CN114717268A - 一种可用于无痕遗传转化菌株的制备 - Google Patents
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Abstract
本发明公开一种可用于无痕遗传转化菌株的制备，属于分子生物学技术领域。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，在不引入其他影响尖孢镰刀菌原本遗传性状的抗性基因的情况下，构建了可用于无痕遗传转化的尖孢镰刀菌ura3缺失突变体，为尖孢镰刀菌遗传特性的研究提供了新的工具。
Description
技术领域
本发明属于分子生物学技术领域，具体涉及一种可用于无痕遗传转化菌株的制备。
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。同时，5-氟乳清酸(5-FOA)可以作为OMP脱羧酶的底物，被转化为毒性中间体5-氟-UMP，它是一种嘧啶类似物，可以掺入核酸链中代替尿嘧啶或胸腺嘧啶，干扰RNA和DNA的加工，影响其功能，最终导致细胞死亡。因此，可以通过添加5-FOA、尿苷、尿嘧啶对ura3基因的缺失突变体进行筛选。
尖孢镰刀菌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。尿嘧啶营养缺陷型尖孢镰刀菌的制备有利于增加其遗传研究工具的数量和灵活性，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。
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，构建了可用于无痕遗传转化的尖孢镰刀菌ura3缺失突变体，为尖孢镰刀菌遗传特性的研究提供了新的工具。本发明的技术方案如下：
尖孢镰刀菌ura3缺失突变体的制备方法，步骤如下：
提取尖孢镰刀菌基因组DNA。利用引物对up-F/R、down-F/R分别扩增尖孢镰刀菌ura3基因的5′端上游片段和3′端下游片段。将上游片段和下游片段进行PCR融合，形成融合片段。将融合片段通过PEG介导转化至尖孢镰刀菌原生质体中，并在含有5-FOA、尿苷和尿嘧啶的培养基上培养，筛选得到尖孢镰刀菌ura3的缺失突变体。
本发明提供了一种尖孢镰刀菌ura3缺失突变体在无痕遗传转化中的应用，是将尖孢镰刀菌ura3缺失突变体作为出发菌，以尿嘧啶营养缺陷作为筛选标记进行目的基因敲除，在不引入新的抗性基因的前提下实现无痕遗传转化。
本发明提供了一种以尿嘧啶营养缺陷为筛选标记进行目的基因敲除的方法，具体步骤如下：
以尖孢镰刀菌DNA作为模板，利用引物扩增获得ura3基因片段、目的基因的上游片段和下游片段。之后对目的基因的上游片段、ura3片段以及下游片段按照摩尔质量比1:3:1进行PCR融合，扩增获得转化所需融合片段。以尖孢镰刀菌ura3缺失突变体为出发菌株，制备尖孢镰刀菌ura3缺失突变体原生质体，PEG介导融合片段转化原生质体。将转化子在不添加尿苷、尿嘧啶和5-FOA的培养基上培养，能够正常生长的菌株被筛选为目的基因敲除的尖孢镰刀菌。
本发明的有益效果为：
本发明的方法能够准确、有效的敲除尖孢镰刀菌的ura3基因，在不引入其他影响尖孢镰刀菌原本遗传性状的抗性基因情况下，构建出遗传性能稳定的尖孢镰刀菌尿嘧啶营养缺陷型菌株，从而能够应用于其它目的基因的定点敲除，实现无抗性标记的遗传转化。
附图说明
图1为尖孢镰刀菌ura3基因敲除策略图；
图2为转化片段的电泳图，其中电泳泳道从左到右的顺序依次为Marker，up片段、down片段和融合片段；
图3为ura3缺失突变体PCR鉴定电泳图，其中，a图和b图从左到右的顺序依次均为Marker，野生型WT，ura3缺失突变体(Δura3)；a图所用引物对为ura3-F/R，b图所用引物对为check-F/R；
图4为尿嘧啶营养缺陷菌株Δura3的验证图，其中，从左到右所用培养基依次为PDA，PDA+5-FOA，PDA+尿苷、尿嘧啶，PDA+5-FOA+尿苷、尿嘧啶；WT为野生型菌株，Δura3为尿嘧啶营养缺陷菌株；
图5为尖孢镰刀菌AMT基因的敲除策略图；
图6为以尿嘧啶营养缺陷菌株作为出发菌株敲除AMT基因的验证图，其中a图：野生菌株和突变菌株中目的基因验证；b图：ura3基因验证；c图：验证突变菌株同源置换臂在目的基因上、下游位置发生同源置换。
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200g，琼脂20g，葡萄糖20g。PDB培养基：土豆200g，葡萄糖20g。MM培养基：KH2PO4 1g，MgSO4·7H2O 0.5g，KCl 0.5g，NaNO3 2g，葡萄糖20g，蔗糖200g，琼脂15g。T-TOP培养基：KH2PO4 1g，MgSO4·7H2O 0.5g，KCl 0.5g，NaNO3 2g，葡萄糖20g，蔗糖200g，琼脂10g。
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，DMSO(作为5-FOA的溶剂)、5-FOA、尿苷、尿嘧啶购自Solarbio公司，崩溃酶购自Sigma公司。TEC：1M PH＝7.5的Tris-HCl，0.5M PH＝8的EDTA，1M CaCl2·2H2O。STC(500mL)：山梨醇72.88g，1M CaCl2·2H2O，1M PH＝7.5的Tris-HCl。PEG：PEG4000120g、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(Fusarium oxysporum f.sp.conglutinans,FOC，58385)2号生理小种，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
制备尖孢镰刀菌ura3缺失突变体，步骤如下：
(1)克隆尖孢镰刀菌ura3基因上下游片段
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，并将其作为该步模板。使用引物对up-F/R(SEQ ID NO:4和5)、down-F/R(SEQ ID NO:6和7)分别扩增尖孢镰刀菌ura3基因(SEQ ID NO:1)的5′端上游片段(up片段，SEQ ID NO:2)和3′端下游片段(down片段，SEQ ID NO:3)。PCR体系：模板2μL，dNTPs 4μL，分别加引物up-F/R、down-F/R各1μL，5×buffer 10μL，ddH2O 31μL，fastpfu酶1μL。PCR程序：95℃预变性2min，95℃变性20s，57℃退火20s，72℃延伸2min，变性至延伸重复35个循环，最后72℃延伸5min。将PCR产物进行琼脂糖凝胶电泳，纯化回收上下游片段。
(2)PCR融合上下游片段
使用步骤(1)所得产物按up片段和down片段的摩尔质量比1:1添加作为模板，不加入引物，加dNTPs 4μL，5×buffer 10μL，fastpfu酶1μL，用ddH2O补足至50μL作为反应体系。PCR程序如下：95℃预变性2min，95℃变性20s，58℃退火10min，72℃延伸2min，变性至延伸重复10个循环，最后72℃延伸5min。
(3)融合片段的扩增
使用步骤(2)所得产物2μL作为模板，引物对up-F/down-R各1μL，dNTPs 4μL，5×buffer10μL，ddH2O 31μL，fastpfu酶1μL。PCR程序如下：95℃预变性2min，95℃变性20s，57℃退火20s，72℃延伸2min，变性至延伸重复35个循环，最后72℃延伸5min得到转化所需融合片段(上、下游片段和融合片段的电泳图见图2)。
(4)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℃、120rpm的摇床崩溃4h。取6mL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15min，倒掉上清，用STC重悬原生质体，调整浓度为1×108cfu/mL，备用。
(5)PEG介导原生质体转化
将2μg融合片段加TEC至体积为60μL，冰置20min，12000rpm、4℃离心2min后吸取上清，每60μL的转化片段对应100μL原生质体。将上清样品加入到100μL原生质体中轻轻混匀，冰置40min。冰置结束后缓慢加入160μL的PEG，用移液枪轻轻吹打充分混匀后，室温放置15min，再加入1mL的STC混匀，3000rpm离心5min，轻轻倒掉上清，沉淀用200μL的STC重悬，置于冰上备用。用移液枪取50μL溶液加入到10mLMM培养基(含0.5mg/mL的尿苷、尿嘧啶)中，然后倒入凝固好的MM培养基平板(含0.5mg/mL的尿苷、尿嘧啶)上，放入25℃培养箱中培养1d。
(6)筛选尖孢镰刀菌ura3缺失突变体
将培养1d的MM培养基平板上覆盖T-Top培养基(含有0.5mg/mL尿苷、尿嘧啶和1.5mg/mL的5-FOA)，继续培养5天。挑取转化子置于PDA培养基(含有0.5mg/mL尿苷、尿嘧啶和1.5mg/mL的5-FOA)上培养3天，提取转化子的DNA作为模板，用引物对ura3-F/R(SEQ IDNO:8和9)和check-F/R(引物序列为SEQ ID NO:10和11；野生型check序列为SEQ ID NO:12，条带大小为4898bp；Δura3的check序列为SEQ ID NO:13，条带大小为3692bp)进行PCR扩增，扩增结果如图3所示，获得尖孢镰刀菌ura3缺失突变体(Δura3)，分离单孢纯化培养。
(7)尖孢镰刀菌ura3缺失突变体的验证
分别在含5-FOA的PDA培养基，含5-FOA、尿苷和尿嘧啶的PDA培养基，含尿苷和尿嘧啶的PDA培养基，以及正常PDA培养基平板上接种野生菌株和突变菌株，在25℃的条件下培养7天，观察生长情况。结果如图4所示。由图4可知，本发明的方法可以构建出遗传性能稳定的尖孢镰刀菌的尿嘧啶营养缺陷型菌株。
实施例2无痕遗传转化应用
使用尖孢镰刀菌ura3缺失突变体作为出发菌株敲除AMT基因(SEQ ID NO:14)，所用引物及遗传转化位置如图5所示。AMT基因是编码甲基转移酶的基因。甲基转移酶又叫转甲基酶，是生物体内普遍存在的重要酶类，其受体范围从DNA、RNA到蛋白质、脂质等，在真菌形态和生理代谢等方面都有着十分重要的作用，可以使真菌适应不同的环境，提高生存能力。
以尖孢镰刀菌的DNA作为模板，分别用引物对ura3-F/R、A-up-F/R(SEQ ID NO:15和16)和A-down-F/R(SEQ ID NO:17和18)进行扩增得到ura3基因片段、AMT基因的上游片段(A-up，SEQ ID NO:19)和下游片段(A-down，SEQ ID NO:20)。之后对目的基因的上游片段、ura3片段以及下游片段按照摩尔质量比1:3:1加入PCR小管作为模板，进行PCR融合，再将其作为模板，使用引物对A-up-F/A-down-R进行扩增得到转化所需片段。原生质体的制备和PEG介导原生质体转化步骤同实施例1。转化子培养1天后覆盖的T-TOP培养基为不添加尿苷、尿嘧啶和5-FOA的普通T-TOP培养基，培养5天后，挑取转化子至普通PDA培养基上培养3天，提取转化子的DNA作为模板，用引物对A-check-F/R(SEQ ID NO:21和22)以及ura3check-F/R(SEQ ID NO:23和24)进行PCR扩增，扩增结果如图6a、图6b所示，再分别同引物对upcheck-F/R(SEQ ID NO:25和26)和downcheck-F/R(SEQ ID NO:27和28)进行扩增，结果如图6c所示，成功构建了两个AMT缺失突变体(MT-1和MT-2)。由图6可知，本发明所构建的尖孢镰刀菌尿嘧啶营养缺陷菌株可在不引入新的抗性基因的前提下完成目的基因的敲除，实现无痕遗传转化。
上述实施例中所涉及的基因序列，如下所示：
ura3基因：
5′-CAAGGCGACTTTCGCCAGTCGAGCTGAGACAGCTACCCATCCTCTCAGTCGACATCTCTACAAGCTCATGGACCTCAAGGCCTCGAATCTTTGCCTCAGCGCCGATGTCGCAACCGCTCGCGAACTGCTCTACTTCGCCGACAAGATTGGTCCCTCCATCGTCGTCCTCAAGACTCATTATGACATGGTGGCCGGCTGGGACTTTGATCCTAGGACAGGTACTGGTGCCAAGCTCGCCTCACTAGCACGCAAGCATGGTTTCCTCATCTTTGAGGATCGCAAGTTTGGTGATATTGGCAATACGGTCGAGTTGCAGTATACTAGCGGTGCTGCGCGCATCATCGAGTGGGCACATATTACCAATGTAAATATGGTTCCAGGCAAGGCCTCGGTTACATCTCTGGCCCACGCTGCCAACCGATGGCTCGAGCGATACCACTACGAGGTCAAGACATCTATCAGCATTGGAACTCCCACCGCCAGTCAACTGGATGAGGATAGCGAGCGTTCAGACAACGAGAACCAGAAGGACACGCCGGAACCTGCTCGCGCTGACAGCGGCCGAAAAGGTAGCATTGTTTCCACCACCACCGTCACCCAACAGTACGAGTCGGCCGATTCACCACGCCTCGTCAAGACGATCCCTGAGGGCGATGAGACAGTATTCCCCGGCATCGATGAAGCGCCGATCGAGAGGGGCCTGCTTATCCTGGCACAGATGTCGAGCCAGGGCAACTTCATGAACAAGGAATATACGCAAGCATGTGTAGAGGCCGCGCGGGAACACAAGAACTTCGTCATGGGCTTTGTCTCACAGGAGTGTCTCAACACAGAACCCGAGGATGACTTCATCCACATGACCCCCGGTTGCCAATTGCCCCCTGAGGGCGCGGATGAGAATGAAGCCATCAAGGGTGATGGCAAGGGTCAACAGTACAACACGCCACAGAAGATCATTGGTATTGCCGGTGCTGACATTGCTATTGTTGGACGCGGTATCATCAAGGC-3′(SEQ ID NO:1)
ura3基因上游片段：
