CN114854779A - 一种番茄抗坏血酸生物合成基因pmi及应用 - Google Patents
一种番茄抗坏血酸生物合成基因pmi及应用 Download PDFInfo
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- CN114854779A CN114854779A CN202210403837.5A CN202210403837A CN114854779A CN 114854779 A CN114854779 A CN 114854779A CN 202210403837 A CN202210403837 A CN 202210403837A CN 114854779 A CN114854779 A CN 114854779A
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Abstract
本发明属于基因工程技术领域，公开了一种番茄抗坏血酸生物合成基因PMI及应用，番茄抗坏血酸生物合成基因PMI为PMI1和PMI2，PMI1和PMI2的ORF的DNA为SEQ ID NO：1和SEQ ID NO：2；利用番茄抗坏血酸生物合成基因PMI编码的蛋白包括SEQ ID NO：3和SEQ ID NO：4。本发明以番茄常规品系AC为对象，克隆抗坏血酸合成Smirnoff途径关键基因，构建相应的遗传转化载体，利用农杆菌介导的遗传转化法转入番茄品系AC中，使目标基因超量或抑制表达；对所获得的转基因番茄进行抗坏血酸含量分析，评价了这些基因在番茄抗坏血酸含量调控中的作用。
Description
技术领域
本发明属于基因工程技术领域，尤其涉及一种番茄抗坏血酸生物合成基因 PMI及应用。
背景技术
目前，最接近的现有技术：
抗坏血酸(AscorbicAcid，AsA)又名维生素C(Vc)，其在生物体内具有很重要的作用，如抗氧化、重要酶的辅因子等。在植物中研究发现，AsA与植物的抗逆性表现正相关，提高内源AsA的含量，可以提高植物的抗逆性，如耐冷等。同时抗坏血酸是人类和动物维持生长发育的一种重要的物质。而灵长类动物(如人类)缺乏AsA生物合成途径中的最后一步酶，因此不能合成AsA，只能从植物中获取足量的AsA(Jain et al.,2000)，而新鲜的果实是抗坏血酸的主要来源。
虽然抗坏血酸在人类所需的营养中具有很重要的作用，但是对于它的生物合成途径的认识并不是十分完整，植物抗坏血酸合成途径的研究起步较动物晚，直到1998年，植物抗坏血酸Smirnoff合成途径才第一次被提出。随后的研究发现植物中抗坏血酸的合成与动物中单一的合成途径不同，可能存在多条合成途径，因此植物抗坏血酸的合成相比动物要复杂的多。
尽管抗坏血酸具有重要的功能，在上世纪30年代就已经鉴定出了其分子结构。随着科学技术的发展，人们发现动物、微生物和植物的抗坏血酸合成途径存在差异。动物和微生物的生物合成途径揭示较早。在1954年lsherwood等提出植物中存在和动物类似的抗坏血酸合成途径，但没有检测到中间产物，故没有得到验证。到90年代，前人从拟南芥突突变体中分离到了抗坏血酸缺陷型，这才使植物抗坏血酸生物合成途径得到重视并且取得很大的进步。1998年，通过同位素示踪、生化分析及早期研究结果，Wheeler等(Wheeler et al.,1998)提出了第一条抗坏血酸途径即D-甘露糖/L-半乳糖途径。自此以后，又有三条途径相继提出，即半乳糖醛酸途径、古洛糖途径以及肌醇途径。
目前，关于PMI转基因相关方面的研究还未见报道。番茄基因组中存在两个同源性较高的PMI，分别命名为PMI1和PMI2。我们构建了PMI1和PMI2 的超量及共抑制表达载体，利用农杆菌介导的方法将他们通过遗传转化的方法导入番茄品系AC中，使目标基因超量或抑制表达。对转基因番茄的叶片及果实进行抗坏血算含量进行测定，通过实验结果的分析，初步鉴定两个PMI在抗坏血酸合成途径中的功能。
番茄是一种全世界普遍栽培的蔬菜作物之一，在蔬菜产业的发展中占有重要的位置。番茄中含有丰富的抗坏血酸，是饮食中维生素的重要来源。由于具有较小的基因组、较短的生长周期且能全年进行生产种植、有成熟的遗传转化体系且是自花授粉植物，因此番茄作为一种模式植物被广泛研究。尽管目前植物抗坏血酸代谢研究已经取得了一些进展，但大部分集中在模式植物拟南芥上。在番茄中，仅有有限的抗坏血酸代谢相关基因被克隆和鉴定，关于番茄中调控抗坏血酸积累的机理研究也仅仅处于起步阶段。
通过基因工程提高控制某一特定代谢物合成的限速酶活性或在植物体内引入新的代谢物合成途径，可以提高转基因植物中目标产物含量或合成外源代谢物。通过遗传改良的方法来提高作物各种重要的农艺性状已经成为以后发展的趋势。因此，为了更有效地提高番茄中抗坏血酸的水平，对抗坏血酸代谢途径中相关酶编码基因的克隆和功能鉴定是关键任务。
综上所述，现有技术存在的问题是：
(1)番茄中仅有少数的抗坏血酸合成代谢基因被克隆和鉴定，关于番茄中调控抗坏血酸积累的机理研究较少。为了更有效地加速番茄中抗坏血酸含量的遗传改良，对抗坏血酸代谢途径中相关基因PMI1和PMI2的功能鉴定是关键任务。
(2)目前缺乏PMIs基因在番茄抗坏血酸合成与代谢调控中如何发挥作用的具体试验证据。因此本发明将PMI1和PMI2基因在番茄中超量表达，并***地测定转基因材料相应基因的表达量及抗坏血酸含量，公布番茄中PMI1和 PMI2基因的具体功能。……
解决上述技术问题的难度：
由于抗坏血酸合成代谢是一个数量性状，主效基因尚未阐明，限制了番茄中抗坏血酸含量的遗传改良。需要通过分子生物学对相关基因进行功能分析，验证PMIs基因在抗坏血酸合成中的作用。
解决上述技术问题的意义：
