CN114107268B - 高等植物acs双酶活关键位点突变制备得到的单酶活突变体 - Google Patents
高等植物acs双酶活关键位点突变制备得到的单酶活突变体 Download PDFInfo
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Abstract
本发明适用于基因工程技术领域，提供了高等植物ACS双酶活关键结构域替换制备得到的单酶突变体，包括保守序列为F‑Q‑D‑Y‑H‑G‑[LM]‑[PSKL]的BOX2保守域、保守序列为M‑S‑S‑F‑[GT]‑L‑[VI]‑S‑S‑Q‑T‑Q的BOX6保守域，以及保守序列为T‑N‑P‑S‑N‑P‑L‑G‑[TA]‑[TAMVIF]的BOX4保守域。将这些保守域序列进行重组替换后，ACS重组蛋白只具有ACS或C‑S裂解酶单酶活性。将拟南芥ACS7基因组中BOX2、BOX6或BOX4保守域的相应位置与小立碗藓相应序列进行重组，重组后的蛋白R6、R10仅具有C‑S裂解酶活性而R12仅具有ACS活性。本发明不仅鉴定了影响高等植物ACS蛋白ACS和C‑S裂解酶活性的关键结构域，更为利用基因工程方法控制乙烯参与的多项生命过程比如果实成熟等提供了关键靶点，在抗逆作物分子育种中也具有广阔的应用前景和重要的经济价值。
Description
技术领域
本发明属于基因工程技术领域，尤其涉及高等植物ACS双酶活关键位点突变制备得到的单酶活突变体。
背景技术
作为重要的植物激素之一，乙烯对高等植物生长发育的诸多方面，包括种子萌发、细胞伸长、根系发育、叶片伸展、开花与果实成熟、器官衰老与脱落等都起着重要的调节作用。同时，乙烯在植物对生物与非生物逆境响应与抵抗过程中的作用也不可或缺。在种子植物中，乙烯的生物合成主要经过三步重要的催化反应：第一步，甲硫氨酸在S-腺苷甲硫氨酸合酶的作用下生成S-腺苷甲硫氨酸（SAM）；第二步，SAM在1- 氨基环丙烷-1-羧酸合酶的催化下发生γ位的C-S健断裂和α，γ位 C-C 环化生成 1-氨基环丙烷-1-羧酸（ACC），第三步，5′-甲硫腺苷（MTA）、ACC在ACC氧化酶（ACC oxidase，ACO）的作用下转化为乙烯。其中，ACS催化SAM形成ACC是高等植物乙烯合成的限速步骤，ACS也被称为乙烯合成的限速酶。
高等植物的ACS由多基因家族编码，其成员的氨基酸序列都包含一个保守的AAT-like（Aspartate aminotransferases-like）结构域，属于磷酸吡哆醛（Pyridoxal-5’-Phosphate，PLP）依赖型蛋白酶的α超家族（superfamily）。除ACS外，这个家族的其他亚族还包括氨基转移酶和C-S裂解酶等。模式植物拟南芥的ACS家族成员共有12个，其中ACS1-2，ACS4-9和ACS11编码乙烯合成关键酶ACS，ACS10和ACS12编码以天冬氨酸和芳香族氨基酸为底物的氨基转移酶，而ACS3是一个没有功能的假基因。高等植物ACS蛋白的保守域有7个，分别被命名为BOX1至BOX7。长期以来，人们一直一致认为乙烯合成关键酶ACS是单酶，只能催化SAM形成ACC，参与植物体内乙烯的生物合成，主要作用于果实成熟、器官衰老等发育过程。但是，申请人发现，高等植物中的乙烯合成关键酶ACS，包括拟南芥AtACS7，除了具有传统认知的ACS活性外，还能以胱氨酸为底物催化生成铵根离子和丙酮酸（图1），表明其也具有C-S裂解酶活性。丙酮酸是糖酵解的终产物和线粒体三羧酸循环的能量底物，是维持呼吸代谢的非常重要的代谢物，在植物抵抗多种逆境胁迫中发挥重要作用。高等植物ACS蛋白双酶活性的发现，使之成为促进植物发育、果实成熟以及抵抗逆境抗性两种不同而又紧密关联的生命过程的关键交叉点，对其双酶活性的平衡和调控是利用生物技术手段进行作物改造的重要靶标。
发明内容
本发明实施例的目的在于提供高等植物ACS蛋白ACS和C-S裂解酶单酶活性的关键位点及其应用，旨在提供一种通过对双酶活性的平衡和调控的生物技术手段进行作物改造的方法。