5′-GGATCTCGACGTTGTACGCCGTGAATTAGGTTAGTTGTCATGTCACCCTTAATCCCTCGCTGGCTCCGGCTTGAAGAACCGCTGAAACGGGTGTTTGCGGCAATAGTTTAGATGTCGTGTAACCCAGCCAGATCACTTGGAGGTTTTTGGAAAATCAGGGTGATGATCGTATCCTAACCGCCTCATACCTGAGAGATGATCCCTTTGTTGAGCTTGGTGGTGGTCTGTGTTGAGATATCACCTGCAGTCAGTATCATAAAAACCATGAACCATTTAATAGCCGCTTCCATGGTGAAAAGCATCAATCCACCATGGCAAAGCCTCCCGCCTCTTGGGCTGTCTTGAACAGATCATTAAGCCAGGATTGGTTATGATCATCTCGCCCTACTTCTCTAGTTTCTGATCCCTGATCCGCAAAGTGCAGTGTAGGCCAAACCCTATCCGGATAGCCAACGTTGGACCTGGTCTCGACAGCAGGTGTATGCTTGTTTAATTCCTCTAAAATCAGATGATTTTTGGGGTTTCGTGTCTTTGTTCATCCACCAAGCTTGTCTTGAATTGGCTTCTCGACTTGTCCGTCTTGGTGCCCCCCACGATACACGTCACTGACGGTATGGATCTAGGTATGGGGGCATGGGTCCTAGGGTTAGGAAGAATTTGTTTCCATCGGTGGTGATATGATCAGGATGAGGAGAGTATGTATTTTTTTCAGTTGAGGATTGTTTCACTGGGCAATGGCGAATGGAGGACTTGTAGACAAGGTGTAGTCATGTAGTTATACATATCGGATTACTTCCGACCAATTCCCCAAAGAAGGATTTATTTGCTTGCCATTTCAATTGGCTGATTCTTCCTCACAGAGTCTTCCACTACAAAGATGGATGGATGGGTGGCTGCCCTTGTCCGCTCCGGCTAAAAGAGAACTGCCAGGATAAGTTGAGTTTCAAGCTTTGGATCGTCCATATCAGTGCCATTTTCTTTGAATTGCTGTCAAACATCCTTGCCCCTGTTCCTGTCTGAGTACATACATACCTACAGCAGTGCCTCTAGTGCGTACATGTTGCATTCGCAGCCTGTTACAGTAAAAAAGAGACACTCTTGTTAATCCCTGCTTGACGCCACGCCTGTGCCTTCAACCTCTGAATGCCTACTTACTTTCTGGCTGCGTTTGAGCCTCGGGCCTTAAACGTGTTAGCTTGTTATGAATCTCGAGGTTCAAGCGCCCTTTCGCTTCATGTTGGAGCTAAGCTAAGGTACCCTAGCACTAGCACCTAGGTGGTAGTGACCCTCTAGCCTTGTCTCTCGCCTCACCTTCAGTGCCTTTCGTTGGTTGGGTCCATAGGTCACTTTTCTTTAAGTCGAGCTGTGGATCGTCACTTTTCACTTTTTACCTTTCACCTCTCACCTACAGTCTCTTCTTAACTAACCCCATCCCATCCCGTGTCATTCTGCGAAAGCTTTTTACTTCAAACCTCCTCCTCCTTCCTCTTAACTCGCTCTCTTTCCTCTCCTCTCCTCATAATTCTCCTCCCACTCCCGATCGACATTCCACCCATTTACGCCTCACATCGGACGACACTTTCAGTCGAGACCCATTCGGCCGAAGCTAATAAGATCTTCACCCCTCGTTCCTATCAGTGGCTCTTGTCTCCGTTTTTCATTTTTCCCCAGCTTCCCCTCTATCAAGCCAAACCGCCACTTGCGCCAGGTAGACTAGATTGTTGTGAACATCTTTGTGAATTGGACAAGTGTCGAGGTTGCCCAAAACTTCCGACTTAGATCCACATACGATTTAGTGTGCTTGAACTCGCTCCC-3′(SEQ ID NO:2)
ura3基因下游片段：
5′-GAGGTTTTTCGCGCGGTAGGCAGGCTGCACTGGAGTTATCGAGACATCTAGCGGGTCTGACATGAATATTATGACTACATGGAGCATCCATGGAGTAACTCACTTACAAGAGAAATGAGACTTGGTTCCCTGATTGGGAATATCATCTAAGCACTAGGCCTCCTCCAACTGCATAAGCCTGGTTCCTCTCTACTGCATCTATGATACCAGGTTCAACACAACCTTACGAGAATGATTGTCGGAAAAGATACGAAATATGAATTGCAATGACGGAATAATTCGGTTCCTTACGTGTGCTTTCAATGCACCTCTCATGTCGACGGTTGTAGCTAATTCACTCCAGCTGCATATATCCATTTCTTCCTTCAATAACGTGGGTCCATTCAAAGATGCACAGGGCTGAATTCCATTCTTGTAATGGATGCCCCACAATCAAAGTGACGCCGTTTTCTTGGAAGCCCGGAGTTAGAGGACTTTGAAAGCTGATCCTCGTCCGCCGAGATCAACATAGACCTTCCCATACTCCACCTTAATATTGACCTCTAGCCTCTAAGTATCTGAATCCCATACTACTTTACTATAACTTTACATATTTGCTCCGGGTATTCCCAAGTGATCAGTCATTCATACCCGTTTCCGAAGAAGCCGACATGACAGCAGCAGCTGAGAACGCTGATGGTCCGCGTCTGAGGAAGAGAAGACGAATTGTCATATCGTGCACTGAATGCCATCGAAGAAAGCAGAGGGTCCGTAACTACAAGACTACGATACACTTGTCTTATACAAGAAATATTTGACTGAAAACATCTTTAGTGCGACCGCGAACAACCATGCGGCAATTGCAAGTCTCGAAATAAAGAGTCAGCTTGTGTTTATGAAACAGGAGCTCCAACAGCCAAACAGAGCAAGCAGACAACAAAAGCCTCGCCGACTTACTCGGATACATTCGCTAAACAAGCCGAGCCGTCACCATCACAGTCTGTTGACTCATCGAATGAGAGGCTAGGCGAGCCTCTGTCATCTAAGGTAGCTGACTGGGGGTACGCTCACAATGGCGCGTCTACCATGGGTATACTTAAACGCATCGAGACGCTCACAGACGGTGAAGATGGGCCCCCAGATCTAACATCTCGTGATTCCTCCGGAGGCTCTCCCGGCAAAGAGCTTGCCTTGAGGGAAAAGTACAGGGCTATAATTCGACAATTACCTGCCCGGAATTACATCGAAAAACTCGTCGAGATGTACATGCGTGGCTTCAATTGGCAGTACTATCCAATCGACCCTGACATCTTTTATGCGCAACTCGACGAGTGGAATTCGTTGCCGTTCTCGGTATTCTCAGATGTCGGTCCCCGCGGCTTGAACCCTGAACTGCGAGCCTTTCCAGCAGTTGTATTTCAAATCATAGCAACGGCCCTTCTCCTATTGCCCGAGAAGCCAGATTCCACATTCGACTCGCTCAAATTTGCTGGAGGTATGAGATTCGAAGACTTGGCTGTCGAGTATAGCGAGTCAGGGCAGGCAGTTCTTGACCTTTTGGGGAAGAAGAACTTGTCTCTGTCTACAGTGCAAGCACAATTTCTCAGGGCGTCATTCCACAAGTTTACGGCAAAGGTTACAGATGCAGTAAGTTCCTGTTAGGAGATGATACCTAGAGAAGAGTCGACTGATGTTGGGTAGTGGCACACCATCGCAATTGCGATTCGCGATGCTCAGGACCTGGGGATGCATCGTGACAGCTTAGATCCCAGACCAAAGGACGCAAG-3′(SEQ ID NO:3)
引物up-F：
5′-GGATCTCGACGTTGTACGCC-3′(SEQ ID NO:4)
引物up-R：
5′-GTGCAGCCTGCCTACCGCGCGAAAAACCTCGGGAGCGAGTTCAAGCACAC-3′(SEQ ID NO:5)引物down-F：
5′-ATACGATTTAGTGTGCTTGAACTCGCTCCCGAGGTTTTTCGCGCGGTAGG-3′(SEQ ID NO:6)引物down-R：
5′-CTTGCGTCCTTTGGTCTGGG-3′(SEQ ID NO:7)
引物ura3-F：
5′-CAAGGCGACTTTCGCCAGTC-3′(SEQ ID NO:8)
引物ura3-R：
5′-GCCTTGATGATACCGCGTCC-3′(SEQ ID NO:9)
引物check-F：
5′-GGAGACGGTCAGGTTGATGC-3′(SEQ ID NO:10)
引物check-R：
5′-GGCGAGCAGGATGTAGATCC-3′(SEQ ID NO:11)
check序列(WT)：
5′-GGAGACGGTCAGGTTGATGCAAAGTCATGTACCTGGGGTTCTGTGGATCTCGACGTTGTACGCCGTGAATTAGGTTAGTTGTCATGTCACCCTTAATCCCTCGCTGGCTCCGGCTTGAAGAACCGCTGAAACGGGTGTTTGCGGCAATAGTTTAGATGTCGTGTAACCCAGCCAGATCACTTGGAGGTTTTTGGAAAATCAGGGTGATGATCGTATCCTAACCGCCTCATACCTGAGAGATGATCCCTTTGTTGAGCTTGGTGGTGGTCTGTGTTGAGATATCACCTGCAGTCAGTATCATAAAAACCATGAACCATTTAATAGCCGCTTCCATGGTGAAAAGCATCAATCCACCATGGCAAAGCCTCCCGCCTCTTGGGCTGTCTTGAACAGATCATTAAGCCAGGATTGGTTATGATCATCTCGCCCTACTTCTCTAGTTTCTGATCCCTGATCCGCAAAGTGCAGTGTAGGCCAAACCCTATCCGGATAGCCAACGTTGGACCTGGTCTCGACAGCAGGTGTATGCTTGTTTAATTCCTCTAAAATCAGATGATTTTTGGGGTTTCGTGTCTTTGTTCATCCACCAAGCTTGTCTTGAATTGGCTTCTCGACTTGTCCGTCTTGGTGCCCCCCACGATACACGTCACTGACGGTATGGATCTAGGTATGGGGGCATGGGTCCTAGGGTTAGGAAGAATTTGTTTCCATCGGTGGTGATATGATCAGGATGAGGAGAGTATGTATTTTTTTCAGTTGAGGATTGTTTCACTGGGCAATGGCGAATGGAGGACTTGTAGACAAGGTGTAGTCATGTAGTTATACATATCGGATTACTTCCGACCAATTCCCCAAAGAAGGATTTATTTGCTTGCCATTTCAATTGGCTGATTCTTCCTCACAGAGTCTTCCACTACAAAGATGGATGGATGGGTGGCTGCCCTTGTCCGCTCCGGCTAAAAGAGAACTGCCAGGATAAGTTGAGTTTCAAGCTTTGGATCGTCCATATCAGTGCCATTTTCTTTGAATTGCTGTCAAACATCCTTGCCCCTGTTCCTGTCTGAGTACATACATACCTACAGCAGTGCCTCTAGTGCGTACATGTTGCATTCGCAGCCTGTTACAGTAAAAAAGAGACACTCTTGTTAATCCCTGCTTGACGCCACGCCTGTGCCTTCAACCTCTGAATGCCTACTTACTTTCTGGCTGCGTTTGAGCCTCGGGCCTTAAACGTGTTAGCTTGTTATGAATCTCGAGGTTCAAGCGCCCTTTCGCTTCATGTTGGAGCTAAGCTAAGGTACCCTAGCACTAGCACCTAGGTGGTAGTGACCCTCTAGCCTTGTCTCTCGCCTCACCTTCAGTGCCTTTCGTTGGTTGGGTCCATAGGTCACTTTTCTTTAAGTCGAGCTGTGGATCGTCACTTTTCACTTTTTACCTTTCACCTCTCACCTACAGTCTCTTCTTAACTAACCCCATCCCATCCCGTGTCATTCTGCGAAAGCTTTTTACTTCAAACCTCCTCCTCCTTCCTCTTAACTCGCTCTCTTTCCTCTCCTCTCCTCATAATTCTCCTCCCACTCCCGATCGACATTCCACCCATTTACGCCTCACATCGGACGACACTTTCAGTCGAGACCCATTCGGCCGAAGCTAATAAGATCTTCACCCCTCGTTCCTATCAGTGGCTCTTGTCTCCGTTTTTCATTTTTCCCCAGCTTCCCCTCTATCAAGCCAAACCGCCACTTGCGCCAGGTAGACTAGATTGTTGTGAACATCTTTGTGAATTGGACAAGTGTCGAGGTTGCCCAAAACTTCCGACTTAGATCCACATACGATTTAGTGTGCTTGAACTCGCTCCCCAGTTCGAGTCACCGTCTTTTCCTATACTTTTGTTCATATCAAATTGATCTTCGAACAAACTTTACCGTGTCATGTCGCCGCATCCTACCCTCAAGGCGACTTTCGCCAGTCGAGCTGAGACAGCTACCCATCCTCTCAGTCGACATCTCTACAAGCTCATGGACCTCAAGGCCTCGAATCTTTGCCTCAGCGCCGATGTCGCAACCGCTCGCGAACTGCTCTACTTCGCCGACAAGATTGGTCCCTCCATCGTCGTCCTCAAGACTCATTATGACATGGTGGCCGGCTGGGACTTTGATCCTAGGACAGGTACTGGTGCCAAGCTCGCCTCACTAGCACGCAAGCATGGTTTCCTCATCTTTGAGGATCGCAAGTTTGGTGATATTGGCAATACGGTCGAGTTGCAGTATACTAGCGGTGCTGCGCGCATCATCGAGTGGGCACATATTACCAATGTAAATATGGTTCCAGGCAAGGCCTCGGTTACATCTCTGGCCCACGCTGCCAACCGATGGCTCGAGCGATACCACTACGAGGTCAAGACATCTATCAGCATTGGAACTCCCACCGCCAGTCAACTGGATGAGGATAGCGAGCGTTCAGACAACGAGAACCAGAAGGACACGCCGGAACCTGCTCGCGCTGACAGCGGCCGAAAAGGTAGCATTGTTTCCACCACCACCGTCACCCAACAGTACGAGTCGGCCGATTCACCACGCCTCGTCAAGACGATCCCTGAGGGCGATGAGACAGTATTCCCCGGCATCGATGAAGCGCCGATCGAGAGGGGCCTGCTTATCCTGGCACAGATGTCGAGCCAGGGCAACTTCATGAACAAGGAATATACGCAAGCATGTGTAGAGGCCGCGCGGGAACACAAGAACTTCGTCATGGGCTTTGTCTCACAGGAGTGTCTCAACACAGAACCCGAGGATGACTTCATCCACATGACCCCCGGTTGCCAATTGCCCCCTGAGGGCGCGGATGAGAATGAAGCCATCAAGGGTGATGGCAAGGGTCAACAGTACAACACGCCACAGAAGATCATTGGTATTGCCGGTGCTGACATTGCTATTGTTGGACGCGGTATCATCAAGGCAAGCGATCCCGAGGAGGAAGCTGAGCGATATCGGTCCGCGGCATGGAAGGCTTACACGGAGCGGGTTCGTTAAGAAGCGAAAATGACTGGAAGTCGGGATGGCCTTGAGGTTTTTCGCGCGGTAGGCAGGCTGCACTGGAGTTATCGAGACATCTAGCGGGTCTGACATGAATATTATGACTACATGGAGCATCCATGGAGTAACTCACTTACAAGAGAAATGAGACTTGGTTCCCTGATTGGGAATATCATCTAAGCACTAGGCCTCCTCCAACTGCATAAGCCTGGTTCCTCTCTACTGCATCTATGATACCAGGTTCAACACAACCTTACGAGAATGATTGTCGGAAAAGATACGAAATATGAATTGCAATGACGGAATAATTCGGTTCCTTACGTGTGCTTTCAATGCACCTCTCATGTCGACGGTTGTAGCTAATTCACTCCAGCTGCATATATCCATTTCTTCCTTCAATAACGTGGGTCCATTCAAAGATGCACAGGGCTGAATTCCATTCTTGTAATGGATGCCCCACAATCAAAGTGACGCCGTTTTCTTGGAAGCCCGGAGTTAGAGGACTTTGAAAGCTGATCCTCGTCCGCCGAGATCAACATAGACCTTCCCATACTCCACCTTAATATTGACCTCTAGCCTCTAAGTATCTGAATCCCATACTACTTTACTATAACTTTACATATTTGCTCCGGGTATTCCCAAGTGATCAGTCATTCATACCCGTTTCCGAAGAAGCCGACATGACAGCAGCAGCTGAGAACGCTGATGGTCCGCGTCTGAGGAAGAGAAGACGAATTGTCATATCGTGCACTGAATGCCATCGAAGAAAGCAGAGGGTCCGTAACTACAAGACTACGATACACTTGTCTTATACAAGAAATATTTGACTGAAAACATCTTTAGTGCGACCGCGAACAACCATGCGGCAATTGCAAGTCTCGAAATAAAGAGTCAGCTTGTGTTTATGAAACAGGAGCTCCAACAGCCAAACAGAGCAAGCAGACAACAAAAGCCTCGCCGACTTACTCGGATACATTCGCTAAACAAGCCGAGCCGTCACCATCACAGTCTGTTGACTCATCGAATGAGAGGCTAGGCGAGCCTCTGTCATCTAAGGTAGCTGACTGGGGGTACGCTCACAATGGCGCGTCTACCATGGGTATACTTAAACGCATCGAGACGCTCACAGACGGTGAAGATGGGCCCCCAGATCTAACATCTCGTGATTCCTCCGGAGGCTCTCCCGGCAAAGAGCTTGCCTTGAGGGAAAAGTACAGGGCTATAATTCGACAATTACCTGCCCGGAATTACATCGAAAAACTCGTCGAGATGTACATGCGTGGCTTCAATTGGCAGTACTATCCAATCGACCCTGACATCTTTTATGCGCAACTCGACGAGTGGAATTCGTTGCCGTTCTCGGTATTCTCAGATGTCGGTCCCCGCGGCTTGAACCCTGAACTGCGAGCCTTTCCAGCAGTTGTATTTCAAATCATAGCAACGGCCCTTCTCCTATTGCCCGAGAAGCCAGATTCCACATTCGACTCGCTCAAATTTGCTGGAGGTATGAGATTCGAAGACTTGGCTGTCGAGTATAGCGAGTCAGGGCAGGCAGTTCTTGACCTTTTGGGGAAGAAGAACTTGTCTCTGTCTACAGTGCAAGCACAATTTCTCAGGGCGTCATTCCACAAGTTTACGGCAAAGGTTACAGATGCAGTAAGTTCCTGTTAGGAGATGATACCTAGAGAAGAGTCGACTGATGTTGGGTAGTGGCACACCATCGCAATTGCGATTCGCGATGCTCAGGACCTGGGGATGCATCGTGACAGCTTAGATCCCAGACCAAAGGACGCAAGCGTCGAGAGTATTCTAGAGAATCAATGGCTGATTCAACGGAGGAGAAGGATCTACATCCTGCTCGCC-3′(SEQ ID NO:12)
check序列(Δura3)：
5’-GGAGACGGTCAGGTTGATGCAAAGTCATGTACCTGGGGTTCTGTGGATCTCGACGTTGTACGCCGTGAATTAGGTTAGTTGTCATGTCACCCTTAATCCCTCGCTGGCTCCGGCTTGAAGAACCGCTGAAACGGGTGTTTGCGGCAATAGTTTAGATGTCGTGTAACCCAGCCAGATCACTTGGAGGTTTTTGGAAAATCAGGGTGATGATCGTATCCTAACCGCCTCATACCTGAGAGATGATCCCTTTGTTGAGCTTGGTGGTGGTCTGTGTTGAGATATCACCTGCAGTCAGTATCATAAAAACCATGAACCATTTAATAGCCGCTTCCATGGTGAAAAGCATCAATCCACCATGGCAAAGCCTCCCGCCTCTTGGGCTGTCTTGAACAGATCATTAAGCCAGGATTGGTTATGATCATCTCGCCCTACTTCTCTAGTTTCTGATCCCTGATCCGCAAAGTGCAGTGTAGGCCAAACCCTATCCGGATAGCCAACGTTGGACCTGGTCTCGACAGCAGGTGTATGCTTGTTTAATTCCTCTAAAATCAGATGATTTTTGGGGTTTCGTGTCTTTGTTCATCCACCAAGCTTGTCTTGAATTGGCTTCTCGACTTGTCCGTCTTGGTGCCCCCCACGATACACGTCACTGACGGTATGGATCTAGGTATGGGGGCATGGGTCCTAGGGTTAGGAAGAATTTGTTTCCATCGGTGGTGATATGATCAGGATGAGGAGAGTATGTATTTTTTTCAGTTGAGGATTGTTTCACTGGGCAATGGCGAATGGAGGACTTGTAGACAAGGTGTAGTCATGTAGTTATACATATCGGATTACTTCCGACCAATTCCCCAAAGAAGGATTTATTTGCTTGCCATTTCAATTGGCTGATTCTTCCTCACAGAGTCTTCCACTACAAAGATGGATGGATGGGTGGCTGCCCTTGTCCGCTCCGGCTAAAAGAGAACTGCCAGGATAAGTTGAGTTTCAAGCTTTGGATCGTCCATATCAGTGCCATTTTCTTTGAATTGCTGTCAAACATCCTTGCCCCTGTTCCTGTCTGAGTACATACATACCTACAGCAGTGCCTCTAGTGCGTACATGTTGCATTCGCAGCCTGTTACAGTAAAAAAGAGACACTCTTGTTAATCCCTGCTTGACGCCACGCCTGTGCCTTCAACCTCTGAATGCCTACTTACTTTCTGGCTGCGTTTGAGCCTCGGGCCTTAAACGTGTTAGCTTGTTATGAATCTCGAGGTTCAAGCGCCCTTTCGCTTCATGTTGGAGCTAAGCTAAGGTACCCTAGCACTAGCACCTAGGTGGTAGTGACCCTCTAGCCTTGTCTCTCGCCTCACCTTCAGTGCCTTTCGTTGGTTGGGTCCATAGGTCACTTTTCTTTAAGTCGAGCTGTGGATCGTCACTTTTCACTTTTTACCTTTCACCTCTCACCTACAGTCTCTTCTTAACTAACCCCATCCCATCCCGTGTCATTCTGCGAAAGCTTTTTACTTCAAACCTCCTCCTCCTTCCTCTTAACTCGCTCTCTTTCCTCTCCTCTCCTCATAATTCTCCTCCCACTCCCGATCGACATTCCACCCATTTACGCCTCACATCGGACGACACTTTCAGTCGAGACCCATTCGGCCGAAGCTAATAAGATCTTCACCCCTCGTTCCTATCAGTGGCTCTTGTCTCCGTTTTTCATTTTTCCCCAGCTTCCCCTCTATCAAGCCAAACCGCCACTTGCGCCAGGTAGACTAGATTGTTGTGAACATCTTTGTGAATTGGACAAGTGTCGAGGTTGCCCAAAACTTCCGACTTAGATCCACATACGATTTAGTGTGCTTGAACTCGCTCCCGAGGTTTTTCGCGCGGTAGGCAGGCTGCACTGGAGTTATCGAGACATCTAGCGGGTCTGACATGAATATTATGACTACATGGAGCATCCATGGAGTAACTCACTTACAAGAGAAATGAGACTTGGTTCCCTGATTGGGAATATCATCTAAGCACTAGGCCTCCTCCAACTGCATAAGCCTGGTTCCTCTCTACTGCATCTATGATACCAGGTTCAACACAACCTTACGAGAATGATTGTCGGAAAAGATACGAAATATGAATTGCAATGACGGAATAATTCGGTTCCTTACGTGTGCTTTCAATGCACCTCTCATGTCGACGGTTGTAGCTAATTCACTCCAGCTGCATATATCCATTTCTTCCTTCAATAACGTGGGTCCATTCAAAGATGCACAGGGCTGAATTCCATTCTTGTAATGGATGCCCCACAATCAAAGTGACGCCGTTTTCTTGGAAGCCCGGAGTTAGAGGACTTTGAAAGCTGATCCTCGTCCGCCGAGATCAACATAGACCTTCCCATACTCCACCTTAATATTGACCTCTAGCCTCTAAGTATCTGAATCCCATACTACTTTACTATAACTTTACATATTTGCTCCGGGTATTCCCAAGTGATCAGTCATTCATACCCGTTTCCGAAGAAGCCGACATGACAGCAGCAGCTGAGAACGCTGATGGTCCGCGTCTGAGGAAGAGAAGACGAATTGTCATATCGTGCACTGAATGCCATCGAAGAAAGCAGAGGGTCCGTAACTACAAGACTACGATACACTTGTCTTATACAAGAAATATTTGACTGAAAACATCTTTAGTGCGACCGCGAACAACCATGCGGCAATTGCAAGTCTCGAAATAAAGAGTCAGCTTGTGTTTATGAAACAGGAGCTCCAACAGCCAAACAGAGCAAGCAGACAACAAAAGCCTCGCCGACTTACTCGGATACATTCGCTAAACAAGCCGAGCCGTCACCATCACAGTCTGTTGACTCATCGAATGAGAGGCTAGGCGAGCCTCTGTCATCTAAGGTAGCTGACTGGGGGTACGCTCACAATGGCGCGTCTACCATGGGTATACTTAAACGCATCGAGACGCTCACAGACGGTGAAGATGGGCCCCCAGATCTAACATCTCGTGATTCCTCCGGAGGCTCTCCCGGCAAAGAGCTTGCCTTGAGGGAAAAGTACAGGGCTATAATTCGACAATTACCTGCCCGGAATTACATCGAAAAACTCGTCGAGATGTACATGCGTGGCTTCAATTGGCAGTACTATCCAATCGACCCTGACATCTTTTATGCGCAACTCGACGAGTGGAATTCGTTGCCGTTCTCGGTATTCTCAGATGTCGGTCCCCGCGGCTTGAACCCTGAACTGCGAGCCTTTCCAGCAGTTGTATTTCAAATCATAGCAACGGCCCTTCTCCTATTGCCCGAGAAGCCAGATTCCACATTCGACTCGCTCAAATTTGCTGGAGGTATGAGATTCGAAGACTTGGCTGTCGAGTATAGCGAGTCAGGGCAGGCAGTTCTTGACCTTTTGGGGAAGAAGAACTTGTCTCTGTCTACAGTGCAAGCACAATTTCTCAGGGCGTCATTCCACAAGTTTACGGCAAAGGTTACAGATGCAGTAAGTTCCTGTTAGGAGATGATACCTAGAGAAGAGTCGACTGATGTTGGGTAGTGGCACACCATCGCAATTGCGATTCGCGATGCTCAGGACCTGGGGATGCATCGTGACAGCTTAGATCCCAGACCAAAGGACGCAAGCGTCGAGAGTATTCTAGAGAATCAATGGCTGATTCAACGGAGGAGAAGGATCTACATCCTGCTCGCC-3′(SEQ ID NO:13)
