CN114107372B - 一种通过基因敲除提高籽粒甲硫氨酸含量的方法 - Google Patents
一种通过基因敲除提高籽粒甲硫氨酸含量的方法 Download PDFInfo
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Abstract
本发明公开了一种通过基因敲除提高籽粒甲硫氨酸含量的方法。通过CRISPR/Cas9技术对玉米ZmMETS2实施基因敲除，发现该基因敲除后玉米籽粒中甲硫氨酸含量得到一定程度的提高。本发明中玉米ZmMETS2的CRISPR/Cas9靶标位点的设计以及该基因的功能能够显著影响甲硫氨酸含量的发现，不仅为该基因在玉米和其他作物中CRISPR研究提供了借鉴，还为玉米和其他作物的甲硫氨酸代谢提供了理论基础，通过提高转基因植物的甲硫氨酸含量，缓解人类在氨基酸营养方面的不足。
Description
技术领域
本发明属于基因工程及分子生物学技术领域，具体涉及一种通过基因敲除提高籽粒甲硫氨酸含量的方法。
背景技术
甲硫氨酸属于人体和单胃动物无法合成的必需氨基酸，必须从饮食中摄取。一方面甲硫氨酸是蛋白质的基本组成氨基酸，其密码子作为启动蛋白质mRNA翻译的起点；另一方面甲硫氨酸通过其主要分解代谢产物S-腺苷蛋氨酸间接调节多种细胞过程，包括DNA和蛋白质甲基化、细胞增殖和分化、凋亡、内环境稳定和基因表达等。特别是在植物种子发育和萌发过程中，甲硫氨酸也非常重要。动植物体内甲硫氨酸的合成途径至少有2种。一种途径是以多尾形式的5甲基四氢叶酸和同型半胱氨酸尾底物、在甲硫氨酸合酶的作用下形成甲硫氨酸和四氢叶酸；另一种途径，在动物中是以胆碱为来源的甜菜碱为底物，在甜菜碱同型半胱氨酸S-甲基转移酶的作用下形成甲硫氨酸，在植物中是以S-甲基蛋氨酸为和同型半胱氨酸为底物，在同型半胱氨酸甲基转移酶作用下形成甲硫氨酸。然而，在农作物，特别是豆类植物的种子中发现甲硫氨酸含量相对较低，从而限制了它们的营养质量，以植物为主的膳食，其甲硫氨酸的含量只有平衡膳食所需量的50-75%。在以素食为主的文化中，或在植物源性食物占主导地位的发展中国家，作物中低的甲硫氨酸含量可能导致人类蛋白质缺乏的非特异性反应，例如降低对疾病的抵抗力，降低血液蛋白质，以及幼儿智力和身体发育迟缓。同时，由于甲硫氨酸是人体甲基的四种主要膳食来源之一，其缺乏也会导致甲基化相关疾病（例如脂肪肝、动脉粥样硬化、神经系统疾病和肿瘤）的发生。因此通过提高作物种子的甲硫氨酸含量可以缓解人类在氨基酸营养方面的不足。
基因组编辑技术是研究植物基因功能和农作物品种改良强有力的手段之一。其中，CRISPR/Cas9是一种用于靶标基因特定DNA修饰的工具，因其高效、便捷、适用范围广而成为当今最热门的基因编辑技术。CRISPR/Cas9系统主要由引导RNA（sgRNA）和Cas9蛋白结构域组成，在识别、切割目标DNA双链后，通过同源重组或非同源末端连接修复途径产生DNA突变。该技术不仅可研究、鉴定和筛选与重要性状相关的基因，还能更准确快速地设计、改造作物品种，同时可有针对性地聚合多个优良性状，以提高植物的抗性、产量或品质。通过基因编辑的手段提高植物氨基酸含量已成为目前生物强化的主要手段之一。
发明内容
本发明的目的是提供通过一种通过基因敲除提高籽粒甲硫氨酸含量的方法。
为了实现本发明目的，本发明利用基因编辑技术，对玉米中编码甲硫氨酸合酶2的基因ZmMETS2（该基因所在位点的编号为GRMZM2G112149，参考玉米基因组B72 RefGen_v2）进行敲除突变，背景材料来源于自交系B104，扩增CDS序列引物对为Primer1(SEQ IDNO.4)/Primer2(SEQ ID NO.5)，其CDS序列为SEQ ID NO.1，编码的氨基酸序列为SEQ IDNO.2，具体优选的敲除突变靶位点为SEQ ID NO.3。
所述的通过基因敲除提高籽粒玉米甲硫氨酸含量的方法步骤如下：
（1）突变靶点：在ZmMETS2外显子对应的基因组区域选择1个靶位点， 3’端具有NGG（N为A，T，C，G任一碱基）特征。