番茄是重要的蔬菜作物，抗坏血酸的含量作为衡量番茄果实品质的重要性状之一，而改良这一性状的前体条件，是必须全面了解与抗坏血酸合成及氧化还原途径中的基因功能，同时也了解其调控机制。目前，抗坏血酸生物合成相关基因的调控机制报道较少。本发明主要公布PMI基因在番茄抗坏血酸合成中的作用，我们把PMI1和PMI2这两个基因在番茄中超量或抑制表达，研究其在调控抗坏血酸生物合成中的作用及调控机制。这将有助于通过遗传改良的方法来提高番茄中抗坏血酸的水平，为番茄品种改良和培育提供新的理论技术和支持。
发明内容
针对现有技术存在的问题，本发明提供了一种番茄抗坏血酸生物合成基因 PMI及应用。本发明以番茄(Solanum lycopersicum)常规品系AC(本实验室保存)为对象，克隆抗坏血酸合成Smirnoff途径关键基因，构建相应的正义和 RNAi遗传转化载体，利用农杆菌介导的遗传转化法转入番茄品系AC中，使目标基因超量或抑制表达。对所获得的转基因番茄进行抗坏血酸含量分析，来评价这些基因在番茄抗坏血酸含量调控中的作用。
本发明是这样实现的，一种番茄抗坏血酸生物合成基因PMI，所述番茄抗坏血酸生物合成基因PMI为PMI1和PMI2，所述PMI1和PMI2的ORF的DNA分别为(Solyc02g086090.2) CTCCCATCGTCACTTGCTTTGTCCAAATCCCCAACTTCCCCTCCTCCTCACA ACACTTTGCCCCTTCAAAAACTAACCCCCCAATGGAAACTATTCAATTCTC TTCAACTTCAACTTCTTTGTTTATTTCAACTTCTTTCTTTTTCTCTCGGGTCT CTCAACAACAATGGAGATGGAGGAAGGTTTTAAGGGGTTACTCAGGTTAA TTGGTTCTGTTAAGAATTACGATTGGGGCCGTACGGCGAAGGAGTCTTGTG TTGGACGTCTGTATAGGCTCAATTCTCGGACGAAAATTGATGAAAAACAGC CATATGCCGAGTTTTGGATGGGAACTCATGACTCTGGACCATCCTACATTGT GGTGGAACGAGGAGGAAGAATTCAGAATGGACATGCTAATGGCGGAGGA ATTAGAGACAAGTGTTCTTTGAAAGATTGGATTCAGAAGAACCCTAGTGTA CTTGGAGAAACTGTTCTTGCCAAATGGGGTACCCAGCTTCCTTTTCTCTTC AAGGTACTCTCAATTGAGAAAGCTTTGTCTATACAAGCTCATCCAGACAAG GATCTGGCAATTCTTTTGCATAAGGAGCAGCCACTCGTTTACAAGGATGAT AACCACAAACCTGAGATGGCTTTGGCATTGACCAAGTTCGAGGCCTTGTG TGGCTTCATAAGTCTTGAGGAGCTTAAAGTGATTGTTCAGACTGTGCCCGA GATTGTTGAAGTAGTTGGTAATGCGCTTGCAGAGCTAGTATTGGACTTGAG CGAGGATGATGAAGAGGAGAAAGGTAAATTAGTGCTCAGAAAATTATTCA CGGAGATTATGTCAGCTAGCAAGGATGTGATCACGGAAGTACTTGCTAAGC TGATTAGTCGTCTGAACATTAAAAACAAGGTAAGGGTGCTGACCGACAAG GAACAACTGGTCCTAGGACTTGAGAAGCAGTATCCATCTGATGTTGGTGTT TTAGCAGCATTCTTGTTTAATTACGTGAAGCTCAATCCTGGTGAAGCTTTAT ATTTAGGGGCAAATGAACCCCATGCATATGTATATGGAGAATGTGTCGAATG TATGGCAACCTCAGATAATGTGGTACGCGCTGGCCTAACTCCCAAGCACCG GGATGTTAGAACTCTCTGTTCAATGCTCACGTATAGACAGGGTAACCCTGA AATTCTGCACGGTACGGCAATAAATCCATACACAGTGAGATACCTCCCTCC TTTTGATGAATTCGAGGTGGATCATTGCATTCTCCCCCCATATTCAACTGTT ACCTTCCCTTCTGCTCCTGGTCCGTCCATGTTTTTGGTCATGGGAGGAGAG GGAACAATGACCACATCAGCAGAAGTGATTGTGGTTGAAGGTGATGTCCT ATTTGCACCTGCAAACACCAATATTACCATTGCAACCTCCTCTGGTTTGCAC TTGTATAGAGCAGGTGTAAACAGCAGATTTTTTGAGGAATGATAGTTGTAG GCTTGTAGCCCCTTATGCTTGCTAATAAAACAGTGAATTTTTCTGTTACACT GGCTGTTCCACCTTGTATTAATAGCATGTTCAGGTATATAACCGCTTAAGTTA AGTGTA
和(Solyc02g063220.2) TAGGAAAGGAAGAAATATTTAAAGTTGGCAGGAAAATGTTTTCCCTTAATT AAATTTGAAACCGCTGCTTTGCGTGTGATAAAGAAAAAAACACCAAAACT TGTAAATCCTCACTAATTTTTCTTGAATGGTACAACACCAACATTCATGTGC TCCTCTGTCAATCTTCTTCTTCTTCAAATCATCATCATCTTTGTTTGAACTGA AGCTACTGTTTCCCGTTCCTTGCTCTGAAGCATGAACCAAGACTACTAAAC CTTTCGCAGAGGAAAAAAACACGAGAGCTTTTTTTTTTTAAACTTAAAATC TGTTTTATGGACACTGACTTGTTGTCGGTGACGGAAGGGAGAGGGAGGCT GGTGAGGTTGATGGGTTGCGTGAAGAATTACGATTGGGGACCACCCGGGA AGGAATCTCGTGTAGCGCGGTTGTATGCTTGCAATAGTGGTAACTATGTTG