AMT基因：
5′-ATGACTACGTCGACGAACCAGCTCGTAATCTTGCCCAAGAAGGTCAAATGGCAGCGCGCCTCCAGACCCAAAGTTCGCACTGGCTGTTTAACCTGGTATGTCCCCCGTTGCAAATGGCTGATTGACGAATCAGAGACTAATATTAGATGTAGCAAGTACGTATTTCTGTTTTAATTGGGCGGTGAATTGCTTTTAATCACTGTGTTATTGTAGAGTTAGGCATTTGAAGTGTGATGAGGAGAAGCCGACATGTCGCCGCTGTCGCGATGGCCGTGTCAAATGCGACGGTTATGCAACGCCCACCAAGAACCAGATCCAGCAACGCGCTTCAATGCCCGTCACTGGATACACCGTCTCATCTCTCAACCCTCGTATAGCCGATACATCCATTGAGAACTGTTATTTTCACCACTTTCACCACTGGACCTCCACTCAACTCACCTGCTGTCCCGGCTCATCAAACTTCTACCTCACTTATGTCCTCCCTCTTGCGCATTCATGCGAACCTATCAAGCATGCCATCATCGCCGTTGGTGCCGCGCATAGGTTCTACATGGCGGGCCAAGAGACGTGCTCTCCACTCCAGCAGCTGAAAAGCTTGGCTATGCAGCAGTACAACAAATCCATCCAGCGTATTATACCCCACATGTCGATGGATTCTGCATTCGACCTGCAGTGTACTCTGGTGTGCTGTTTGTTATTCGTTGCATTTGAGGGTATCACTGGTCGATATGCCGAGTCGATCCGACATATCCGTGCTGGAACGAAACTGCTCTCGTCGCCTCTACTGATCAATAGTGCGAAGGAACAGAAGATGGCGAGGAAGCTTACGGAGATGTTTGCGAATCTTGGTGTTGAGGCTTCGATGTTTTTGGAGGATACCATCATTCCGGATATCAACTCTGGGTGCCTTGACGTTTTGCAGTGCGGCGATGGCTCGGATCAGCCATTCCAGGACCTCGATGAAGCAGCTAGCTTCCTGCGCAAACTCGACGTCGAGACCGTCGACATTATTGCGCAAACGCCATATACATATTCGATGAACGACGATGGCCGAATCGAGATGTGTACCCTCGAAGGGCCCGAGAAGCAGGAACTGGAAGCGAAATGGGAAGAGATGGACGCCAAGTTTGAGGTTTGGAATTCGCGATTCGAATTGACAAAGAAACACATTGCAACATGGGAATACCAAGAACTGACATCCCCCCAACTCACATACCTCAACATGCAACAAACCTTCTGGCGAATGGGCATGTCATGCGGTCCCGAAGACCTCTCCGAACCAACCCCCGACACCGCCGAAGCATTCCTCAACGCCGCCGAGATCTTCGCCCAGCGCTTAATAACACCCGGCCAACCAAGTTTCTCCCTCGACGGCGATTTGATTTCTGGTCTTTCGGTAGTTGTATGGTCGTGCACTGAAGGGCCGCATCGTGCTAGAGCGCTGGATCTGCTGCGACGCTTGAATAGACGTGAGGGTATTTGGGATAGTAAGGAGATCGTGGAGATGCATGAGGCTGCGTTGGCGTTAGATGATCCCAAGTTATGGTACAATATGGAGATTCCGGGTGGTGTGCCGGGGTATGTTGCTGAGTTGGCGAAGATTTCGCCGAGGTTTGGATTACATCGCTCGGGACTGTTGATACCGGATTTTGAGTGCATTTAAGTCAGATTAGTAAGGAGAGTAATGCTTCTAGTAATATCCCGTGCGCCTGATAGCCACTGCTCGAGTAAAATAAACATAATCATTACATTACATTACTTGCAGAATTGATGAGTCTTGGCTGTGAGAAATTAACTTGTTGTGATGCTTATCTTACGATCCCCAGCGTCATCTTGTCAGATTCAACTACTACGTCATTGGCTGCACACGAGATACAAATGCCCCTCTTCCCAACGCCTGATGCAAATAACTTTCTACATCCAGTGAACTTGACCTGCTGACGAAATCAATCGACCAAAGCCTCTGCTTCAATTGATCAACATGTCTCAGCTCGAGCGCTCCCTCAAAGCAACCAGTCTCTCAGAGACACTTAGTACCTATGATGAATGGGCAAACACCTACAACCAGGATGTCGAGAAAGAAGAATACGTCGCTCCCCAACTCGCGTCTCAAGATTTAATCACCCATCTCGGCACTCAAATCTCCTCAGCTAAAATTCTCGACGCCGGCTGCGGAACGGGCCTCGTCGGCGAAGCGCTCACAAAACTCGGTGCTTCAAACCTTCACGGAATCGACCTAAGTCCCGGTATGCTTGAAGTCGCTGAGCGAACAGGCGTGTACAAATCACTCAATGTCGCGAACCTCGCTGAAGCGCTTGATATCCCCTCACAGAGTTATGATGCGGTCATTTGTGTTGGGACTATGACGGAGGGTCATGTTGGACCCGAGGCTTTTGATGAGTTTGTGAGAGTTAGCAAGGCTGGTGGCGTTATTGTCTCGACGATTCGGGAGTCGGTTTGGCAGGCGAAGGGGTATGAGGCCAAGGTCAACGGACTAGCTGATGAGGGGAGGGTAAAGGTTGTAGCGAGGAAGAGGGAACATCAGAGGATTGGAGCTGGTGTTTATGCGTATTTTGTTGTTCTAGAGGTTCAGTAG-3′(SEQ ID NO:14)
引物A-up-F：
5′-GCCAAGGCCGAGTTATCCTG-3′(SEQ ID NO:15)
引物A-up-R：
5′-GACTGGCGAAAGTCGCCTTGCAGTGTGGAGACGCCAAGAG-3′(SEQ ID NO:16)
引物A-down-F：
5′-GGACGCGGTATCATCAAGGCGCCTGATTTCTGGCGGTTGC-3′(SEQ ID NO:17)
引物A-down-R：
5′-CTTCGCCCTCAAAGCTTCCC-3′(SEQ ID NO:18)
AMT基因上游片段：
5′-GCCAAGGCCGAGTTATCCTGTAGATCTCCTTACCACTATGCAGGTGTTGTTCGGCATTCGAGGAGAAAGTTCCATTGTGGTCTTTTGAGGGGTTTGCTCCGGTGAGTGTTCGACAACCCTGAGCCCCCTTAGCAAGAGTGACGTAATCAATCACTTCACCATTCAGGCTAATTGTCAAACTCCTGGCCAAAACGACCAGTGATACTGCTTGTTGAGCTCTTTTCGACTGGCTCATTCAACTCTTTCTTTCGCGGTATGAACGTTAATACAGCCTTTCCGTCTTCCCGAGAGTCGTAGTCCGCGGAGCGCACGTGCTCTGCCACGAAACAACACCTTATTGATCTCGGGAGTCTGTTTCTCAAGGCTATCCTGGGCTTGGCCTAGCGAACAAACTCAAAAGTATAACTTCGATTTGGCGGTGATTACCAAAAGCTACAAAGCTGAAGTGGGGCCAGTATAGTCCCGGCCGTAGTCATCTTATACGGGATAATGTTTATGGTTGTCTATAGGCTTTTACATACGCTTAAACACCACCAAGTTGATGTGAATAACTACCTGCGACAGTTTTGAGAGCTTCAAGTACGGGGACTGCAGCGTTGCAGAGTACTCACCGTTCCGAGCCAACCGTTCCGAGTTCCGGTACTAGTTCCGACAATGCGGAACGGACAGCAGATCATGATACTCCCTTCATCCTTGGCAGTATCAGCCCTTAAACGATCTTGAAGACTGTTTCGTCCAGAGATGCAGTGTCATGTTGTTTTACAATAGTTCCGCTAGTCACATATCCTTCTCATCACGGACAATTTTCCCAAACAGGCTCGCCGTTCAGCGATACCTCACAATGCAGCGTGATTCCAGCACAAATCGAGCATCAAATTACAATTGGGATGTTCTCGACCTGCAAAGCCCCTGGGAGCCGCGTAATTCATCTCGATTTCTGCCTTTTTAATTCAGCCTTATCGGCACTCAACAGCCGAAAACGTGCATGCCAAGATGCTCCGTTTCAGGTGAATTGGGAGGCACTGTCAAATATGCATGATTCTTCCAGCGAAACAAACGAAGTGTATCATTGAGGCTTACGCAGTTGCTACCAAAAGTGACTTTGCCCGGTATGACACAGACAGTTCCAGATGCTGTGAGACCCACGTTTCAGCCGAGCTTTCTTTCGTGATTGGCTGCGTCGTAAAAGCAGGGATCAAGACCCTGCTAGCGCTGTACTGCGCCTCTTGGCGTCTCCACACTG-3′(SEQ ID NO:19)
AMT基因下游片段：
5′-GCCTGATTTCTGGCGGTTGCATCGCCGGAAACATCTGGGGCGCCAGACAAGGATGGTGATGTTGTAGGCTGACTACGATCTGGCTCACGTCAGATCACGGTGCCATTGAGAATAATGCGCGATATACGACAAGCAAGTAGATTGGACATCACCGAGGTGATAGATTCAATTCGTTATCGAACGTGTTACTTCAAGACTGGATGAAGATTAACGAGAATGCATGTCAAGCTGAGTCACGAAGGTGAGATATTGTGTACCCATGAGATGACCCGAATGAAACAGGGTATGCGGTCGTGTTACATAAGGCCCATGTCTATAAATGAGAGGCTCTTGTATAAGACAATACTGCATATCCCTGAGGAATAGCAACTAGACTTGAGGTTCTTTTGTCTTTCAAAGTGAGTCAATCCCATGACTCAGGCGCAATTAATCCGCGTCGAGAATCGATCACCACTTCACTATCGCGAATCTGGGTCGTGAGTCATGAGCCATCGCCAAATAGGAAGGTGAAAGTGGAGGCGCCATGCATGCTTACCCTGCGCTTAGATACGTGCCGGATTCCCGATGTGATAAACGATTATCTACGACTACATAAACCATAAAGACAAACCTCAATCATCTTTTTTTTTCTATCAATCATCGTCATCATGGACATCTCAAGCGTGACCAAGGCGTTCCAATCGGAACGTCTTATCTACCGCTCCCCTGAAGACAATGACAAAGATAAAGAATTCTTCTTCAAGCATATTGAGAACGACCCCGTTGCAAAAGCCCTCGCAGATCCCTCCATTCTTCGTCCCAAGAGCAAAAAAGGCGTTGAAGACTTCCTGACAGAGTTCCAAAAGTCCGTTCTGGCGGTTGTCCTGTGTCTTTCTCCCGAAGAAGCCAAACAACAGAACATTGAAAGCTCAGAACCAGTCCCCATCGGCTTCATATGTATCGGCTGGGGCGGCACTCCCAAGAATCGCGAGCAGCATCGGGGTACCCATATTGGTATTGTCATCGCGGACGCGTTTAGGAACAAAGGATATGGAGGGGAGAGCATCAATTGGGCGCTGGATTGGGCTTTTAGGTTTGGTGGCTATCATCGTGTTGGCATTGGCACTTTTGGGTTCAATGAGCGGGGGCAGCATTTGTATAAGAAGTTGGGATTTGTGGAGGAGGGGAGGAGTAGAGAGGCGATTTGGTTTGATAGGCAATGGTGGGATATGATTGAGTATGGAATGCTGGAGCACGAATGGGAAGCTTTGAGGGCGAAG-3′(SEQ ID NO:20)
引物A-check-F：
5′-GCCTCCAGACCCAAAGTTCG-3′(SEQ ID NO:21)
引物A-check-R：
5′-CCGCACTGCAAAACGTCAAG-3′(SEQ ID NO:22)
引物ura3check-F：
5′-TCGAATCTTTGCCTCAGCGC-3′(SEQ ID NO:23)
引物ura3check-R：
5′-GCCTTGATGATACCGCGTCC-3′(SEQ ID NO:24)
引物upcheck-F：
5′-CGATTAGCGAGGCCAAGACG-3′(SEQ ID NO:25)
引物upcheck-R：
5′-TCGATGCCGGGGAATACTGT-3′(SEQ ID NO:26)
引物downcheck-F：
5′-TCGATGCCGGGGAATACTGT-3′(SEQ ID NO:27)
引物downcheck-R：
5′-GCAGTTGGGATCTGTGGCTG-3′(SEQ ID NO:28)
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。
序列表
<110> 天津农学院
<120> 一种可用于无痕遗传转化菌株的制备
<150> 202111279781.9
<151> 2021-11-01
<160> 28
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1008