（2）构建包含上述靶位点的CRISPR/Cas9载体，即重组质粒ZmMETS2-sgRNA-POsCas9。（方法可参照Li CX, Liu CL et al., 2017, Plant Biotechnology Journal,15:1566-1576）。
（3）该CRISP/Cas9载体可通过使用Ti质粒、植物病毒载体、直接DNA转化、微注射、电穿孔等常规生物技术方法导入植物细胞中得到重组菌株ZmMETS2-sgRNA-POsCas9（Weissbach，1998，Method for Plant Molecular
Biology VIII，Academy Press，New York，第411-463页；Geiserson和Corey，1998，Plant Molecular Biology，2nd Edition）。
（4）将重组菌株ZmMETS2-sgRNA-POsCas9采用农杆菌介导方法转化玉米自交系（品种B104），获得基因敲除的转基因T0代植株。
（5）T0代植株自交，从T1代植株中筛选出能够稳定遗传的已发生基因突变但不含有重组载体ZmMETS2-sgRNA-POsCas9的转基因阴性植株（ZmMETS2-KO）。
（6）按照食品中氨基酸的测定（GB 5009.124-2016 食品安全国家标准），使用氨基酸分析仪对基因敲除的转基因玉米的干籽粒进行甲硫氨酸含量的检测，检测器官为收获的干籽粒。
所述CRISPR/Cas9敲除载体的引物包括引物Primer3(SEQ ID NO.6)和Primer4(SEQ ID NO.7)。用于扩增自交系B104中ZmMETS2基因CDS序列引物对为Primer1(SEQ IDNO.4)和Primer2(SEQ ID NO.5)、用于检测转基因植株靶位点是否突变的扩增引物对为Primer7(SEQ ID NO.10)和Primer8(SEQ ID NO.11)、用于植株转录水平检测的qRT-PCR扩增引物对为Primer9(SEQ ID NO.12)和Primer10(SEQ ID NO.13)。
本发明的有益效果：本发明利用CRISPR/Cas9技术对玉米中编码甲硫氨酸合酶2的基因ZmMETS2进行基因编辑，并且发现了该基因的敲除一定程度提高了玉米籽粒中甲硫氨酸的含量。玉米ZmMETS2基因编码的甲硫氨酸合酶2能够以同型半胱氨酸为底物，生成甲硫氨酸。其功能的缺失导致其他合成甲硫氨酸途径的关键基因转录水平显著地提高，最终导致甲硫氨酸的积累。这不仅为在玉米和其他作物中研究氨基酸代谢提供了理论基础，还可以通过基因工程手段，提高转基因植物的甲硫氨酸含量，缓解人类在氨基酸营养方面的不足。
附图说明
图1中A为本发明实施例1中野生型玉米自交系B104的ZmMETS2基因CDS序列PCR扩增结果；图1中B为本发明在ZmMETS2基因外显子上设计的1个CRISPR/Cas9敲除靶位点的位置（T1）。
图2为本发明实施例1中对ZmMETS2T0代转基因植株材料的Bar基因PCR扩增部分结果；其中1、2、3、4、5为阳性转基因植株，WT为野生型B104。
图3为本发明实施例1中野生型B104（A）和基因编辑植株ZmMETS2-KO（B）在第一个靶标位点的测序峰图结果。
图4为本发明实施例2中qRT-PCR检测转基因玉米籽粒中甲硫氨酸合成基因的相对表达量。白色柱子代表野生型玉米叶片（B104）中的对应基因的转录水平，将B104中的表达水平定义为1；黑色柱子代表转基因株系（CR）叶片中对应基因的转录水平。
图5为本发明实施例3中玉米ZmMETS2蛋白以三尾和四尾形式的5甲基四氢叶酸为底物的甲硫氨酸合酶酶学反应，检测手段为液相色谱-质谱联用体系；图中，酵母甲硫氨酸合酶METE的酶学反应速度为白色柱子，ZmMETS2的酶学反应速度为黑色柱子，5-M-Glu3，三尾形式的5甲基四氢叶酸；5-M-Glu4，四尾形式的5甲基四氢叶酸。
图6为本发明实施例4中转基因玉米干籽粒中甲硫氨酸含量的检测结果，白色柱子代表野生型玉米干籽粒（B104）中的甲硫氨酸水平，其他3个柱子（CR1/CR2/CR3）代表不同的转基因株系干籽粒材料的甲硫氨酸水平。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册（Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001），或按照制造厂商说明书建议的条件。