ACCAAGAGAAGCCTTATGCCGAATTTTGGATGGGTACTCACGATTCTGGGC CTTCCTATGTTGTGGAAGGAACTGAGAATGGGTTGGTTAATGGTAAAGGAG AGGGACACAAGTTAACATTGAAGAATTGGATTCAAAACAACCCTAATGTTC TTGGAGAGAAGGTTGTGAAGAAATGGGGTACCAACCTTCCTTTTCTCTTCA AGGTACTATCTGTTGCAAAAGCTTTGTCCATACAGGCCCATCCAGACAAGG ATTTAGCCTCTCGTCTGCATAGTGAGCTTCCGGATGTTTATAAGGATGACAA TCACAAACCGGAGATGGCATTGGCGTTGACGGAATTTGAGGCATTGTGTG GATTTATAAGTCTCGAGGAGCTTAAGTTGATTGTTCAAACTGTGCCAGAGATTGTTGAATTGGTCGGTACAGCACACACAGAGCAGGTATTGGAATTGAAC GAGGATGATGGGAAGGAAAAAGGTAAATTCGTCCTACAATCAGTATTTACT GAGCTGATGTCAGCAAACAAGGATGTGGTTGCTGAAGTGATAGCCAAGCTGATTAGTCGCCTACACGTTAAAAATCAGGCAAGGGAGCTGACAGAGAAAG AACAAGTGGTGCTTAGACTTGAGAAGCAGTATCCAGCTGATATTGGTGTCT TGGCTGCATTCTTGTTAAATTATGTGAAACTCAATCCTGGTGAAGCCTTATA TTTAGGAGCAAATGAACCTCATGCTTATTTATATGGTGATTGTATTGAATGCA TGGCAACATCGGACAACGTTGTTCGCGCTGGCCTAACTCCAAAACACCGA GATGTTAAAACACTATGCTCAATGCTCACTTACAGACAGGGTTTTCCTGAA ATTCTGCAGGGCACCGCAGTAAATCCTCATGTTATGAGGTACATTCCTCCTT TTGATGAATTTGAAGTTGATCGTTGTATTCTTCCCGAACAATCAACTACTGA ATTTCCATCTATTCCCGGTCCATCCATTTTTATGGTCGTGGAGGGAGAGGGA ACATTAACCTCATCATCAGACGAGATTATCCATGAAGGTGATGTCCTTTTTG CACCTGCAAACACCAACATTACTGTCTCGACATCTTCTGGTTTGCAATTATA TAGAACAGGAATAAACAGCAGGTTTTTTGAGGAGTGAAGGTTGTATACGTA TTCTAGTACATAAAATTGTTCATTAATTTTTCTCTATAGGAAAATCGGCTGATCCAACCTTGTAATATTAGCCATTTCTGTAAATCAGGTATACAAATAAATATGA CTTGTTATAGTTGCCTCATTGTACTAGTTCAGTGTTTCACAATCTAAGTGCA ATAGGTGGAATTAGTATAGGGATTAGGATTTAAGGTTTTGTAAGATACTTTC AAATGCTTTTAATCCAAAACAAAAAGAGCACAGGTATAATTCCCAACAAA AGAAAAAGAGATTGGAGCAGACAATACAGTAATCTTGTGTGCTGTCAAC；所述Solyc02g086090.2片段为1591bp；所述Solyc02g063220.2片段为1945bp；所述PMI1和PMI2核酸水平的一致性为79％。
本发明的另一目的在于提供一种利用所述番茄抗坏血酸生物合成基因PMI编码的蛋白，所述蛋白分别包括(PMI1酶蛋白)MEMEEGFKGLLRLIGSVKNYDWGRTAKESCVGRLYRLNSRTKIDEKQPY AEFWMGTHDSGPSYIVVERGGRIQNGHANGGGIRDKCSLKDWIQKNPSVLG ETVLAKWGTQLPFLFKVLSIEKALSIQAHPDKDLAILLHKEQPLVYKDDNHKP EMALALTKFEALCGFISLEELKVIVQTVPEIVEVVGNALAELVLDLSEDDEEE KGKLVLRKLFTEIMSASKDVITEVLAKLISRLNIKNKVRVLTDKEQLVLGLEK QYPSDVGVLAAFLFNYVKLNPGEALYLGANEPHAYVYGECVECMATSDNVV RAGLTPKHRDVRTLCSMLTYRQGNPEILHGTAINPYTVRYLPPFDEFEVDHCIL PPYSTVTFPSAPGPSMFLVMGGEGTMTTSAEVIVVEGDVLFAPANTNITIATSS GLHLYRAGVNSRFFEE*
和(PM2酶蛋白) MDTDLLSVTEGRGRLVRLMGCVKNYDWGPPGKESRVARLYACNSGNYVDQ EKPYAEFWMGTHDSGPSYVVEGTENGLVNGKGEGHKLTLKNWIQNNPNVL GEKVVKKWGTNLPFLFKVLSVAKALSIQAHPDKDLASRLHSELPDVYKDDN HKPEMALALTEFEALCGFISLEELKLIVQTVPEIVELVGTAHTEQVLELNEDDG KEKGKFVLQSVFTELMSANKDVVAEVIAKLISRLHVKNQARELTEKEQVVLR LEKQYPADIGVLAAFLLNYVKLNPGEALYLGANEPHAYLYGDCIECMATSDN VVRAGLTPKHRDVKTLCSMLTYRQGFPEILQGTAVNPHVMRYIPPFDEFEVDR CILPEQSTTEFPSIPGPSIFMVVEGEGTLTSSSDEIIHEGDVLFAPANTNITVSTSSGLQLYRTGINSRFFEE；