本发明实施例是这样实现的，高等植物ACS蛋白BOX2的保守序列为F-Q-D-Y-H-G-[LM]-[PSKL]，该保守域中的Q位点对ACS蛋白ACS活性的维持尤其至关重要。在拟南芥ACS7蛋白中，该Q位点位于其所有编码氨基酸的第98位，故申请人将ACS7蛋白中的该位点命名为Q98。用不同物种生物包括人类、老鼠、酵母、细菌、蚊子、大麦、拟南芥、西红柿和苹果等的ACS、C-S裂解酶和氨基转移酶序列做系统进化树，结果表明，只有单独形成一个独立分支的、真正的ACS蛋白具有相应于拟南芥ACS7蛋白 Q98的Q位点，其他非ACS蛋白均不在这个特定分支，也不在相应位置具有Q位点（图2）。另外，如果将ACS蛋白的该位点Q突变成其他氨基酸，可以使该ACS蛋白丧失ACS活性。例如，如果将位于拟南芥第四号染色体13275200-13277188位置区间的ACS7基因组中第13275698-13275700位、其对应mRNA或cDNA序列中第399-401位的CAA碱基突变为GCA碱基，从而使ACS7蛋白的第98位氨基酸从Q突变成A，突变后的ACS7Q98A蛋白无论在体内还是体外都仅具有C-S裂解酶活性，而ACS活性消失（图3、图4）。
进一步地，单酶突变体ACS7Q98A的氨基酸序列如SEQ ID NO.1 所示。
高等植物ACS蛋白保守域BOX4的保守序列为T-N-P-S-N-P-L-G-[TA]-[TAMVIF]，该保守序列C末端的最后两个氨基酸[TA]-[TAMVIF]对ACS活性的维持也非常重要。在拟南芥ACS7蛋白中，这两个保守位点分别对应其所有编码氨基酸中第221位的A和第222位的T，故申请人将拟南芥ACS7蛋白中这两个保守位点分别命名为A221和T222。如果将这两个保守位点突变成其他氨基酸，可以使该ACS蛋白丧失ACS活性。例如，如果将位于拟南芥第四号染色体13275200-13277188位置区间的ACS7基因组中第13276263-13276265位、第13276266-13276268位及其对应cDNA序列第768-770位、第771-773位的GCG碱基和ACG碱基分别突变为ACT和 CTT，或者将其对应mRNA序列第768-770位、第771-773位的GCG碱基和ACG碱基分别突变为ACU和 CUU,可以将ACS7蛋白的第221位和222位氨基酸由AT突变成TL， 突变后的ACS7A221T T222L 蛋白也仅具有C-S裂解酶活性，而ACS活性消失(图5)。
进一步地，单酶突变体ACS7A221T T222L的氨基酸序列如SEQ ID NO.2所示。
拟南芥ACS7蛋白第245位氨基酸（D），虽然不位于上述任何一个保守BOX，但是申请人发现，将拟南芥、水稻、苹果、番茄、小立碗藓的ACS、C-S裂解酶和氨基转移酶序列一起做MEME保守域分析，结果表明ACS蛋白具有九个保守域，即ACS-motif 1-9，其中拟南芥ACS7蛋白第245位氨基酸（D）及其在其他物种ACS蛋白中的相应位点位于第五个ACS-motif（图6），其保守序列为D-F-x(3)-K-N-I-H-L-[IV]-S-D-E-I-[YF]-[SA]-G-[TS]-V-F， 其中拟南芥ACS7蛋白第245位氨基酸对应上述保守序列中的第13个氨基酸（D），申请人将拟南芥ACS7蛋白中的该保守位点命名为D245。申请人还发现，拟南芥ACS7蛋白的D245位点及其在其他ACS蛋白中的相应位点（D）对C-S裂解酶活性的维持非常重要，如果将该位点的氨基酸D突变成其他氨基酸，会导致C-S裂解酶活性丧失而保留ACS活性。例如，如果将位于拟南芥第四号染色体13275200-13277188位置区间的拟南芥ACS7基因组中第13276335-13276337位或其对应cDNA序列中第840-842位的GAC碱基突变为AAT碱基，或者将其对应mRNA序列中第840-842位的GAC碱基突变为AAU碱基，可以使ACS7蛋白第245位氨基酸从D突变为N，突变后的ACS7D245N蛋白仅具有ACS活性，而无C-S裂解活性（图7）。
进一步地，单酶突变体ACS7D245N的氨基酸序列如SEQ ID NO.3所示。
进一步的技术方案，三种所述拟南芥ACS7单酶活性突变体的制备方法，包括以下步骤：
步骤（1）构建含有编码所述拟南芥突变型ACS7的核苷酸序列的载体，
利用基因合成的方法，合成ACS7基因的上下游引物以及包含突变位点信息的引物序列如下：
ACS7-AF： GGATCCATGGGTCTTCCTCTAATGATGGAGAGATCATCAAACAACAACA