实施例1 玉米ZmMETS2基因敲除载体的构建和转基因植株的获得与鉴定
（1）采集玉米自交系B104叶片，Trizol法提取玉米总RNA，用RevertAid FirstStrand cDNA Synthesis Kit(Thermo)获得玉米cDNA。以玉米cDNA为模板，Primer1(SEQ IDNO.4)/Primer2(SEQ ID NO.5)为扩增引物，扩增GRMZM2G112149基因的CDS全长（图1A）。
（2）参照测序序列结果，在ZmMETS2外显子对应的基因组区域设计1个靶标位点（图1B的T1，设计网站：http://cas9.cbi.pku.edu.cn/index.jsp），3’端具有NGG(N为A、T、C、G任一碱基)，序列为SEQ ID NO .3。
（3）参照基因敲除载体构建的方法（Li CX, Liu CL et al., 2017, PlantBiotechnology Journal, 15:1566-1576），使用的引物Primer3(SEQ ID NO.6)和Primer4(SEQ ID NO.7)包含了靶标序列，用于构建包含靶位点的CRISPR/Cas9载体，即重组质粒ZmMETS2-sgRNA-POsCas9。
（4）将重组质粒ZmMETS2-sgRNA-POsCas9送至北京博美兴奥科技有限公司转化，获得ZmMETS2基因编辑的转基因T0代植株。
（5）利用重组质粒ZmMETS2-sgRNA-POsCas9载体上的标记基因BAR鉴定T0代植株是否带有转基因，设计用于BAR基因检测的上游引物Primer5(SEQ ID NO.8)和下游引物Primer6(SEQ ID NO.9)。PCR反应体系：DNA模板1 μl，Primer5(SEQ ID NO.8) 0.5 μl，Primer6(SEQ ID NO.9) 0.5 μl，Taq-Mix（购于北京康润诚业生物科技有限公司）10 μl，ddH2O 8 μl；PCR扩增程序：95℃预变性1 min；95℃，30 s，55℃退火，30 s，72℃延伸，60 s，35个循环。72℃终延伸10 min，4℃保温。检测结果如图2所示，1、2、3、4、5为阳性转基因植株。
（6）在ZmMETS2基因组序列上设计引物Primer7(SEQ ID NO.10)和Primer8(SEQ IDNO.11)用于检测T1代植株是否在靶点位置发生突变。以上述T1代植株DNA为模板进行扩增，PCR反应体系：DNA模板1 μl，Primer7(SEQ ID NO.10) 0.5 μl，Primer8(SEQ ID NO.11)0.5 μl，Fast-Pfu（购于北京全式金生物技术有限公司）0.5 μl，dNTP 2μl，5X Buffer 5 μl，ddH2O 16 μl；PCR扩增程序：95℃预变性1 min；95℃，20 s，55℃退火，20 s，72℃延伸，2min，40个循环。72℃终延伸10 min，4℃保温。测序结果见图3，图3A为野生型B104的靶标位置的序列，图3B中靶点位置发生了碱基的缺失（CCTGAGG），且导致氨基酸翻译提前终止，ZmMETS2基因被敲除。
ZmMETS2基因敲除：纯合突变-缺失
AGAAAGTTGCCACTGACCTGAGGTCTAGC(WT)
AGAAAGTTGCCACTGA-------TCTAGC (ZmMETS2-KO)；
实施例2 转录水平上验证甲硫氨酸合成途径在基因编辑植株中的激活
植物体内甲硫氨酸的合成途径至少有2种。一种途径是以多尾形式的5甲基四氢叶酸和同型半胱氨酸尾底物、在甲硫氨酸合酶的作用下形成甲硫氨酸和四氢叶酸，甲硫氨酸合酶ZmMETS2还有同源基因ZmMETS1 和ZmMETS3；另一种途径是以S-甲基蛋氨酸为和同型半胱氨酸为底物，在同型半胱氨酸甲基转移酶作用下形成甲硫氨酸，其编码基因ZmHMT。因此ZmMETS2基因编辑后，利用qRT-PCR检测试剂盒（购自北京全式金生物技术有限公司）检测转基因植株甲硫氨酸合成途径关键基因的转录水平。