所述PMI1酶蛋白由434个氨基酸组成；所述PMI2酶蛋白由435个氨基酸组成；PMI1酶蛋白和PMI2酶蛋白氨基酸水平的相似性为70％。
本发明的另一目的在于提供一种利用所述番茄抗坏血酸生物合成基因PMI 构建的表达载体pMV2(载体图详见图7)。
本发明的另一目的在于提供一种所述表达载体的构建方法，所述表达载体的构建方法包括：
第一步，利用Primer 5设计全长基因引物，在PCR仪中以番茄AC的cDNA 为模板扩增获得目的片段；将PCR产物链接到pEASY-B(pEASY-B为公开商业载体，详细载体信息见北京全式金生物技术有限公司(www.transgen.com.cn)) 载体上，连接产物热激法转化大肠杆菌，50mg/L Km抗性平板筛选阳性克隆，挑选阳性克隆，在37℃摇床上以200r/min震荡培养过夜，用基因特异性引物经 PCR检测后挑选阳性克隆测序验证；
pEASY-B的序列为：
AGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGG AAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCC GGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTGC CCTTAAGGGCAGCTTCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGG AAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGC ACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGT GTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCT CGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACC TCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACG TTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGA TTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACA AAATTCAGGGCGCAAGGGCTGCTAAAGGAAGCGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGA ATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGG CGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGACAG GATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGC TATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTT TGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCG TTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGAT CTCCTGTCATCCCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCC GGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATC AGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGC GAGGATCTCGTCGTGACCCACGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGA CTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCG AATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGAC GAGTTCTTCTGAATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTT TGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTA CATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTA AAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAG AATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGC CATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGC ACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGAC ACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACA ATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTG ATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTA GTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAA GCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCT AGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTA GAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACC AGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAA ATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTA ATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAA GGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACC TACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGA ACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACT TGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCC TGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTT GAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAG
第二步，挑选序列比对正确的克隆摇菌抽提质粒；序列如前文所述基因注册号：Solyc02g086090.2和Solyc02g063220.2。Xbal和KpnI双酶切重组质粒 1.5h；
第三步，切胶利用胶回收试剂盒回收得到目的片段，利用T4连接酶将目的片段连接到用Xbal和Kpn I双酶切后的pMV2载体上；连接产物热激法转化大肠杆菌，挑选阳性克隆，用35S结合基因反向特异性引物对单克隆进行PCR阳性检测，挑取阳性克隆摇菌提质粒，双酶切验证正确后通过电击法转入农杆菌感受态C58细胞中；
第四步，挑选阳性克隆，在28℃摇床上以150r/min震荡培养过夜，利用载体上35S加基因反向特异性引物同样对菌液进行PCR阳性检测；阳性克隆加甘油后混匀保存在-70℃低温冰箱。
进一步，第一步中，PCR扩增方法包括：
采用10μL反应体系，以EF1a为内参，经罗氏荧光定量PCR仪LC480进行测定分析，体系包含
Premix Ex TaqTM(2×)5μL，正反向引物各0.5μL，模板4μL；
每个样品重复三次，反应程序：95℃预变性5min；95℃ 10s，58℃ 1min，经45个循环后进行熔解曲线分析，确定PMI1和PMI2基因的特异性。
本发明的另一目的在于提供一种所述的番茄抗坏血酸生物合成基因PMI在 PMIs超量转基因植株叶片相对表达量及抗坏血酸含量测定上的应用，所述PMIs 超量转基因植株叶片相对表达量及抗坏血酸含量测定上的应用方法包括：
筛选出T0代相对表达量较高的超量转基因植株，T1代再次进行检测确认；以3个超量转基因株系和非转基因株系幼嫩叶片为材料，相对表达量经qPCR 进行测定分析；
抗坏血酸含量采用HCl抽提的方法用酶标仪测定；取转化植株幼嫩叶片液氮速冻，样品在液氮中研磨成粉末，分装0.1g样品于2ml离心管中，每个样品 3次重复；
测定时每管中加入1ml预冷的0.2mol/L HCl溶液进行抽提，抽提半个小时左右，每隔5min颠倒混匀，4℃ 12000r/min离心10min；
取500μL上清液加入50μL 0.2mol/L NaH2PO4(pH 5.6)，用0.2mol/L NaOH调节pH值在5与6；
混匀后取100μL上清，依次加入140μL 0.12mol/L NaH2PO4和10μL 25 mmol/L DTT，室温避光反应30min。取上清95μL加入0.1ml 0.2mol/L NaH2PO4，酶标仪测定吸收波长为265nm处的值；
最后加5μL 40U/ml AO酶，反应后测吸收波长值；酶标仪测定抗坏血酸标准溶液，并绘制标准曲线。
综上所述，本发明的优点及积极效果为：
本发明从番茄中克隆了抗坏血酸合成相关基因，将其在番茄中超量表达，***地对相应的转基因材料进行相应表达量及抗坏血酸含量的测定，初步鉴定了PMIs在番茄抗坏血酸合成与代谢调控中的作用。
与现有技术相比，本发明的优点包括：
1)克隆了甘露糖-6磷酸异构酶基因家族中的两个成员PMI1和PMI2，其 ORF分别为1591bp和1945bp，分别编码434和435个氨基酸，核酸水平的一致性为79％，氨基酸水平的相似性为70％。
2)PMI1及PMI2组织表达谱分析结果表明PMIs在番茄各组织中为组成型表达，但在各器官中的表达水平存在较大差异。PMI1在叶中的表达量最高，其次是根和花，在茎和红熟果实中表达量最低；PMI2在花中的表达量最高，其次是叶，在果实不同时期呈现先降低后升高的趋势。
3)本发明测定了番茄品系AC的根、茎、叶、花和各个发育时期果实中抗坏血酸含量，结果表明总抗坏血酸含量存在较大差异，在根中含量最低，叶片中含量最高；随着果实的发育，总抗坏血酸含量呈现出逐渐增加的趋势，以红熟期果实抗坏血酸含量为最高。
4)本发明构建了PMI1和PMI2超量表达载体，利用农杆菌介导的遗传转化方法将它们转入番茄常规品系AC中。获得的番茄转化植株经PCR检测，结果表明外源基因已***番茄基因组中。
5)靶基因在超量转基因株系叶片中的表达量明显高于非转基因材料，PMI1 超量转基因株系O2-6﹑O11-7和O12-2表达量分别为非转基因材料157.7倍﹑ 338.9倍和156.4倍；PMI2超量转基因株系O4-2﹑O12-11和O18-9表达量分别为非转基因材料11.6倍﹑33.3倍和20.3倍。超量表达PMI1的倍数比超量表达PMI2的倍数更高，这可能是由于PMI1在叶片中的本底表达量比PMI2低。
