-F/R扩增ku70基因的上游片段和下游片段、lig4基因的上游片段和下游片段；用引物对ura3-F/R扩增ura3基因；对上游片段、ura3基因和下游片段进行融合分别得到ku70和lig4的同源置换片段；将同源置换片段通过PEG介导转化至尖孢镰刀菌ura3基因缺失突变体的原生质体中，并在普通无抗性T-TOP培养基上培养，筛选得到尖孢镰刀菌ku70和lig4的缺失突变体。
3.根据权利要求2所述的制备方法，其特征在于，尖孢镰刀菌ku70、lig4基因序列分别如SEQ ID NO:1和SEQ ID NO:2所示，ku70的5′端上游片段和3′端下游片段分别如SEQ IDNO:3和SEQ ID NO:4所示，lig4的5′端上游片段和3′端下游片段分别如SEQ ID NO:5和SEQID NO:6所示。
4.根据权利要求2所述的制备方法，其特征在于，尖孢镰刀菌ku70、lig4基因序列分别如SEQ ID NO:1和SEQ ID NO:2所示，ku70的5′端上游片段和3′端下游片段分别如SEQ IDNO:3和SEQ ID NO:4所示，lig4的5′端上游片段和3′端下游片段分别如SEQ ID NO:5和SEQID NO:6所示。
5.根据权利要求4所述的应用，其特征在于，以尿嘧啶营养缺陷作为筛选标记构建的尖孢镰刀菌（∆ura3）为出发菌进行ku70或lig4基因敲除。
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310809937.2A CN116676328A (zh) | 2023-07-04 | 2023-07-04 | 一种提高尖孢镰刀菌基因敲除效率的方法及应用 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310809937.2A CN116676328A (zh) | 2023-07-04 | 2023-07-04 | 一种提高尖孢镰刀菌基因敲除效率的方法及应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116676328A true CN116676328A (zh) | 2023-09-01 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310809937.2A Pending CN116676328A (zh) | 2023-07-04 | 2023-07-04 | 一种提高尖孢镰刀菌基因敲除效率的方法及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116676328A (zh) |
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2023
- 2023-07-04 CN CN202310809937.2A patent/CN116676328A/zh active Pending
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