T0代籽粒种植后分离出ZmMETS2基因敲除且不带有CRISPR/Cas9重组质粒的阴性转基因代植株，为稳定遗传的ZmMETS2基因敲除T1代株系。将ZmMETS2基因敲除的T1代植株自交，获得T2代种子。种子萌发30天后取叶片液氮冻存、研磨成粉末，使用总RNA提取试剂盒（购自北京原平皓生物技术有限公司）提取总RNA，用RevertAid First Strand cDNASynthesis Kit(Thermo)获得cDNA。通过qRT-PCR检测试剂盒（购自北京全式金生物技术有限公司）检测。ZmMETS2基因的PCR反应体系：DNA模板1 μl，Primer9 (SEQ ID NO.12) 0.4 μl，Primer10 (SEQ ID NO.13) 0.4 μl，Dye II 0.4 μl， 2X Buffer 10 μl，ddH2O 7.8 μl；PCR扩增程序：94℃预变性30 s；94℃，5 s，60℃退火，30 s，40个循环。4℃保温。ZmMETS1、ZmMETS3和HMT使用同样的程序，引物分别为Primer11 (SEQ ID NO.14) 和Primer12 (SEQID NO.15)、Primer13 (SEQ ID NO.16)和Primer14 (SEQ ID NO.17)、Primer15 (SEQ IDNO.18)和Primer16 (SEQ ID NO.19)。检测结果如图4所示，与野生型B104相比，转基因植株ZmMETS1、ZmMETS2、 ZmMETS3和ZmHMT基因的转录水平大幅上升。
实施例3 验证三尾和四尾形式的5-甲基四氢叶酸为底物的ZmMETS2蛋白酶促反应实验
（1）将酵母甲硫氨酸合酶METE基因和玉米ZmMETS2基因CDS序列分别连接至PET28a载体，转化入BL21大肠杆菌中。使用0.3 mM的异丙基硫代半乳糖苷16摄氏度诱导过夜。
（3）收集菌体，匀浆，用Ni-NTA亲和柱(Qiagen) 纯化上清。洗脱蛋白后用透析袋透析，超滤管浓缩蛋白，将蛋白置换在蛋白缓冲溶液（150 mM NaCl，25 mM Tris-HCl，pH 7.2）中，使得浓度约1 mg mL-1。
（4）甲硫氨酸合酶以多尾形式的5甲基四氢叶酸及同型半胱氨酸为底物，30度反应30分钟，产物为甲硫氨酸和四氢叶酸。本体系中通过5甲基四氢叶酸含量的变化体现甲硫氨酸合酶活性。300微升反应体系中包括磷酸钾 50 mM，Tris-HCl 50 mM，pH 8.0，同型半胱氨酸 0.05 mM，三尾或者四尾形式的5甲基四氢叶酸 0.05 mM，纯化的酶10微升。反应到时间后，加入700微升反应终止溶液（50%甲醇、0.1%抗坏血酸钠、0.5%β-巯基乙醇和20mM醋酸铵）。
（5）采用液相色谱-质谱联用检测三尾或者四尾形式的5甲基四氢叶酸，比较反应前后的含量，计算甲硫氨酸合酶的活性。在三尾形式的5甲基四氢叶酸尾作为底物的时候，玉米ZmMETS2活性是酵母METE的66%，而在四尾形式的5甲基四氢叶酸尾作为底物的时候，玉米ZmMETS2活性是酵母METE的112%（图5）。因此ZmMETS2具有甲硫氨酸合酶活性，与酵母甲硫氨酸合酶METE类似，其活性随着底物尾数形式的不同而有变化。
实施例4ZmMETS2基因敲除的玉米甲硫氨酸含量测定
（7）按照食品中氨基酸的测定（GB 5009.124-2016 食品安全国家标准），使用氨基酸分析仪对基因敲除的转基因玉米的干籽粒进行甲硫氨酸含量的检测，检测器官为收获的干籽粒。
结果如图6所示，ZmMETS2转基因玉米干籽粒中甲硫氨酸含量比对照（野生型）都有提高，增加约15-20%。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 中国农业科学院生物技术研究所
<120> 一种通过基因敲除提高籽粒甲硫氨酸含量的方法
<160> 13
<170> SIPOSequenceListing 1.0
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<211> 2298
<212> DNA
<213> 玉米(Zea mays L.)