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 1
caaggcgact ttcgccagtc gagctgagac agctacccat cctctcagtc gacatctcta 60
caagctcatg gacctcaagg cctcgaatct ttgcctcagc gccgatgtcg caaccgctcg 120
cgaactgctc tacttcgccg acaagattgg tccctccatc gtcgtcctca agactcatta 180
tgacatggtg gccggctggg actttgatcc taggacaggt actggtgcca agctcgcctc 240
actagcacgc aagcatggtt tcctcatctt tgaggatcgc aagtttggtg atattggcaa 300
tacggtcgag ttgcagtata ctagcggtgc tgcgcgcatc atcgagtggg cacatattac 360
caatgtaaat atggttccag gcaaggcctc ggttacatct ctggcccacg ctgccaaccg 420
atggctcgag cgataccact acgaggtcaa gacatctatc agcattggaa ctcccaccgc 480
cagtcaactg gatgaggata gcgagcgttc agacaacgag aaccagaagg acacgccgga 540
acctgctcgc gctgacagcg gccgaaaagg tagcattgtt tccaccacca ccgtcaccca 600
acagtacgag tcggccgatt caccacgcct cgtcaagacg atccctgagg gcgatgagac 660
agtattcccc ggcatcgatg aagcgccgat cgagaggggc ctgcttatcc tggcacagat 720
gtcgagccag ggcaacttca tgaacaagga atatacgcaa gcatgtgtag aggccgcgcg 780
ggaacacaag aacttcgtca tgggctttgt ctcacaggag tgtctcaaca cagaacccga 840
ggatgacttc atccacatga cccccggttg ccaattgccc cctgagggcg cggatgagaa 900
tgaagccatc aagggtgatg gcaagggtca acagtacaac acgccacaga agatcattgg 960
tattgccggt gctgacattg ctattgttgg acgcggtatc atcaaggc 1008
<210> 2
<211> 1815
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 2
ggatctcgac gttgtacgcc gtgaattagg ttagttgtca tgtcaccctt aatccctcgc 60
tggctccggc ttgaagaacc gctgaaacgg gtgtttgcgg caatagttta gatgtcgtgt 120
aacccagcca gatcacttgg aggtttttgg aaaatcaggg tgatgatcgt atcctaaccg 180
cctcatacct gagagatgat ccctttgttg agcttggtgg tggtctgtgt tgagatatca 240
cctgcagtca gtatcataaa aaccatgaac catttaatag ccgcttccat ggtgaaaagc 300
atcaatccac catggcaaag cctcccgcct cttgggctgt cttgaacaga tcattaagcc 360
aggattggtt atgatcatct cgccctactt ctctagtttc tgatccctga tccgcaaagt 420
gcagtgtagg ccaaacccta tccggatagc caacgttgga cctggtctcg acagcaggtg 480
tatgcttgtt taattcctct aaaatcagat gatttttggg gtttcgtgtc tttgttcatc 540
caccaagctt gtcttgaatt ggcttctcga cttgtccgtc ttggtgcccc ccacgataca 600
cgtcactgac ggtatggatc taggtatggg ggcatgggtc ctagggttag gaagaatttg 660
tttccatcgg tggtgatatg atcaggatga ggagagtatg tatttttttc agttgaggat 720
tgtttcactg ggcaatggcg aatggaggac ttgtagacaa ggtgtagtca tgtagttata 780
catatcggat tacttccgac caattcccca aagaaggatt tatttgcttg ccatttcaat 840
tggctgattc ttcctcacag agtcttccac tacaaagatg gatggatggg tggctgccct 900
tgtccgctcc ggctaaaaga gaactgccag gataagttga gtttcaagct ttggatcgtc 960
catatcagtg ccattttctt tgaattgctg tcaaacatcc ttgcccctgt tcctgtctga 1020
gtacatacat acctacagca gtgcctctag tgcgtacatg ttgcattcgc agcctgttac 1080
agtaaaaaag agacactctt gttaatccct gcttgacgcc acgcctgtgc cttcaacctc 1140
tgaatgccta cttactttct ggctgcgttt gagcctcggg ccttaaacgt gttagcttgt 1200
tatgaatctc gaggttcaag cgccctttcg cttcatgttg gagctaagct aaggtaccct 1260
agcactagca cctaggtggt agtgaccctc tagccttgtc tctcgcctca ccttcagtgc 1320
ctttcgttgg ttgggtccat aggtcacttt tctttaagtc gagctgtgga tcgtcacttt 1380
tcacttttta cctttcacct ctcacctaca gtctcttctt aactaacccc atcccatccc 1440
gtgtcattct gcgaaagctt tttacttcaa acctcctcct ccttcctctt aactcgctct 1500
ctttcctctc ctctcctcat aattctcctc ccactcccga tcgacattcc acccatttac 1560
gcctcacatc ggacgacact ttcagtcgag acccattcgg ccgaagctaa taagatcttc 1620
acccctcgtt cctatcagtg gctcttgtct ccgtttttca tttttcccca gcttcccctc 1680
tatcaagcca aaccgccact tgcgccaggt agactagatt gttgtgaaca tctttgtgaa 1740
ttggacaagt gtcgaggttg cccaaaactt ccgacttaga tccacatacg atttagtgtg 1800
cttgaactcg ctccc 1815
<210> 3
<211> 1766
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 3
gaggtttttc gcgcggtagg caggctgcac tggagttatc gagacatcta gcgggtctga 60
catgaatatt atgactacat ggagcatcca tggagtaact cacttacaag agaaatgaga 120
cttggttccc tgattgggaa tatcatctaa gcactaggcc tcctccaact gcataagcct 180
ggttcctctc tactgcatct atgataccag gttcaacaca accttacgag aatgattgtc 240
ggaaaagata cgaaatatga attgcaatga cggaataatt cggttcctta cgtgtgcttt 300
caatgcacct ctcatgtcga cggttgtagc taattcactc cagctgcata tatccatttc 360
ttccttcaat aacgtgggtc cattcaaaga tgcacagggc tgaattccat tcttgtaatg 420
gatgccccac aatcaaagtg acgccgtttt cttggaagcc cggagttaga ggactttgaa 480
agctgatcct cgtccgccga gatcaacata gaccttccca tactccacct taatattgac 540
ctctagcctc taagtatctg aatcccatac tactttacta taactttaca tatttgctcc 600
gggtattccc aagtgatcag tcattcatac ccgtttccga agaagccgac atgacagcag 660
cagctgagaa cgctgatggt ccgcgtctga ggaagagaag acgaattgtc atatcgtgca 720
ctgaatgcca tcgaagaaag cagagggtcc gtaactacaa gactacgata cacttgtctt 780
atacaagaaa tatttgactg aaaacatctt tagtgcgacc gcgaacaacc atgcggcaat 840
tgcaagtctc gaaataaaga gtcagcttgt gtttatgaaa caggagctcc aacagccaaa 900
cagagcaagc agacaacaaa agcctcgccg acttactcgg atacattcgc taaacaagcc 960
gagccgtcac catcacagtc tgttgactca tcgaatgaga ggctaggcga gcctctgtca 1020
tctaaggtag ctgactgggg gtacgctcac aatggcgcgt ctaccatggg tatacttaaa 1080
cgcatcgaga cgctcacaga cggtgaagat gggcccccag atctaacatc tcgtgattcc 1140
tccggaggct ctcccggcaa agagcttgcc ttgagggaaa agtacagggc tataattcga 1200
caattacctg cccggaatta catcgaaaaa ctcgtcgaga tgtacatgcg tggcttcaat 1260
tggcagtact atccaatcga ccctgacatc ttttatgcgc aactcgacga gtggaattcg 1320
ttgccgttct cggtattctc agatgtcggt ccccgcggct tgaaccctga actgcgagcc 1380
tttccagcag ttgtatttca aatcatagca acggcccttc tcctattgcc cgagaagcca 1440
gattccacat tcgactcgct caaatttgct ggaggtatga gattcgaaga cttggctgtc 1500
gagtatagcg agtcagggca ggcagttctt gaccttttgg ggaagaagaa cttgtctctg 1560
tctacagtgc aagcacaatt tctcagggcg tcattccaca agtttacggc aaaggttaca 1620
gatgcagtaa gttcctgtta ggagatgata cctagagaag agtcgactga tgttgggtag 1680
tggcacacca tcgcaattgc gattcgcgat gctcaggacc tggggatgca tcgtgacagc 1740
ttagatccca gaccaaagga cgcaag 1766
<210> 4
<211> 20
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 4
ggatctcgac gttgtacgcc 20
<210> 5
<211> 50
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 5
gtgcagcctg cctaccgcgc gaaaaacctc gggagcgagt tcaagcacac 50
<210> 6
<211> 50
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 6
atacgattta gtgtgcttga actcgctccc gaggtttttc gcgcggtagg 50
<210> 7
<211> 20
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 7
cttgcgtcct ttggtctggg 20
<210> 8
<211> 20
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 8
caaggcgact ttcgccagtc 20
<210> 9
<211> 20
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 9
gccttgatga taccgcgtcc 20
<210> 10
<211> 20