Q98A-BF： GTTCCGTGAAAACGCATTGTTTGCAGACTACCACGGTCTCAAAACTT
Q98A-AR： AAGTTTTGAGACCGTGGTAGTCTGCAAACAATGCGTTTTCACGGAAC
A221T&T222L-BF：GGAGTGCTCATAACTAATCCTTCGAACCCATTGGGCACTCTTGTCCAAAAGAAG
A221T&T222L-AR： CTTCTTTTGGACAAGAGTGCCCAATGGGTTCGAAGGATTAGTTATGAGCACTCC
D245N-BF： CAAGAATATTCACTTGGTCTCAAATGAGATCTACTCCGGCTCCGTCT
D245N-AR： AGACGGAGCCGGAGTAGATCTCATTTGAGACCAAGTGAATATTCTTG
ACS7-BR： GAATTCTCAAAACCTCCTTCGTCGGTCCATG
ACS7-A221T&T222L-BR： GCGGCCGCTCAAAACCTCCTTCGTCGGTCCATG；
步骤（2）将测序验证无误的载体，取100 ng转化到BL21感受态细胞中，涂布在含有硫酸卡那霉素的LB平板上，37℃培养过夜，然后挑取单克隆；
步骤（3）培养并收集转化后细胞，按照His-Trap FF column说明书提取纯化各ACS7突变体单酶。
进一步的技术方案，所述步骤（1）中的基因合成方法包括以下步骤：
i. 利用PCR首先对含有突变位点的上下游片段进行扩增，以ACS7-AF与Q98A-AR为引物扩增出ACS7Q98A-A片段，以ACS7-BR与Q98A-BF为引物扩增出ACS7Q98A-B片段；用ACS7-AF与A221T&T222L-AR扩增出ACS7 A221T&T222L-A片段；用ACS7-A221T&T222L-BR与A221T&T222L-BF扩增出ACS7 A221T&T222L-B片段；以ACS7-AF与D245N-AR扩增出ACS7D245N-A片段，以ACS7-BR与D245N-BF扩增出ACS7D245N-B片段；
ii. 以A、B片段互为引物分别通过融合PCR获得ACS7Q98A、ACS7D245N和ACS7A221T T222L 目的片段；
iii. 将上述PCR产物纯化后，按照pMD_18-T Vector Cloning Kit 试剂盒将产物连接到pMD_18-T载体；经测序验证正确后，用BamH I 和 EcoR I双酶切pMD_18-T载体和表达载体pET28a后将目标片段连接至pET28a，获得含有ACS7Q98A和ACS7D245N单酶活性突变体核苷酸序列的表达载体；用BamH I 和 Not I双酶切pMD_18-T载体和表达载体pET28a后将目标片段连接至pET28a，获得ACS7A221T T222L单酶活性突变体核苷酸序列的表达载体。
进一步的技术方案，在步骤i中，扩增的反应体系为：在50 μl反应体系中，以含有野生型ACS7基因的质粒为模板，10 μM 的引物各1μl，以HiFi Hot Start为DNA聚合酶；扩增的条件为：95℃ 5 min；98℃ 20s； 58℃ 15s；72 ℃ 1 min；72℃ 10 min； 共25个循环。
进一步的技术方案，在步骤ii中，扩增的反应体系为：在50 μl反应体系中，分别根据A和B片段的浓度和长度，按照总体积15 μl原则，计算A和B片段各自应加入的体积，以HiFi Hot Start为DNA聚合酶；扩增条件为：95℃ 5 min；98℃ 20s； 58℃ 15s；72℃ 2min；72℃ 10 min； 共11个循环，然后，分别以该步骤获得的纯化产物为模板，以HiFi HotStart为DNA聚合酶，以ACS7上下游ACS7-AF和ACS7-BR为引物通过融合PCR分别获得ACS7Q98A、ACS7D245N产物，以ACS7上下游ACS7-AF和ACS7-A221T&T222L-BR为引物通过融合PCR获得ACS7A221T T222L 目标产物。
进一步的技术方案，包括ACS蛋白ACS和C-S裂解酶双酶的活性测定，其步骤如下：