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atggcgtccc atattgttgg ataccctcgc atgggcccca agagggagct caagtttgcc 60
ttggagtctt tctgggatgg gaagagcagc gccgaggatt tggagaaagt tgccactgac 120
ctgaggtcta gcatctggaa gcaaatgtca gaagctggga tcaagtacat tcccagcaat 180
accttctcgt actacgacca ggttcttgat accacggcca tgcttggcgc tgtcccagag 240
cgctactctt ggactggagg cgagattggc ttgagcacct acttctctat ggccagggga 300
aatgccactg tccctgccat ggagatgacc aagtggtttg atacaaacta ccactttatt 360
gtccctgaac ttggtccaag caccaagttc acatacgctt ctcacaaggc tgtttctgag 420
tacaaggagg caaaggcgct cggcattgat acagtcccag tgcttgttgg accagtctca 480
tacttgctcc tctctaagcc tgccaagggt gtggagaaat ctttctctct tctttcactt 540
cttggtagca ttcttcccat ctacaaggag gttgttgctg agctgaaggc agctggtgct 600
tcatggattc agcttgatga gcctaccctt gttaaagacc ttgatgctca cgaattggcc 660
gcattctctt cagcatatgc tgaactggag tcatcgttct ctggattgaa tgtgcttatc 720
gagacatact tcgctgatat tcctgctgag tcctacaaga ccctcacatc attgagtggt 780
gtgactgctt acggtttcga tcttatccgt ggagccaaga cccttgatct tatcaggagc 840
agcttcccct ctgggaagta cctcttcgct ggtgttgtag atggacgcaa catttgggct 900
gatgatcttg ctgcatctct tagcactctt cattctcttg aggctgttgc tggcaaggac 960
aaacttgtgg tgtcaacctc ctgctcactg atgcacaccg ctgttgacct tgtaaatgag 1020
actaagctgg atgatgagat taagtcatgg cttgcatttg ctgcccaaaa ggttgttgag 1080
gttaatgccc ttgccaaggc tttggcaggc caaaaggatg aggtctactt tgcagccaat 1140
gctgctgctc aggcctcaag gagatcatcg cccagggtga caaacgagga ggtccagaag 1200
gctgcagctg ctttgagggg atctgaccac cgccgttcta ccactgtttc tgctagattg 1260
gatgctcagc agaaaaagct caaccttcct gtccttccca caaccacaat tggttcattc 1320
cctcagactg tggaactcag gagggttcgc cgtgaataca aggcaaagaa gatcaccgag 1380
gacgaataca tcagtgccat caaggaagaa atcagcaagg tcgtcaagat ccaagaggag 1440
cttgacattg atgtgcttgt gcatggagag ccagagagaa atgacatggt tgagtacttc 1500
ggtgagcaat tatctggttt tgcgttcact gccaacggat gggtgcaatc ctatggatca 1560
cgctgtgtga agccacccat tatctacggt gatgtcagcc ggccgaaccc catgactgtt 1620
ttctggtcca agatggcaca gagcatgacc cctcgtccca tgaagggaat gttgactggt 1680
ccggtcacaa tcctcaactg gtcattcgtc aggaacgacc agcctaggtt tgagacatgc 1740
taccaaatag ctcttgcaat caaaaaggag gttgaggatc ttgaggctgc tggtattcag 1800
gtgatccaga tcgatgaggc agctctaagg gagggtctgc cactacgcaa gtcagagcat 1860
gcattctacc tggactgggc tgtccactct ttcaggatca ccaactgcgg agtccaggac 1920
accacccaga tccacaccca catgtgctac tccaacttca acgacatcat ccactccatc 1980
atcgacatgg atgccgatgt gatcacgatc gagaactccc ggtctgacga gaagctactg 2040
tccgtcttcc gtgagggtgt gaagtacgga gctggcattg gccctggtgt ctacgacatc 2100
cactctccta ggattccctc cacagaggag atcgcagacc gcgtcgagaa gatgctcgcc 2160
gtgctcgaca ccaacatcct ctgggtgaac cctgactgtg gtctcaagac acgcaagtac 2220
acggaggtca agcccgccct gaccaacatg gtctcggcca ccaagctcat ccgcacccag 2280
cttgccagcg cgaaatga 2298
<210> 2
<211> 765
<212> PRT
<213> 玉米(Zea mays L.)