6)本发明采用酶标仪对PMI1和PMI2超量转基因株系叶片中总抗坏血酸的含量进行了测定，PMI1超量表达显著提高了番茄叶片中总抗坏血酸含量， PMI2超量表达也提高了番茄叶片中总抗坏血酸含量，PMI1上调表达后，促进了APX1上调表达，抑制了GalUR﹑GalDH和MIOX的表达，抗坏血酸合成步骤中绝大部分基因的表达没有受到影响；PMI2上调表达后促进了抗坏血酸合成代谢相关基因GLDH﹑GMP﹑MDHAR﹑GME1﹑GME2﹑DHAR﹑APX1﹑GGP ﹑APX6﹑PMI1﹑PMI2﹑GR﹑GalUR﹑GalDH和MIOX的表达，其中GGP的表达量上至非转基因的5.0倍，但明显抑制了其上游GME1和GME2的表达，分别下降至非转基因的37.0％和16.1％。
附图说明
图1是本发明实施例提供的技术路线图。
图2是本发明实施例提供的番茄不同组织中PMIs的组织表达谱分析及番茄不同组织中总抗坏血酸含量图。
图3是本发明实施例提供的转基因植株的PCR检测图。
图中：M：分子量标记；N：阴性对照；P：阳性对照(重组质粒)；A:1 到5为PMI1超量转基因植株；B:1到15为PMI2超量转基因植株；C:1到14 为PMIs共抑制转基因植株；PCR检测所用引物为35S加基因反向引物。
图4是本发明实施例提供的转基因转基因株系叶片中靶基因的表达分析图。
图中：A:O2、O11、O12、O17、O18为PMI1超量株系；B:O4、O5、O6、 O12、O14、O16、O17、O18、O22、O26为PMI2超量株系；WT野生型。
图5是本发明实施例提供的超量表达PMI1转基因株系果实的表达量检测 (A,C)；超量表达PMI2转基因株系果实AsA含量测定(B,D)。
图中：WT野生型；O2-6、O11-7、O12-2为PMI1超量植株；O4-2、O12-11、 O18-9为PMI2超量植株。
图6是本发明实施例提供的PMIs超量转基因株系叶片中AsA合成相关基因的表达分析图。
图中：WT野生型；O12-2：PMI1超量植株；O4-2：PMI2超量植株。
图7是本发明实施例提供的pMV2载体图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
抗坏血酸(AsA)是一种抗氧化剂，在植物的多个生理过程中发挥着重要的作用。抗坏血酸是广泛存在于新鲜水果蔬菜及许多生物中的一种重要的维生素，作为一种生物活性物质，它参与许多新陈代谢活动。抗坏血酸的生物合成途径及其代谢调控的基因工程研究取得了突破进展。但在番茄中，仅有少数的抗坏血酸合成代谢基因被克隆和鉴定，通过克隆关键基因调控番茄中抗坏血酸合成代谢的还有待深入。现有技术中，没有从番茄中克隆抗坏血酸合成相关基因，将其在番茄中超量表达，***地对相应的转基因材料进行相应表达量及抗坏血酸含量的测定，对初步鉴定PMIs在番茄抗坏血酸合成与代谢调控中的作用缺乏具体的理论依据。
针对现有技术存在的问题，本发明提供了一种番茄抗坏血酸生物合成基因 PMI及应用，下面结合附图对本发明作详细的描述。
本发明实施例提供的番茄抗坏血酸生物合成基因PMI，所述番茄抗坏血酸生物合成基因PMI为PMI1和PMI2，所述PMI1和PMI2的ORF的DNA为 SEQ ID NO：1和SEQ ID NO：2；所述SEQ ID NO：1片段为1591bp；所述 SEQ ID NO：2片段为1945bp；所述PMI1和PMI2核酸水平的一致性为79％。
本发明提供一种利用所述番茄抗坏血酸生物合成基因PMI编码的蛋白，所述蛋白的包括SEQ ID NO：3和SEQ ID NO：4；所述SEQ ID NO：3由SEQ ID NO：1编码，为434个氨基酸。所述SEQ ID NO：4由SEQ ID NO：2编码，为435 个氨基酸；SEQ ID NO：3和SEQ ID NO：4氨基酸水平的相似性为70％。
本发明提供一种利用所述番茄抗坏血酸生物合成基因PMI构建的表达载体。
本发明提供一种所述表达载体的构建方法，所述表达载体的构建方法包括：
第一步，利用Primer 5设计全长基因引物，在PCR仪中以番茄AC的cDNA 为模板扩增获得目的片段；将PCR产物链接到pEASY-B载体上，连接产物热激法转化大肠杆菌，50mg/LKm抗性平板筛选阳性克隆，挑选阳性克隆，在37℃摇床上以200r/min震荡培养过夜，用基因特异性引物经PCR检测后挑选阳性克隆测序验证。
第二步，挑选序列比对正确的克隆摇菌抽提质粒；Xbal和KpnI双酶切重组质粒1.5h。
第三步，切胶利用胶回收试剂盒回收得到目的片段，利用T4连接酶将目的片段连接到用Xbal和Kpn I双酶切后的pMV2载体上；连接产物热激法转化大肠杆菌，挑选阳性克隆，用35S结合基因反向特异性引物对单克隆进行PCR阳性检测，挑取阳性克隆摇菌提质粒，双酶切验证正确后通过电击法转入农杆菌感受态C58细胞中。
第四步，挑选阳性克隆，在28℃摇床上以150r/min震荡培养过夜，利用载体上35S加基因反向特异性引物同样对菌液进行PCR阳性检测；阳性克隆加甘油后混匀保存在-70℃低温冰箱。
在本发明实施例中，第一步中，PCR扩增方法包括：
采用10μL反应体系，以EF1a为内参，经罗氏荧光定量PCR仪LC480进行测定分析，体系包含
Premix Ex TaqTM(2×)5μL，正反向引物各0.5μL，模板4μL。
每个样品重复三次，反应程序：95℃预变性5min；95℃ 10s，58℃ 1min，经45个循环后进行熔解曲线分析，确定PMI1和PMI2基因的特异性。
本发明提供一种PPs超量转基因植株叶片相对表达量及抗坏血酸含量测定上的应用方法包括：
筛选出T0代相对表达量较高的超量转基因植株，T1代再次进行检测确认；以3个超量转基因株系和非转基因株系幼嫩叶片为材料，相对表达量经qPCR 进行测定分析。
抗坏血酸含量采用HCl抽提的方法用酶标仪测定；取转化植株幼嫩叶片液氮速冻，样品在液氮中研磨成粉末，分装0.1g样品于2ml离心管中，每个样品 3次重复。