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 10
ggagacggtc aggttgatgc 20
<210> 11
<211> 20
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 11
ggcgagcagg atgtagatcc 20
<210> 12
<211> 4898
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 12
ggagacggtc aggttgatgc aaagtcatgt acctggggtt ctgtggatct cgacgttgta 60
cgccgtgaat taggttagtt gtcatgtcac ccttaatccc tcgctggctc cggcttgaag 120
aaccgctgaa acgggtgttt gcggcaatag tttagatgtc gtgtaaccca gccagatcac 180
ttggaggttt ttggaaaatc agggtgatga tcgtatccta accgcctcat acctgagaga 240
tgatcccttt gttgagcttg gtggtggtct gtgttgagat atcacctgca gtcagtatca 300
taaaaaccat gaaccattta atagccgctt ccatggtgaa aagcatcaat ccaccatggc 360
aaagcctccc gcctcttggg ctgtcttgaa cagatcatta agccaggatt ggttatgatc 420
atctcgccct acttctctag tttctgatcc ctgatccgca aagtgcagtg taggccaaac 480
cctatccgga tagccaacgt tggacctggt ctcgacagca ggtgtatgct tgtttaattc 540
ctctaaaatc agatgatttt tggggtttcg tgtctttgtt catccaccaa gcttgtcttg 600
aattggcttc tcgacttgtc cgtcttggtg ccccccacga tacacgtcac tgacggtatg 660
gatctaggta tgggggcatg ggtcctaggg ttaggaagaa tttgtttcca tcggtggtga 720
tatgatcagg atgaggagag tatgtatttt tttcagttga ggattgtttc actgggcaat 780
ggcgaatgga ggacttgtag acaaggtgta gtcatgtagt tatacatatc ggattacttc 840
cgaccaattc cccaaagaag gatttatttg cttgccattt caattggctg attcttcctc 900
acagagtctt ccactacaaa gatggatgga tgggtggctg cccttgtccg ctccggctaa 960
aagagaactg ccaggataag ttgagtttca agctttggat cgtccatatc agtgccattt 1020
tctttgaatt gctgtcaaac atccttgccc ctgttcctgt ctgagtacat acatacctac 1080
agcagtgcct ctagtgcgta catgttgcat tcgcagcctg ttacagtaaa aaagagacac 1140
tcttgttaat ccctgcttga cgccacgcct gtgccttcaa cctctgaatg cctacttact 1200
ttctggctgc gtttgagcct cgggccttaa acgtgttagc ttgttatgaa tctcgaggtt 1260
caagcgccct ttcgcttcat gttggagcta agctaaggta ccctagcact agcacctagg 1320
tggtagtgac cctctagcct tgtctctcgc ctcaccttca gtgcctttcg ttggttgggt 1380
ccataggtca cttttcttta agtcgagctg tggatcgtca cttttcactt tttacctttc 1440
acctctcacc tacagtctct tcttaactaa ccccatccca tcccgtgtca ttctgcgaaa 1500
gctttttact tcaaacctcc tcctccttcc tcttaactcg ctctctttcc tctcctctcc 1560
tcataattct cctcccactc ccgatcgaca ttccacccat ttacgcctca catcggacga 1620
cactttcagt cgagacccat tcggccgaag ctaataagat cttcacccct cgttcctatc 1680
agtggctctt gtctccgttt ttcatttttc cccagcttcc cctctatcaa gccaaaccgc 1740
cacttgcgcc aggtagacta gattgttgtg aacatctttg tgaattggac aagtgtcgag 1800
gttgcccaaa acttccgact tagatccaca tacgatttag tgtgcttgaa ctcgctcccc 1860
agttcgagtc accgtctttt cctatacttt tgttcatatc aaattgatct tcgaacaaac 1920
tttaccgtgt catgtcgccg catcctaccc tcaaggcgac tttcgccagt cgagctgaga 1980
cagctaccca tcctctcagt cgacatctct acaagctcat ggacctcaag gcctcgaatc 2040
tttgcctcag cgccgatgtc gcaaccgctc gcgaactgct ctacttcgcc gacaagattg 2100
gtccctccat cgtcgtcctc aagactcatt atgacatggt ggccggctgg gactttgatc 2160
ctaggacagg tactggtgcc aagctcgcct cactagcacg caagcatggt ttcctcatct 2220
ttgaggatcg caagtttggt gatattggca atacggtcga gttgcagtat actagcggtg 2280
ctgcgcgcat catcgagtgg gcacatatta ccaatgtaaa tatggttcca ggcaaggcct 2340
cggttacatc tctggcccac gctgccaacc gatggctcga gcgataccac tacgaggtca 2400
agacatctat cagcattgga actcccaccg ccagtcaact ggatgaggat agcgagcgtt 2460
cagacaacga gaaccagaag gacacgccgg aacctgctcg cgctgacagc ggccgaaaag 2520
gtagcattgt ttccaccacc accgtcaccc aacagtacga gtcggccgat tcaccacgcc 2580
tcgtcaagac gatccctgag ggcgatgaga cagtattccc cggcatcgat gaagcgccga 2640
tcgagagggg cctgcttatc ctggcacaga tgtcgagcca gggcaacttc atgaacaagg 2700
aatatacgca agcatgtgta gaggccgcgc gggaacacaa gaacttcgtc atgggctttg 2760
tctcacagga gtgtctcaac acagaacccg aggatgactt catccacatg acccccggtt 2820
gccaattgcc ccctgagggc gcggatgaga atgaagccat caagggtgat ggcaagggtc 2880
aacagtacaa cacgccacag aagatcattg gtattgccgg tgctgacatt gctattgttg 2940
gacgcggtat catcaaggca agcgatcccg aggaggaagc tgagcgatat cggtccgcgg 3000
catggaaggc ttacacggag cgggttcgtt aagaagcgaa aatgactgga agtcgggatg 3060
gccttgaggt ttttcgcgcg gtaggcaggc tgcactggag ttatcgagac atctagcggg 3120
tctgacatga atattatgac tacatggagc atccatggag taactcactt acaagagaaa 3180
tgagacttgg ttccctgatt gggaatatca tctaagcact aggcctcctc caactgcata 3240
agcctggttc ctctctactg catctatgat accaggttca acacaacctt acgagaatga 3300
ttgtcggaaa agatacgaaa tatgaattgc aatgacggaa taattcggtt ccttacgtgt 3360
gctttcaatg cacctctcat gtcgacggtt gtagctaatt cactccagct gcatatatcc 3420
atttcttcct tcaataacgt gggtccattc aaagatgcac agggctgaat tccattcttg 3480
taatggatgc cccacaatca aagtgacgcc gttttcttgg aagcccggag ttagaggact 3540
ttgaaagctg atcctcgtcc gccgagatca acatagacct tcccatactc caccttaata 3600
ttgacctcta gcctctaagt atctgaatcc catactactt tactataact ttacatattt 3660
gctccgggta ttcccaagtg atcagtcatt catacccgtt tccgaagaag ccgacatgac 3720
agcagcagct gagaacgctg atggtccgcg tctgaggaag agaagacgaa ttgtcatatc 3780
gtgcactgaa tgccatcgaa gaaagcagag ggtccgtaac tacaagacta cgatacactt 3840
gtcttataca agaaatattt gactgaaaac atctttagtg cgaccgcgaa caaccatgcg 3900
gcaattgcaa gtctcgaaat aaagagtcag cttgtgttta tgaaacagga gctccaacag 3960
ccaaacagag caagcagaca acaaaagcct cgccgactta ctcggataca ttcgctaaac 4020
aagccgagcc gtcaccatca cagtctgttg actcatcgaa tgagaggcta ggcgagcctc 4080
tgtcatctaa ggtagctgac tgggggtacg ctcacaatgg cgcgtctacc atgggtatac 4140
ttaaacgcat cgagacgctc acagacggtg aagatgggcc cccagatcta acatctcgtg 4200
attcctccgg aggctctccc ggcaaagagc ttgccttgag ggaaaagtac agggctataa 4260
ttcgacaatt acctgcccgg aattacatcg aaaaactcgt cgagatgtac atgcgtggct 4320
tcaattggca gtactatcca atcgaccctg acatctttta tgcgcaactc gacgagtgga 4380
attcgttgcc gttctcggta ttctcagatg tcggtccccg cggcttgaac cctgaactgc 4440
gagcctttcc agcagttgta tttcaaatca tagcaacggc ccttctccta ttgcccgaga 4500
agccagattc cacattcgac tcgctcaaat ttgctggagg tatgagattc gaagacttgg 4560
ctgtcgagta tagcgagtca gggcaggcag ttcttgacct tttggggaag aagaacttgt 4620
ctctgtctac agtgcaagca caatttctca gggcgtcatt ccacaagttt acggcaaagg 4680
ttacagatgc agtaagttcc tgttaggaga tgatacctag agaagagtcg actgatgttg 4740
ggtagtggca caccatcgca attgcgattc gcgatgctca ggacctgggg atgcatcgtg 4800
acagcttaga tcccagacca aaggacgcaa gcgtcgagag tattctagag aatcaatggc 4860
tgattcaacg gaggagaagg atctacatcc tgctcgcc 4898
<210> 13
<211> 3692
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 13
ggagacggtc aggttgatgc aaagtcatgt acctggggtt ctgtggatct cgacgttgta 60
cgccgtgaat taggttagtt gtcatgtcac ccttaatccc tcgctggctc cggcttgaag 120