a. 将纯化的拟南芥 ACS7 突变体蛋白或相应的负对照、ACS 反应缓冲液和 S-腺苷甲硫氨酸一起孵育，用 100 mM HgCl2 终止反应，然后加入新鲜配制的 ACC assaysolution 将生成的 ACC 转化为乙烯，利用气象色谱仪分析计算乙烯生成量；
b. 将纯化的拟南芥 ACS7突变体蛋白或相应的负对照、底物、75 mM磷酸钾缓冲液pH7.6和100 μM PLP一起孵育，反应结束后加入氯仿抽提蛋白；
c.采用分光光度法检测丙酮酸与 2，4-二硝基苯肼反应生成丙酮酸-2，4-二硝基苯腙的量，来测定反应产物丙酮酸；
d.将上述抽提蛋白后的反应液抽滤，用氨基酸分析仪检测分析，来测定反应产物铵根离子。
进一步的技术方案，在步骤a中的ACS 反应缓冲液包括50 mM EPPS， 10 μM PLP，2mM DTT组成，且其pH值为8.5。
进一步的技术方案，在步骤a中，ACC assay solution为饱和 NaOH：次氯酸钠=2：1（v：v）的溶液。
进一步的技术方案，包括拟南芥ACS7的单酶突变体ACS7Q98A、ACS7A221T T222L和ACS7D245N序列在果实成熟以及抗逆作物育种方面的应用。
本发明实施例不仅解析了高等植物ACS蛋白ACS、C-S裂解单酶活性的关键位点，更为利用基因工程方法控制乙烯参与的多项生命过程为成熟等提供了关键靶点，在抗逆作物分子育种中也具有广阔的应用前景和重要的经济价值。
附图说明
图1为本发明的拟南芥AtACS7同时具有ACS和C-S裂解酶双重活性示意图；
图2为不同物种ACS、C-S裂解酶和氨基转移酶序列的系统进化分析示意图；
图3为本发明的ACS7Q98A 体外纯化蛋白只具有C-S裂解酶而无ACS活性示意图；
图4为本发明的ACS7Q98A 在植物体内也只具有C-S裂解酶活性而无ACS活性示意图；
图5为本发明的ACS7A221T T222L 只具有C-S裂解酶活性而无ACS活性示意图；
图6为不同物种ACS蛋白的MEME结构域分析示意图；
图7为本发明的ACS7D245N 纯化蛋白只具有ACS活性而无C-S裂解酶活性示意图。
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，以下结合附图及实施例，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。
以下结合具体实施例对本发明的具体实现进行详细描述。
本发明所述三种拟南芥ACS7单酶活性突变体的制备方法，包括如下步骤：
（1）构建含有编码所述拟南芥突变型ACS7的核苷酸序列的载体
利用基因合成的方法，合成ACS7基因的上下游引物以及包含突变位点信息的引物序列如下：
ACS7-AF：GGATCCATGGGTCTTCCTCTAATGATGGAGAGATCATCAAACAACAACA
Q98A-BF：GTTCCGTGAAAACGCATTGTTTGCAGACTACCACGGTCTCAAAACTT
Q98A-AR：AAGTTTTGAGACCGTGGTAGTCTGCAAACAATGCGTTTTCACGGAAC
A221T&T222L-BF：GGAGTGCTCATAACTAATCCTTCGAACCCATTGGGCACTCTTGTCCAAAAGAAG
A221T&T222L-AR： CTTCTTTTGGACAAGAGTGCCCAATGGGTTCGAAGGATTAGTTATGAGCACTCC
D245N-BF：CAAGAATATTCACTTGGTCTCAAATGAGATCTACTCCGGCTCCGTCT
D245N-AR：AGACGGAGCCGGAGTAGATCTCATTTGAGACCAAGTGAATATTCTTG
ACS7-BR：GAATTCTCAAAACCTCCTTCGTCGGTCCATG
ACS7-A221T&T222L-BR：GCGGCCGCTCAAAACCTCCTTCGTCGGTCCATG