测定时每管中加入1ml预冷的0.2mol/L HCl溶液进行抽提，抽提半个小时左右，每隔5min颠倒混匀，4℃ 12000r/min离心10min。
取500μL上清液加入50μL 0.2mol/L NaH2PO4(pH 5.6)，用0.2mol/L NaOH调节pH值在5与6。
混匀后取100μL上清，依次加入140μL 0.12mol/L NaH2PO4和10μL 25 mmol/L DTT，室温避光反应30min。取上清95μL加入0.1ml 0.2mol/L NaH2PO4，酶标仪测定吸收波长为265nm处的值。
最后加5μL 40U/ml AO酶，反应后测吸收波长值；酶标仪测定抗坏血酸标准溶液，并绘制标准曲线。
下面结合具体实施例对本发明作进一步描述。
实施例
1)植物材料：
番茄材料为(Solanum lycopersicum)常规品系AC(本实验室保存)，用于基因克隆及遗传转化。AC春季盆栽，坐果成熟后收集成株的根、茎、叶、花及不同发育时期的果实样品用液氮速冻，保存在-70℃超低温冰箱，用于分析PMIs 的组织表达模式。
2)PMIs组织表达谱及各组织抗坏血酸含量测定分析：
利用Primer5软件设计PMI基因的qPCR引物。采用10μL反应体系,以EF1a为内参(Lovdal and Lillo,2009)，经罗氏荧光定量PCR仪LC480进行测定分析，其体系包含
Premix Ex TaqTM(2×)5μL，正反向引物各0.5μL，模板4μL。每个样品重复三次，反应程序：95℃预变性5min；95℃ 10s，58℃ 1 min，经45个循环后进行熔解曲线分析，以便确定引物的特异性。利用公式2- △△Ct计算基因相对表达量(Svensson et al.,2006)。采用通用的高效液相色谱法测定番茄各组织抗坏血酸含量。
3)PMIs基因扩增和序列分析：
以Unigene Solyc02g086090.2和Solyc02g063220.2为探针，在 SGN(http://solgenomics.net/index.pl)获得番茄全长cDNA序列。利用NCBI、 ClustalW、Softberry、Multalin等软件对PMI1与PMI2进行相应生物信息学分析。利用Primer 5.0软件设计包含完整编码区的基因特异性引物。以番茄品系AC叶片cDNA为模板进行PCR扩增。
4)PMI1与PMI2超量表达载体的构建：
首先利用Primer 5设计全长基因引物，在PCR仪中以番茄AC的cDNA为模板扩增获得目的片段。将PCR产物链接到pEASY-B载体上，连接产物热激法转化大肠杆菌，50mg/L Km抗性平板筛选阳性克隆，挑选阳性克隆，在37℃摇床上以200r/min震荡培养过夜，用基因特异性引物经PCR检测后挑选阳性克隆测序验证。挑选序列比对正确的克隆摇菌抽提质粒。Xbal和KpnI双酶切重组质粒1.5h。切胶利用胶回收试剂盒回收得到目的片段，利用T4连接酶将目的片段连接到用Xbal和Kpn I双酶切后的pMV2载体上。连接产物热激法转化大肠杆菌，挑选阳性克隆，用35S结合基因反向特异性引物对单克隆进行PCR阳性检测，挑取阳性克隆摇菌提质粒，双酶切验证正确后通过电击法转入农杆菌感受态C58细胞中。挑选阳性克隆，在28℃摇床上以150r/min震荡培养过夜，利用载体上35S加基因反向特异性引物同样对菌液进行PCR阳性检测。阳性克隆加甘油后混匀保存在-70℃低温冰箱，用于下一步的番茄遗传转化。
5)PMIs超量转基因植株叶片相对表达量及抗坏血酸含量测定：
筛选出T0代相对表达量较高的超量转基因植株，T1代再次进行检测确认。以3个超量转基因株系和非转基因株系幼嫩叶片为材料，相对表达量经qPCR 进行测定分析。
抗坏血酸含量采用HCl抽提的方法用酶标仪测定。取转化植株幼嫩叶片液氮速冻，样品在液氮中研磨成粉末，分装0.1g样品于2ml离心管中，每个样品 3次重复。测定时每管中加入1ml预冷的0.2mol/L HCl溶液进行抽提，抽提半个小时左右，每隔5min颠倒混匀，4℃12000r/min离心10min。取500μL 上清液加入50μL 0.2mol/L NaH2PO4(pH 5.6)，用0.2mol/L NaOH调节pH值在5与6之间。混匀后取100μL上清，依次加入140μL 0.12mol/L NaH2PO4(pH7.5)和10μL 25mmol/L DTT，室温避光反应30min。取上清95μL加入0.1ml 0.2 mol/LNaH2PO4(pH 5.6)，酶标仪测定吸收波长为265nm处的值，最后加5μL 40 U/ml AO酶，反应后测其值。酶标仪测定抗坏血酸标准溶液，并绘制标准曲线。
在本发明实施例中，本发明提供的PMI1与PMI2核酸序列比对如下：
在本发明实施例中，本发明提供的PMI1与PMI2蛋白序列比对如下：
在本发明实施例中，图2是本发明实施例提供的番茄不同组织中PMIs的组织表达谱分析及番茄不同组织中总抗坏血酸含量图。由实施例步骤2)PMIs组织表达谱及各组织抗坏血酸含量测定分析所得。采用通用的高效液相色谱法测定番茄各组织抗坏血酸含量。
图3是本发明实施例提供的转基因植株的PCR检测图。首先进行步骤3) PMIs基因扩增和序列分析，获得PMIs基因的全长序列；然后经由步骤4)进行 PMI1与PMI2超量表达载体的构建；超量转基因植株叶片相对表达量检测由实施例步骤5)PMIs超量转基因植株叶片相对表达量检测所得。相对表达量经 qPCR进行测定分析。
图中：M：分子量标记；N：阴性对照；P：阳性对照(重组质粒)；A:1到 5为PMI1超量转基因植株；B:1到15为PMI2超量转基因植株；C:1到14为 PMIs共抑制转基因植株；PCR检测所用引物为35S加基因反向引物。
图4是本发明实施例提供的转基因转基因株系叶片中靶基因的表达分析图。由实施例步骤5)PMIs超量转基因植株叶片相对表达量检测所得。相对表达量经qPCR进行测定分析。