aaccgctgaa acgggtgttt gcggcaatag tttagatgtc gtgtaaccca gccagatcac 180
ttggaggttt ttggaaaatc agggtgatga tcgtatccta accgcctcat acctgagaga 240
tgatcccttt gttgagcttg gtggtggtct gtgttgagat atcacctgca gtcagtatca 300
taaaaaccat gaaccattta atagccgctt ccatggtgaa aagcatcaat ccaccatggc 360
aaagcctccc gcctcttggg ctgtcttgaa cagatcatta agccaggatt ggttatgatc 420
atctcgccct acttctctag tttctgatcc ctgatccgca aagtgcagtg taggccaaac 480
cctatccgga tagccaacgt tggacctggt ctcgacagca ggtgtatgct tgtttaattc 540
ctctaaaatc agatgatttt tggggtttcg tgtctttgtt catccaccaa gcttgtcttg 600
aattggcttc tcgacttgtc cgtcttggtg ccccccacga tacacgtcac tgacggtatg 660
gatctaggta tgggggcatg ggtcctaggg ttaggaagaa tttgtttcca tcggtggtga 720
tatgatcagg atgaggagag tatgtatttt tttcagttga ggattgtttc actgggcaat 780
ggcgaatgga ggacttgtag acaaggtgta gtcatgtagt tatacatatc ggattacttc 840
cgaccaattc cccaaagaag gatttatttg cttgccattt caattggctg attcttcctc 900
acagagtctt ccactacaaa gatggatgga tgggtggctg cccttgtccg ctccggctaa 960
aagagaactg ccaggataag ttgagtttca agctttggat cgtccatatc agtgccattt 1020
tctttgaatt gctgtcaaac atccttgccc ctgttcctgt ctgagtacat acatacctac 1080
agcagtgcct ctagtgcgta catgttgcat tcgcagcctg ttacagtaaa aaagagacac 1140
tcttgttaat ccctgcttga cgccacgcct gtgccttcaa cctctgaatg cctacttact 1200
ttctggctgc gtttgagcct cgggccttaa acgtgttagc ttgttatgaa tctcgaggtt 1260
caagcgccct ttcgcttcat gttggagcta agctaaggta ccctagcact agcacctagg 1320
tggtagtgac cctctagcct tgtctctcgc ctcaccttca gtgcctttcg ttggttgggt 1380
ccataggtca cttttcttta agtcgagctg tggatcgtca cttttcactt tttacctttc 1440
acctctcacc tacagtctct tcttaactaa ccccatccca tcccgtgtca ttctgcgaaa 1500
gctttttact tcaaacctcc tcctccttcc tcttaactcg ctctctttcc tctcctctcc 1560
tcataattct cctcccactc ccgatcgaca ttccacccat ttacgcctca catcggacga 1620
cactttcagt cgagacccat tcggccgaag ctaataagat cttcacccct cgttcctatc 1680
agtggctctt gtctccgttt ttcatttttc cccagcttcc cctctatcaa gccaaaccgc 1740
cacttgcgcc aggtagacta gattgttgtg aacatctttg tgaattggac aagtgtcgag 1800
gttgcccaaa acttccgact tagatccaca tacgatttag tgtgcttgaa ctcgctcccg 1860
aggtttttcg cgcggtaggc aggctgcact ggagttatcg agacatctag cgggtctgac 1920
atgaatatta tgactacatg gagcatccat ggagtaactc acttacaaga gaaatgagac 1980
ttggttccct gattgggaat atcatctaag cactaggcct cctccaactg cataagcctg 2040
gttcctctct actgcatcta tgataccagg ttcaacacaa ccttacgaga atgattgtcg 2100
gaaaagatac gaaatatgaa ttgcaatgac ggaataattc ggttccttac gtgtgctttc 2160
aatgcacctc tcatgtcgac ggttgtagct aattcactcc agctgcatat atccatttct 2220
tccttcaata acgtgggtcc attcaaagat gcacagggct gaattccatt cttgtaatgg 2280
atgccccaca atcaaagtga cgccgttttc ttggaagccc ggagttagag gactttgaaa 2340
gctgatcctc gtccgccgag atcaacatag accttcccat actccacctt aatattgacc 2400
tctagcctct aagtatctga atcccatact actttactat aactttacat atttgctccg 2460
ggtattccca agtgatcagt cattcatacc cgtttccgaa gaagccgaca tgacagcagc 2520
agctgagaac gctgatggtc cgcgtctgag gaagagaaga cgaattgtca tatcgtgcac 2580
tgaatgccat cgaagaaagc agagggtccg taactacaag actacgatac acttgtctta 2640
tacaagaaat atttgactga aaacatcttt agtgcgaccg cgaacaacca tgcggcaatt 2700
gcaagtctcg aaataaagag tcagcttgtg tttatgaaac aggagctcca acagccaaac 2760
agagcaagca gacaacaaaa gcctcgccga cttactcgga tacattcgct aaacaagccg 2820
agccgtcacc atcacagtct gttgactcat cgaatgagag gctaggcgag cctctgtcat 2880
ctaaggtagc tgactggggg tacgctcaca atggcgcgtc taccatgggt atacttaaac 2940
gcatcgagac gctcacagac ggtgaagatg ggcccccaga tctaacatct cgtgattcct 3000
ccggaggctc tcccggcaaa gagcttgcct tgagggaaaa gtacagggct ataattcgac 3060
aattacctgc ccggaattac atcgaaaaac tcgtcgagat gtacatgcgt ggcttcaatt 3120
ggcagtacta tccaatcgac cctgacatct tttatgcgca actcgacgag tggaattcgt 3180
tgccgttctc ggtattctca gatgtcggtc cccgcggctt gaaccctgaa ctgcgagcct 3240
ttccagcagt tgtatttcaa atcatagcaa cggcccttct cctattgccc gagaagccag 3300
attccacatt cgactcgctc aaatttgctg gaggtatgag attcgaagac ttggctgtcg 3360
agtatagcga gtcagggcag gcagttcttg accttttggg gaagaagaac ttgtctctgt 3420
ctacagtgca agcacaattt ctcagggcgt cattccacaa gtttacggca aaggttacag 3480
atgcagtaag ttcctgttag gagatgatac ctagagaaga gtcgactgat gttgggtagt 3540
ggcacaccat cgcaattgcg attcgcgatg ctcaggacct ggggatgcat cgtgacagct 3600
tagatcccag accaaaggac gcaagcgtcg agagtattct agagaatcaa tggctgattc 3660
aacggaggag aaggatctac atcctgctcg cc 3692
<210> 14
<211> 2599
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 14
atgactacgt cgacgaacca gctcgtaatc ttgcccaaga aggtcaaatg gcagcgcgcc 60
tccagaccca aagttcgcac tggctgttta acctggtatg tcccccgttg caaatggctg 120
attgacgaat cagagactaa tattagatgt agcaagtacg tatttctgtt ttaattgggc 180
ggtgaattgc ttttaatcac tgtgttattg tagagttagg catttgaagt gtgatgagga 240
gaagccgaca tgtcgccgct gtcgcgatgg ccgtgtcaaa tgcgacggtt atgcaacgcc 300
caccaagaac cagatccagc aacgcgcttc aatgcccgtc actggataca ccgtctcatc 360
tctcaaccct cgtatagccg atacatccat tgagaactgt tattttcacc actttcacca 420
ctggacctcc actcaactca cctgctgtcc cggctcatca aacttctacc tcacttatgt 480
cctccctctt gcgcattcat gcgaacctat caagcatgcc atcatcgccg ttggtgccgc 540
gcataggttc tacatggcgg gccaagagac gtgctctcca ctccagcagc tgaaaagctt 600
ggctatgcag cagtacaaca aatccatcca gcgtattata ccccacatgt cgatggattc 660
tgcattcgac ctgcagtgta ctctggtgtg ctgtttgtta ttcgttgcat ttgagggtat 720
cactggtcga tatgccgagt cgatccgaca tatccgtgct ggaacgaaac tgctctcgtc 780
gcctctactg atcaatagtg cgaaggaaca gaagatggcg aggaagctta cggagatgtt 840
tgcgaatctt ggtgttgagg cttcgatgtt tttggaggat accatcattc cggatatcaa 900
ctctgggtgc cttgacgttt tgcagtgcgg cgatggctcg gatcagccat tccaggacct 960
cgatgaagca gctagcttcc tgcgcaaact cgacgtcgag accgtcgaca ttattgcgca 1020
aacgccatat acatattcga tgaacgacga tggccgaatc gagatgtgta ccctcgaagg 1080
gcccgagaag caggaactgg aagcgaaatg ggaagagatg gacgccaagt ttgaggtttg 1140
gaattcgcga ttcgaattga caaagaaaca cattgcaaca tgggaatacc aagaactgac 1200
atccccccaa ctcacatacc tcaacatgca acaaaccttc tggcgaatgg gcatgtcatg 1260
cggtcccgaa gacctctccg aaccaacccc cgacaccgcc gaagcattcc tcaacgccgc 1320
cgagatcttc gcccagcgct taataacacc cggccaacca agtttctccc tcgacggcga 1380
tttgatttct ggtctttcgg tagttgtatg gtcgtgcact gaagggccgc atcgtgctag 1440
agcgctggat ctgctgcgac gcttgaatag acgtgagggt atttgggata gtaaggagat 1500
cgtggagatg catgaggctg cgttggcgtt agatgatccc aagttatggt acaatatgga 1560
gattccgggt ggtgtgccgg ggtatgttgc tgagttggcg aagatttcgc cgaggtttgg 1620
attacatcgc tcgggactgt tgataccgga ttttgagtgc atttaagtca gattagtaag 1680