利用PCR首先对含有突变位点的上下游片段进行扩增，以ACS7-AF与Q98A-AR为引物扩增出ACS7Q98A-A片段，以ACS7-BR与Q98A-BF为引物扩增出ACS7Q98A-B片段；用ACS7-AF与A221T&T222L-AR扩增出ACS7 A221T&T222L-A片段；用ACS7-A221T&T222L-BR与A221T&T222L-BF扩增出ACS7 A221T&T222L-B片段；以ACS7-AF与D245N-AR扩增出ACS7D245N-A片段，以ACS7-BR与D245N-BF扩增出ACS7D245N-B片段。扩增反应体系为，在50 μl反应体系中，以含有野生型ACS7基因的质粒为模板，10 μM 的引物各1 μl，以HiFi Hot Start（北京普凯瑞生物科技有限公司，货号KK2601）为DNA聚合酶。扩增条件为，95℃ 5 min；98℃ 20s； 58℃ 15s；72 ℃ 1min；72℃ 10 min； 共25个循环。
以A、B片段互为引物分别获得ACS7Q98A、ACS7D245N和ACS7A221TT222L 目的片段。扩增反应体系为，在50 μl反应体系中，分别根据A和B片段的浓度和长度，按照总体积15 μl原则，计算A和B片段各自应加入的体积，以HiFi Hot Start为DNA聚合酶。扩增条件为，95℃ 5min；98℃ 20s； 58℃ 15s；72℃ 2 min；72℃ 10 min； 共11个循环。然后，分别以该步骤获得的纯化产物为模板，以HiFi Hot Start为DNA聚合酶，以ACS7上下游ACS7-AF和ACS7-BR为引物通过融合PCR分别获得ACS7Q98A、ACS7D245N产物，以ACS7上下游ACS7-AF和ACS7-A221T&T222L-BR为引物通过融合PCR获得ACS7A221T T222L 目标产物。
将上述PCR产物纯化后，按照pMD_18-T Vector Cloning Kit 试剂盒将产物连接到pMD_18-T载体。经测序验证正确后，用BamH I 和 EcoR I 双酶切pMD_18-T载体和表达载体pET28a后将目标片段连接至pET28a，获得含有ACS7Q98A和ACS7D245N单酶活性突变体核苷酸序列的表达载体。用BamH I 和 Not I 双酶切pMD_18-T载体和表达载体pET28a后将目标片段连接至pET28a，获得ACS7A221TT222L 单酶活性突变体核苷酸序列的表达载体。
（2） 将测序验证无误的载体，取100 ng转化到BL21感受态细胞中，涂布在含有硫酸卡那霉素的LB平板上，37℃培养过夜，然后挑取单克隆。
（3） 培养并收集转化后细胞，按照His-Trap FF column （GE Healthcare，货号17-5255-01）说明书提取纯化各ACS7突变体单酶。
其次，本发明所涉及的ACS蛋白ACS和C-S裂解酶双酶活性测定方法如下。其中ACS活性的测定方法为：将纯化的拟南芥 ACS7 突变体蛋白或相应的负对照（转化空载体pET28a 菌株的相同流程蛋白提取物）、ACS 反应缓冲液（50 mM EPPS，pH8.5，10 μM PLP，2mM DTT）和 S-腺苷甲硫氨酸（SAM）一起孵育，用 100 mM HgCl2 终止反应，然后加入新鲜配制的 ACC assay solution [饱和 NaOH：次氯酸钠=2：1（v：v）]将生成的 ACC 转化为乙烯，利用气象色谱仪（Agilent 7890A）分析计算乙烯生成量。C-S裂解酶活性测定的基本反应体系为：将纯化的拟南芥 ACS7突变体蛋白或相应的负对照、底物L-胱氨酸（L-cystine）、75mM磷酸钾缓冲液 pH7.6和100 μM PLP一起孵育，反应结束后加入氯仿抽提蛋白。对于反应产物丙酮酸的测定，采用分光光度法检测丙酮酸与 2，4-二硝基苯肼反应生成丙酮酸-2，4-二硝基苯腙的量。对于反应产物铵根离子（NH4 +）的测定，则将上述抽提蛋白后的反应液抽滤，用氨基酸分析仪（A300，MembraPure GmbH）检测分析。
如图1所示，氨基酸分析仪测定结果显示拟南芥AtACS7可以以胱氨酸为底物催化C-S裂解反应，造成反应体系中胱氨酸含量的显著下降并生成铵根离子；B. 化学显色法证明ACS7可催化底物胱氨酸发生C-S键断裂生成丙酮酸。同时GC法检测证明AtACS7具有正常的ACS活性。His-ACL1蛋白作为C-S裂解酶催化反应的正对照，以空载体pET28a为负对照（control）。