<400> 2
Met Ala Ser His Ile Val Gly Tyr Pro Arg Met Gly Pro Lys Arg Glu
1 5 10 15
Leu Lys Phe Ala Leu Glu Ser Phe Trp Asp Gly Lys Ser Ser Ala Glu
20 25 30
Asp Leu Glu Lys Val Ala Thr Asp Leu Arg Ser Ser Ile Trp Lys Gln
35 40 45
Met Ser Glu Ala Gly Ile Lys Tyr Ile Pro Ser Asn Thr Phe Ser Tyr
50 55 60
Tyr Asp Gln Val Leu Asp Thr Thr Ala Met Leu Gly Ala Val Pro Glu
65 70 75 80
Arg Tyr Ser Trp Thr Gly Gly Glu Ile Gly Leu Ser Thr Tyr Phe Ser
85 90 95
Met Ala Arg Gly Asn Ala Thr Val Pro Ala Met Glu Met Thr Lys Trp
100 105 110
Phe Asp Thr Asn Tyr His Phe Ile Val Pro Glu Leu Gly Pro Ser Thr
115 120 125
Lys Phe Thr Tyr Ala Ser His Lys Ala Val Ser Glu Tyr Lys Glu Ala
130 135 140
Lys Ala Leu Gly Ile Asp Thr Val Pro Val Leu Val Gly Pro Val Ser
145 150 155 160
Tyr Leu Leu Leu Ser Lys Pro Ala Lys Gly Val Glu Lys Ser Phe Ser
165 170 175
Leu Leu Ser Leu Leu Gly Ser Ile Leu Pro Ile Tyr Lys Glu Val Val
180 185 190
Ala Glu Leu Lys Ala Ala Gly Ala Ser Trp Ile Gln Leu Asp Glu Pro
195 200 205
Thr Leu Val Lys Asp Leu Asp Ala His Glu Leu Ala Ala Phe Ser Ser
210 215 220
Ala Tyr Ala Glu Leu Glu Ser Ser Phe Ser Gly Leu Asn Val Leu Ile
225 230 235 240
Glu Thr Tyr Phe Ala Asp Ile Pro Ala Glu Ser Tyr Lys Thr Leu Thr
245 250 255
Ser Leu Ser Gly Val Thr Ala Tyr Gly Phe Asp Leu Ile Arg Gly Ala
260 265 270
Lys Thr Leu Asp Leu Ile Arg Ser Ser Phe Pro Ser Gly Lys Tyr Leu
275 280 285
Phe Ala Gly Val Val Asp Gly Arg Asn Ile Trp Ala Asp Asp Leu Ala
290 295 300
Ala Ser Leu Ser Thr Leu His Ser Leu Glu Ala Val Ala Gly Lys Asp
305 310 315 320
Lys Leu Val Val Ser Thr Ser Cys Ser Leu Met His Thr Ala Val Asp
325 330 335
Leu Val Asn Glu Thr Lys Leu Asp Asp Glu Ile Lys Ser Trp Leu Ala
340 345 350
Phe Ala Ala Gln Lys Val Val Glu Val Asn Ala Leu Ala Lys Ala Leu
355 360 365
Ala Gly Gln Lys Asp Glu Val Tyr Phe Ala Ala Asn Ala Ala Ala Gln
370 375 380
Ala Ser Arg Arg Ser Ser Pro Arg Val Thr Asn Glu Glu Val Gln Lys
385 390 395 400
Ala Ala Ala Ala Leu Arg Gly Ser Asp His Arg Arg Ser Thr Thr Val
405 410 415
Ser Ala Arg Leu Asp Ala Gln Gln Lys Lys Leu Asn Leu Pro Val Leu