图中：A:O2、O11、O12、O17、O18为PMI1超量株系；B:O4、O5、O6、 O12、O14、O16、O17、O18、O22、O26为PMI2超量株系；WT野生型。
图5是本发明实施例提供的超量表达PMI1转基因株系果实的表达量检测(A,C)；超量表达PMI2转基因株系果实AsA含量测定(B,D)。由实施例步骤 5)PMIs超量转基因植株叶片抗坏血酸含量测定所得。抗坏血酸含量采用HCl 抽提的方法用酶标仪测定。
图中：WT野生型；O2-6、O11-7、O12-2为PMI1超量植株；O4-2、O12-11、 O18-9为PMI2超量植株。
图6是本发明实施例提供的PMIs超量转基因株系叶片中AsA合成相关基因的表达分析图。由实施例步骤5)PMIs超量转基因植株叶片相对表达量检测所得。相对表达量经qPCR进行测定分析。
图中：WT野生型；O12-2：PMI1超量植株；O4-2：PMI2超量植株。
图7是本发明提供的pMV2载体图。
证明部分(具体实施例/实验/药理学分析/能够证明本发明创造性的正面实验数据、证据材料、鉴定报告、商业数据、研发证据、商业合作证据等)
实施结果表明，在番茄中超量表达PMI1时番茄果实中抗坏血酸含量，从 80mg/100gFW上升至119mg/100gFW,涨幅达48％(图5B)。在番茄中超量表达PMI2时，番茄果实中抗坏血酸含量从80mg/100gFW上升至112mg/100gFW，提高幅度达40％(图5D)。因此，PMIs在番茄果实抗坏血酸合成积累中发挥重要促进作用。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
序列表
<110> 武汉楚为生物科技有限公司
<120> 一种番茄抗坏血酸生物合成基因PMI及应用
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Claims (6)
1.一种番茄抗坏血酸生物合成基因PMI，其特征在于，所述番茄抗坏血酸生物合成基因PMI为PMI1和PMI2，所述PMI1和PMI2的ORF的DNA为SEQ ID NO：1和SEQ ID NO：2；所述SEQ IDNO：1片段为1591bp；所述SEQ ID NO：2片段为1945bp；所述PMI1和PMI2核酸水平的一致性为79％。
2.一种利用权利要求1所述番茄抗坏血酸生物合成基因PMI编码的蛋白，其特征在于，所述蛋白的包括SEQ ID NO：3和SEQ ID NO：4；所述SEQ ID NO：3由SEQ ID NO：1编码，为273个氨基酸；
所述SEQ ID NO：4由SEQ ID NO：2编码，为268个氨基酸；SEQ ID NO：3和SEQ ID NO：4氨基酸水平的相似性为80％。
3.一种利用权利要求1所述番茄抗坏血酸生物合成基因PMI构建的表达载体。
4.一种如权利要求3所述表达载体的构建方法，其特征在于，所述表达载体的构建方法包括：
第一步，利用Primer 5设计全长基因引物，在PCR仪中以番茄AC的cDNA为模板扩增获得目的片段；将PCR产物链接到pEASY-B载体上，连接产物热激法转化大肠杆菌，50mg/L Km抗性平板筛选阳性克隆，挑选阳性克隆，在37℃摇床上以200r/min震荡培养过夜，用基因特异性引物经PCR检测后挑选阳性克隆测序验证；
第二步，挑选序列比对正确的克隆摇菌抽提质粒；Xbal和KpnI双酶切重组质粒1.5h；
第三步，切胶利用胶回收试剂盒回收得到目的片段，利用T4连接酶将目的片段连接到用Xbal和Kpn I双酶切后的pMV2载体上；连接产物热激法转化大肠杆菌，挑选阳性克隆，用35S结合基因反向特异性引物对单克隆进行PCR阳性检测，挑取阳性克隆摇菌提质粒，双酶切验证正确后通过电击法转入农杆菌感受态C58细胞中；
第四步，挑选阳性克隆，在28℃摇床上以150r/min震荡培养过夜，利用载体上35S加基因反向特异性引物同样对菌液进行PCR阳性检测；阳性克隆加甘油后混匀保存在-70℃低温冰箱。
5.如权利要求4所述的表达载体的构建方法，其特征在于，第一步中，PCR扩增方法包括：
采用10μL反应体系，以EF1a为内参，经罗氏荧光定量PCR仪LC480进行测定分析，体系包含
Premix Ex TaqTM(2×)5μL，正反向引物各0.5μL，模板4μL；
每个样品重复三次，反应程序：95℃预变性5min；95℃10s，58℃1min，经45个循环后进行熔解曲线分析，确定PMI1和PMI2基因的特异性。
6.一种如权利要求1所述的番茄抗坏血酸生物合成基因PMIs在PMIs超量转基因植株叶片相对表达量及抗坏血酸含量测定上的应用，其特征在于，所述PMIs超量转基因植株叶片相对表达量及抗坏血酸含量测定上的应用方法包括：
筛选出T0代相对表达量较高的超量转基因植株，T1代再次进行检测确认；以3个超量转基因株系和非转基因株系幼嫩叶片为材料，相对表达量经qPCR进行测定分析；
抗坏血酸含量采用HCl抽提的方法用酶标仪测定；取转化植株幼嫩叶片液氮速冻，样品在液氮中研磨成粉末，分装0.1g样品于2ml离心管中，每个样品3次重复；
测定时每管中加入1ml预冷的0.2mol/L HCl溶液进行抽提，抽提半个小时左右，每隔5min颠倒混匀，4℃12000r/min离心10min；
取500μL上清液加入50μL 0.2mol/L NaH2PO4(pH 5.6)，用0.2mol/L NaOH调节pH值在5与6；
混匀后取100μL上清，依次加入140μL 0.12mol/L NaH2PO4和10μL 25mmol/L DTT，室温避光反应30min。取上清95μL加入0.1ml 0.2mol/L NaH2PO4，酶标仪测定吸收波长为265nm处的值；
最后加5μL 40U/ml AO酶，反应后测吸收波长值；酶标仪测定抗坏血酸标准溶液，并绘制标准曲线。
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