用不同物种生物包括人类、老鼠、酵母、细菌、蚊子、大麦、拟南芥、西红柿和苹果等的ACS、C-S裂解酶和氨基转移酶序列做系统进化树，结果表明，只有单独形成一个独立分支的、真正的ACS蛋白具有相应于拟南芥ACS7蛋白 Q98的Q位点，其他非ACS蛋白均不在这个特定分支，也不在相应位置具有Q位点（如图2），表明拟南芥ACS7的Q98位点及其在其他物种中的相应位点对ACS活性的维持至关重要。例如，将突变后的ACS7Q98A进行C-S裂解酶和ACS活性的测定，发现ACS7Q98A蛋白无论在体内还是体外都仅具有C-S裂解酶活性，而ACS活性消失。如图3所示，A.化学显色法证明ACS7Q98A蛋白可催化底物胱氨酸发生C-S键断裂生成丙酮酸. B. ACS7Q98A 蛋白丧失ACS活性。拟南芥ACS7纯化蛋白作为ACS和C-S裂解酶催化反应的正对照，空载体pET28a为负对照（control）。
将35S:AtACS7 Q98A -eYFP 通过农杆菌介导的瞬时转化方法注射烟草叶片，两天后分别测定所注射叶片中的ACC和丙酮酸含量，以35S:AtACS7-eYFP 和35S:eYFP分别作为正负对照。结果表明，AtACS7 Q98A 过表达叶片中的丙酮酸含量较空载体负对照中的显著升高，而ACC含量并无显著差异，说明ACS7Q98A在植物体内只具有C-S裂解酶活性而无ACS活性（如图4）；
将纯化后的ACS7A221T T222L蛋白进行C-S裂解酶和ACS活性的测定，野生型ACS7蛋白（WT）活性设为100%。结果显示，ACS7A221T T222L蛋白只具有C-S裂解酶活性，而无ACS活性（图5）。
将拟南芥、水稻、苹果、番茄、小立碗藓的ACS、C-S裂解酶和氨基转移酶序列一起做MEME保守域分析，结果表明ACS蛋白具有九个保守域，即ACS-motif 1-9，其中拟南芥ACS7蛋白第245位氨基酸（D）及其在其他物种ACS蛋白中的相应位点位于第五个ACS-motif（如图6）。拟南芥ACS7蛋白的D245位点及其在其他ACS蛋白中的相应位点（D）对C-S裂解酶活性的维持非常重要，如果将该位点的氨基酸D突变成其他氨基酸，会导致C-S裂解酶活性丧失而保留ACS活性。例如，将拟南芥ACS7D245N纯化蛋白进行体外双酶活性测定，以野生型ACS7蛋白（WT）作为对照。结果发现，突变后的ACS7D245N 蛋白只具有ACS活性而无C-S裂解酶活性（图7）。
高等植物中的乙烯合成关键酶ACS，包括拟南芥AtACS7，除了具有传统认知的ACS活性外，还能以胱氨酸为底物催化生成铵根离子和丙酮酸，表明其也具有C-S裂解酶活性。丙酮酸是糖酵解的终产物和线粒体三羧酸循环的能量底物，是维持呼吸代谢的非常重要的代谢物，在植物抵抗多种逆境胁迫中发挥重要作用。高等植物ACS蛋白双酶活性的发现，使之成为促进植物发育、果实成熟以及抵抗逆境抗性两种不同而又紧密关联的生命过程的关键交叉点，对其双酶活性的平衡和调控是利用生物技术手段进行作物改造的重要靶标。
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
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Phe Met Glu Gln Ile Arg Gly Gly Lys Ala Arg Phe Asp Pro Asp Arg
115 120 125
Ile Val Leu Thr Ala Gly Ala Thr Ala Ala Asn Glu Leu Leu Thr Phe
130 135 140
Ile Leu Ala Asp Pro Asn Asp Ala Leu Leu Val Pro Thr Pro Tyr Tyr
145 150 155 160
Pro Gly Phe Asp Arg Asp Leu Arg Trp Arg Thr Gly Val Lys Ile Val
165 170 175
Pro Ile His Cys Asp Ser Ser Asn His Phe Gln Ile Thr Pro Glu Ala
180 185 190
Leu Glu Ser Ala Tyr Gln Thr Ala Arg Asp Ala Asn Ile Arg Val Arg
195 200 205