420 425 430
Pro Thr Thr Thr Ile Gly Ser Phe Pro Gln Thr Val Glu Leu Arg Arg
435 440 445
Val Arg Arg Glu Tyr Lys Ala Lys Lys Ile Thr Glu Asp Glu Tyr Ile
450 455 460
Ser Ala Ile Lys Glu Glu Ile Ser Lys Val Val Lys Ile Gln Glu Glu
465 470 475 480
Leu Asp Ile Asp Val Leu Val His Gly Glu Pro Glu Arg Asn Asp Met
485 490 495
Val Glu Tyr Phe Gly Glu Gln Leu Ser Gly Phe Ala Phe Thr Ala Asn
500 505 510
Gly Trp Val Gln Ser Tyr Gly Ser Arg Cys Val Lys Pro Pro Ile Ile
515 520 525
Tyr Gly Asp Val Ser Arg Pro Asn Pro Met Thr Val Phe Trp Ser Lys
530 535 540
Met Ala Gln Ser Met Thr Pro Arg Pro Met Lys Gly Met Leu Thr Gly
545 550 555 560
Pro Val Thr Ile Leu Asn Trp Ser Phe Val Arg Asn Asp Gln Pro Arg
565 570 575
Phe Glu Thr Cys Tyr Gln Ile Ala Leu Ala Ile Lys Lys Glu Val Glu
580 585 590
Asp Leu Glu Ala Ala Gly Ile Gln Val Ile Gln Ile Asp Glu Ala Ala
595 600 605
Leu Arg Glu Gly Leu Pro Leu Arg Lys Ser Glu His Ala Phe Tyr Leu
610 615 620
Asp Trp Ala Val His Ser Phe Arg Ile Thr Asn Cys Gly Val Gln Asp
625 630 635 640
Thr Thr Gln Ile His Thr His Met Cys Tyr Ser Asn Phe Asn Asp Ile
645 650 655
Ile His Ser Ile Ile Asp Met Asp Ala Asp Val Ile Thr Ile Glu Asn
660 665 670
Ser Arg Ser Asp Glu Lys Leu Leu Ser Val Phe Arg Glu Gly Val Lys
675 680 685
Tyr Gly Ala Gly Ile Gly Pro Gly Val Tyr Asp Ile His Ser Pro Arg
690 695 700
Ile Pro Ser Thr Glu Glu Ile Ala Asp Arg Val Glu Lys Met Leu Ala
705 710 715 720
Val Leu Asp Thr Asn Ile Leu Trp Val Asn Pro Asp Cys Gly Leu Lys
725 730 735
Thr Arg Lys Tyr Thr Glu Val Lys Pro Ala Leu Thr Asn Met Val Ser
740 745 750
Ala Thr Lys Leu Ile Arg Thr Gln Leu Ala Ser Ala Lys
755 760 765
<210> 3
<211> 23
<212> DNA
<213> 玉米(Zea mays L.)
<400> 3
agaaagttgc ctctgacctc agg 23
<210> 4
<211> 27
<212> DNA
<213> 玉米(Zea mays L.)
<400> 4
atggattaca aggatgacga cgataag 27
<210> 5
<211> 25
<212> DNA
<213> 玉米(Zea mays L.)
<400> 5
catttcgcgc tggcaagctg ggtgc 25
<210> 6
<211> 34
<212> DNA
<213> 玉米(Zea mays L.)
<400> 6
cagtggtctc agccagaaag ttgccactga cctg 34
<210> 7
<211> 34
<212> DNA
<213> 玉米(Zea mays L.)