gagagtaatg cttctagtaa tatcccgtgc gcctgatagc cactgctcga gtaaaataaa 1740
cataatcatt acattacatt acttgcagaa ttgatgagtc ttggctgtga gaaattaact 1800
tgttgtgatg cttatcttac gatccccagc gtcatcttgt cagattcaac tactacgtca 1860
ttggctgcac acgagataca aatgcccctc ttcccaacgc ctgatgcaaa taactttcta 1920
catccagtga acttgacctg ctgacgaaat caatcgacca aagcctctgc ttcaattgat 1980
caacatgtct cagctcgagc gctccctcaa agcaaccagt ctctcagaga cacttagtac 2040
ctatgatgaa tgggcaaaca cctacaacca ggatgtcgag aaagaagaat acgtcgctcc 2100
ccaactcgcg tctcaagatt taatcaccca tctcggcact caaatctcct cagctaaaat 2160
tctcgacgcc ggctgcggaa cgggcctcgt cggcgaagcg ctcacaaaac tcggtgcttc 2220
aaaccttcac ggaatcgacc taagtcccgg tatgcttgaa gtcgctgagc gaacaggcgt 2280
gtacaaatca ctcaatgtcg cgaacctcgc tgaagcgctt gatatcccct cacagagtta 2340
tgatgcggtc atttgtgttg ggactatgac ggagggtcat gttggacccg aggcttttga 2400
tgagtttgtg agagttagca aggctggtgg cgttattgtc tcgacgattc gggagtcggt 2460
ttggcaggcg aaggggtatg aggccaaggt caacggacta gctgatgagg ggagggtaaa 2520
ggttgtagcg aggaagaggg aacatcagag gattggagct ggtgtttatg cgtattttgt 2580
tgttctagag gttcagtag 2599
<210> 15
<211> 20
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 15
gccaaggccg agttatcctg 20
<210> 16
<211> 40
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 16
gactggcgaa agtcgccttg cagtgtggag acgccaagag 40
<210> 17
<211> 40
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 17
ggacgcggta tcatcaaggc gcctgatttc tggcggttgc 40
<210> 18
<211> 20
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 18
cttcgccctc aaagcttccc 20
<210> 19
<211> 1243
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 19
gccaaggccg agttatcctg tagatctcct taccactatg caggtgttgt tcggcattcg 60
aggagaaagt tccattgtgg tcttttgagg ggtttgctcc ggtgagtgtt cgacaaccct 120
gagccccctt agcaagagtg acgtaatcaa tcacttcacc attcaggcta attgtcaaac 180
tcctggccaa aacgaccagt gatactgctt gttgagctct tttcgactgg ctcattcaac 240
tctttctttc gcggtatgaa cgttaataca gcctttccgt cttcccgaga gtcgtagtcc 300
gcggagcgca cgtgctctgc cacgaaacaa caccttattg atctcgggag tctgtttctc 360
aaggctatcc tgggcttggc ctagcgaaca aactcaaaag tataacttcg atttggcggt 420
gattaccaaa agctacaaag ctgaagtggg gccagtatag tcccggccgt agtcatctta 480
tacgggataa tgtttatggt tgtctatagg cttttacata cgcttaaaca ccaccaagtt 540
gatgtgaata actacctgcg acagttttga gagcttcaag tacggggact gcagcgttgc 600
agagtactca ccgttccgag ccaaccgttc cgagttccgg tactagttcc gacaatgcgg 660
aacggacagc agatcatgat actcccttca tccttggcag tatcagccct taaacgatct 720
tgaagactgt ttcgtccaga gatgcagtgt catgttgttt tacaatagtt ccgctagtca 780
catatccttc tcatcacgga caattttccc aaacaggctc gccgttcagc gatacctcac 840
aatgcagcgt gattccagca caaatcgagc atcaaattac aattgggatg ttctcgacct 900
gcaaagcccc tgggagccgc gtaattcatc tcgatttctg cctttttaat tcagccttat 960
cggcactcaa cagccgaaaa cgtgcatgcc aagatgctcc gtttcaggtg aattgggagg 1020
cactgtcaaa tatgcatgat tcttccagcg aaacaaacga agtgtatcat tgaggcttac 1080
gcagttgcta ccaaaagtga ctttgcccgg tatgacacag acagttccag atgctgtgag 1140
acccacgttt cagccgagct ttctttcgtg attggctgcg tcgtaaaagc agggatcaag 1200
accctgctag cgctgtactg cgcctcttgg cgtctccaca ctg 1243
<210> 20
<211> 1259
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 20
gcctgatttc tggcggttgc atcgccggaa acatctgggg cgccagacaa ggatggtgat 60
gttgtaggct gactacgatc tggctcacgt cagatcacgg tgccattgag aataatgcgc 120
gatatacgac aagcaagtag attggacatc accgaggtga tagattcaat tcgttatcga 180
acgtgttact tcaagactgg atgaagatta acgagaatgc atgtcaagct gagtcacgaa 240
ggtgagatat tgtgtaccca tgagatgacc cgaatgaaac agggtatgcg gtcgtgttac 300
ataaggccca tgtctataaa tgagaggctc ttgtataaga caatactgca tatccctgag 360
gaatagcaac tagacttgag gttcttttgt ctttcaaagt gagtcaatcc catgactcag 420
gcgcaattaa tccgcgtcga gaatcgatca ccacttcact atcgcgaatc tgggtcgtga 480
gtcatgagcc atcgccaaat aggaaggtga aagtggaggc gccatgcatg cttaccctgc 540
gcttagatac gtgccggatt cccgatgtga taaacgatta tctacgacta cataaaccat 600
aaagacaaac ctcaatcatc tttttttttc tatcaatcat cgtcatcatg gacatctcaa 660
gcgtgaccaa ggcgttccaa tcggaacgtc ttatctaccg ctcccctgaa gacaatgaca 720
aagataaaga attcttcttc aagcatattg agaacgaccc cgttgcaaaa gccctcgcag 780
atccctccat tcttcgtccc aagagcaaaa aaggcgttga agacttcctg acagagttcc 840
aaaagtccgt tctggcggtt gtcctgtgtc tttctcccga agaagccaaa caacagaaca 900
ttgaaagctc agaaccagtc cccatcggct tcatatgtat cggctggggc ggcactccca 960
agaatcgcga gcagcatcgg ggtacccata ttggtattgt catcgcggac gcgtttagga 1020
acaaaggata tggaggggag agcatcaatt gggcgctgga ttgggctttt aggtttggtg 1080
gctatcatcg tgttggcatt ggcacttttg ggttcaatga gcgggggcag catttgtata 1140
agaagttggg atttgtggag gaggggagga gtagagaggc gatttggttt gataggcaat 1200
ggtgggatat gattgagtat ggaatgctgg agcacgaatg ggaagctttg agggcgaag 1259
<210> 21
<211> 20
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 21
gcctccagac ccaaagttcg 20
<210> 22
<211> 20
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 22
ccgcactgca aaacgtcaag 20
<210> 23
<211> 20
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 23
tcgaatcttt gcctcagcgc 20
<210> 24
<211> 20
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 24
gccttgatga taccgcgtcc 20
<210> 25
<211> 20
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 25
cgattagcga ggccaagacg 20
<210> 26
<211> 20
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 26
tcgatgccgg ggaatactgt 20
<210> 27
<211> 20
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 27
tcgatgccgg ggaatactgt 20
<210> 28
<211> 20
<212> DNA
<213> 尖孢镰刀菌(Fusarium oxysporum)
<400> 28
gcagttggga tctgtggctg 20
Claims (5)
1.尖孢镰刀菌ura3缺失突变体的制备方法，其特征在于，步骤如下：
提取尖孢镰刀菌基因组DNA；利用引物对up-F/R、down-F/R分别扩增尖孢镰刀菌ura3基因的5′端上游片段和3′端下游片段；将上游片段和下游片段进行PCR融合，形成融合片段；将融合片段通过PEG介导转化至尖孢镰刀菌原生质体中，并在含有5-FOA、尿苷和尿嘧啶的培养基上培养，筛选得到尖孢镰刀菌ura3的缺失突变体。
2.根据权利要求1所述的制备方法，其特征在于，尖孢镰刀菌ura3基因序列如SEQ IDNO:1所示，5′端上游片段序列如SEQ ID NO:2所示，3′端下游片段序列如SEQ ID NO:3所示，引物up-F序列如SEQ ID NO:4所示，引物up-R序列如SEQ ID NO:5所示，引物down-F序列如SEQ ID NO:6所示，引物down-R序列如SEQ ID NO:7所示。
3.权利要求1或2所述方法制备的尖孢镰刀菌ura3缺失突变体在无痕遗传转化中的应用。
4.根据权利要求3所述的应用，其特征在于，所述无痕遗传转化为，以尖孢镰刀菌ura3缺失突变体为出发菌，以尿嘧啶营养缺陷作为筛选标记进行目的基因敲除。
5.一种以尿嘧啶营养缺陷为筛选标记进行目的基因敲除的方法，其特征在于，步骤如下：以尖孢镰刀菌DNA作为模板，利用引物扩增获得ura3基因片段、目的基因的上游片段和下游片段；之后对目的基因的上游片段、ura3片段以及下游片段按照摩尔质量比1:3:1进行PCR融合，扩增获得转化所需融合片段；以权利要求1或2所述方法制备的尖孢镰刀菌ura3缺失突变体为出发菌株，制备尖孢镰刀菌ura3缺失突变体原生质体，PEG介导融合片段转化原生质体；将转化子在不添加尿苷、尿嘧啶和5-FOA的培养基上培养，能够正常生长的菌株被筛选为目的基因敲除的尖孢镰刀菌。
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