Gly Val Leu Ile Thr Asn Pro Ser Asn Pro Leu Gly Ala Thr Val Gln
210 215 220
Lys Lys Val Leu Glu Asp Leu Leu Asp Phe Cys Val Arg Lys Asn Ile
225 230 235 240
His Leu Val Ser Asn Glu Ile Tyr Ser Gly Ser Val Phe His Ala Ser
245 250 255
Glu Phe Thr Ser Val Ala Glu Ile Val Glu Asn Ile Asp Asp Val Ser
260 265 270
Val Lys Glu Arg Val His Ile Val Tyr Ser Leu Ser Lys Asp Leu Gly
275 280 285
Leu Pro Gly Phe Arg Val Gly Thr Ile Tyr Ser Tyr Asn Asp Asn Val
290 295 300
Val Arg Thr Ala Arg Arg Met Ser Ser Phe Thr Leu Val Ser Ser Gln
305 310 315 320
Thr Gln His Met Leu Ala Ser Met Leu Ser Asp Glu Glu Phe Thr Glu
325 330 335
Lys Tyr Ile Arg Ile Asn Arg Glu Arg Leu Arg Arg Arg Tyr Asp Thr
340 345 350
Ile Val Glu Gly Leu Lys Lys Ala Gly Ile Glu Cys Leu Lys Gly Asn
355 360 365
Ala Gly Leu Phe Cys Trp Met Asn Leu Gly Phe Leu Leu Glu Lys Lys
370 375 380
Thr Lys Asp Gly Glu Leu Gln Leu Trp Asp Val Ile Leu Lys Glu Leu
385 390 395 400
Asn Leu Asn Ile Ser Pro Gly Ser Ser Cys His Cys Ser Glu Val Gly
405 410 415
Trp Phe Arg Val Cys Phe Ala Asn Met Ser Glu Asn Thr Leu Glu Ile
420 425 430
Ala Leu Lys Arg Ile His Glu Phe Met Asp Arg Arg Arg Arg Phe
435 440 445
Claims (1)
1.高等植物ACS双酶活关键位点突变制备得到的单酶活突变体，其特征在于，所述单酶活突变体为拟南芥ACS7Q98A C-S裂解酶单酶活性突变体、拟南芥ACS7A221T T222L C-S裂解酶单酶活性突变体或拟南芥ACS7D245N ACS单酶活性突变体；
其中，所述拟南芥ACS7Q98A C-S裂解酶单酶活性突变体的氨基酸序列如SEQ ID NO.1所示，所述拟南芥ACS7A221T T222L C-S裂解酶单酶活性突变体的氨基酸序列如SEQ ID NO.2所示，所述拟南芥ACS7D245N ACS单酶活性突变体的氨基酸序列如SEQ ID NO.3所示。
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| CN101985624A (zh) * | 2010-11-13 | 2011-03-16 | 上海交通大学 | 小苍兰ACC合成酶FhACS1蛋白编码序列 |
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| Title |
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| 陈秋红 ; 韦鹏飞 ; 喻泱华 ; 谭攀 ; 郭崇炎 ; 王志龙 ; .蓝莓Vc ACS基因的克隆与表达分析.安徽农学通报.2018,(第21期),全文. * |
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