<400> 7
cagtggtctc aaaacaggtc agtggcaact ttct 34
<210> 8
<211> 26
<212> DNA
<213> 玉米(Zea mays L.)
<400> 8
gtctgcacca tcgtcaacca ctacat 26
<210> 9
<211> 24
<212> DNA
<213> 玉米(Zea mays L.)
<400> 9
tgaagtccag ctgccagaaa ccca 24
<210> 10
<211> 24
<212> DNA
<213> 玉米(Zea mays L.)
<400> 10
ccaagatggc acagagcatg accc 24
<210> 11
<211> 24
<212> DNA
<213> 玉米(Zea mays L.)
<400> 11
cggacagtag cttctcgtca gacc 24
<210> 12
<211> 30
<212> DNA
<213> 玉米(Zea mays L.)
<400> 12
taagatttta ctgttatata gaaagatggc 30
<210> 13
<211> 26
<212> DNA
<213> 玉米(Zea mays L.)
<400> 13
gggacaataa agtggtagtt tgtatc 26
Claims (3)
1.一种通过基因敲除提高籽粒甲硫氨酸含量的方法，其特征在于，所述方法是对玉米ZmMETS2基因进行敲除突变，获得籽粒甲硫氨酸含量提高的玉米植株。
2.根据权利要求1所述通过基因敲除提高籽粒甲硫氨酸含量的方法，其特征在于，所述基因敲除的过程为：构建含有玉米ZmMETS2基因靶标序列的重组基因CRISPR/Cas9敲除载体，转化野生型玉米自交系，对玉米的基因ZmMETS2进行突变；所述靶标序列的核苷酸序列如序列表SEQ ID NO.3所示。
3.根据权利要求2所述通过基因敲除提高籽粒甲硫氨酸含量的方法，其特征在于，所述CRISPR/Cas9敲除载体的引物包括Primer3和Primer4，其核苷酸序列如序列表SEQ ID NO.6和SEQ ID NO.7所示。
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| CN101291590A (zh) * | 2005-10-31 | 2008-10-22 | 巴斯夫欧洲公司 | 用于制备l-甲硫氨酸的微生物和方法 |
| CN101454460A (zh) * | 2006-05-24 | 2009-06-10 | 赢创德固赛有限责任公司 | 制备l-甲硫氨酸的方法 |
| CN109055289A (zh) * | 2018-07-27 | 2018-12-21 | 浙江工业大学 | 一种高产l-甲硫氨酸的重组大肠杆菌及其应用 |
| CN109652351A (zh) * | 2018-12-18 | 2019-04-19 | 江南大学 | 一种高产5-甲基四氢叶酸重组枯草芽孢杆菌及其应用 |
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| US8252555B2 (en) * | 2006-12-29 | 2012-08-28 | Evonik Degussa Ag | Nucleic acid encoding a cobalamin-dependent methionine synthase polypeptide |
| US9879273B2 (en) * | 2014-03-14 | 2018-01-30 | Boyce Thompson Institute For Plant Research, Inc. | Compositions and methods for increasing methionine content in plants |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101291590A (zh) * | 2005-10-31 | 2008-10-22 | 巴斯夫欧洲公司 | 用于制备l-甲硫氨酸的微生物和方法 |
| CN101454460A (zh) * | 2006-05-24 | 2009-06-10 | 赢创德固赛有限责任公司 | 制备l-甲硫氨酸的方法 |
| CN109055289A (zh) * | 2018-07-27 | 2018-12-21 | 浙江工业大学 | 一种高产l-甲硫氨酸的重组大肠杆菌及其应用 |
| CN109652351A (zh) * | 2018-12-18 | 2019-04-19 | 江南大学 | 一种高产5-甲基四氢叶酸重组枯草芽孢杆菌及其应用 |
Non-Patent Citations (1)
| Title |
|---|
| PREDICTED: Zea mays 5-methyltetrahydropteroyltriglutamate--homocysteine methyltransferase 1 (LOC541714), transcript variant X2, mRNA;NCBI;《NCBI Reference Sequence: XM_008646413.4》;20200831;origin * |
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| US20230242930A1 (en) | 2023-08-03 |
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