CN116555301B - 一种SlMETS1基因及其在调控番茄生长发育中的应用 - Google Patents
一种SlMETS1基因及其在调控番茄生长发育中的应用 Download PDFInfo
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Abstract
本发明提供了一种SlMETS1基因及其在调控番茄生长发育中的应用。本发明提供的SlMETS1基因具有调控番茄生长发育的功能。使用CRISPR/Cas9基因编辑技术获得了SlMETS1基因敲除番茄,其种子单粒重显著上升、发芽势显著下降、子叶长度显著变短、育性显著下降、花柱长度显著缩短、花粉活力显著下降。本发明发现的SlMETS1的新功能具有较好的应用潜力,为研究植物生长发育提供了基因资源和新思路;得到的SlMETS1基因敲除番茄(mets突变体)有利于杂交育种时免去去雄步骤,具有广泛的应用前景和较高的使用价值。
Description
技术领域
本发明涉及植物分子生物学领域,具体说是一种SlMETS1基因及其在调控番茄生长发育中的应用。
背景技术
番茄(SolanumlycopersicumL.)是我国主要的园艺蔬菜作物之一,由于番茄是一种严格的自花授粉植物,具有明显的杂种优势,在番茄的生产中常使用杂交育种方法。番茄的杂交育种需要人工去雄,然而人工去雄存在人工成本高,时间耗费久,难以保证杂交收种纯度等风险。现有技术常通过雄性不育系做母本进行杂种种子生产,然而雄性不育系具有培育成本较高、品种稳定性较差及技术难度较高的缺点,在生产中存在阻碍。
番茄甲硫氨酸合成酶1基因SlMETS1可以在叶酸依赖的单碳代谢途径中催化甲硫氨酸(Met)的合成,充当叶酸途径和植物免疫之间的纽带(González et al., 2019; Yanet al., 2019)。SlMETS1催化合成的Met可以转化为S-腺苷蛋氨酸(SAM),它是多胺和乙烯合成的前体。
在调控植物抗逆性方面,SlMETS1可以通过叶酸途径参与植物防御反应的调节,SlMETS1表达的降低会以某种方式激活更有效的免疫反应,而SlMETS1的过度表达抑制了植物的免疫力,并伴随着全基因组DNA甲基化的增加,损害植物的免疫力并导致植物的疾病易感性增强(González et al., 2019)。
SlMETS1对染色质沉默的影响与DNA甲基化(CG、CHG和CHH)和组蛋白-3赖氨酸-9二甲基化水平降低有关。SlMETS1突变导致S-腺苷甲硫氨酸与S-腺苷同型半胱氨酸的比率显著降低(Yan et al., 2019)。叶酰聚谷氨酸合酶(FPGS1)可以通过SlMETS1酶介导叶酸的聚谷氨酰化使SlMETS1合成Met,是全基因组DNA甲基化和基因沉默的关键因素(González etal., 2019)。除此之外,在芳香族CK 6 -苄氨基嘌呤(BAP)介导的分生组织(RAM)大小调控水平上,SlMETS1和ACO2 作为乙烯生物合成途径中的酶都是即时根生长响应所必需的,也表明了细胞分裂素(CK)和乙烯在根系生长的调节作用上紧密相连(Zd'arska et al.,2013)。除分生组织和根系生长之外,有关SlMETS1基因调控生长发育的功能和在番茄杂交授粉中的应用还有待进一步研究。
发明内容
针对现有技术中存在的缺陷,本发明的目的在于提供一种SlMETS1基因及其在调控番茄生长发育中的应用。本发明所述SlMETS1基因具有调控番茄生长发育的功能,通过敲除该基因得到的mets突变体可以免去杂交育种中的去雄步骤。
为达到以上目的,本发明采取的技术方案是:
一种番茄SlMETS1基因,其核苷酸序列如SEQ ID NO.1所示。
SEQ ID NO.1为:
atgttaactgttgctaagaaattgagttcaatatatatgagactagttttgcctacaccttcttcatcgacttttttatcatttggttcatcagtttctgttttctcaccgcctcgtggcacccaattcttgcgtttaaattttcgattcagaacgatggcgtctcatgttgttggatatcctcgtatggggcccaagagagagcttaagtttgcgttggaatcattttgggatgggaagagcaattctgaggatttggagaaagtggcagctgatcttaggttatctatttggaagcagatggctgatgctggcattaaatatattccaagcaacaccttctcgtattatgaccaagttttggacacaactgctatgctaggtgcagttccaccaagatacggttggaatggtggtgagattggttttgatgtctacttcccaatggctaggggaaacgcctctgtacctgccatggaaatgacaaaatggtttgacaccaactaccactatattgttcctgaattaggtccagatgttaagttttcctatgcgtctcacaaggcagttagtgaatataaggaagctaaatctctcggcattgacacagtccctgtcctcgtaggtccagtttccttcctcttgttatcaaaagcagcaaaaggtgttgaaaagtcgtttcctcttctatcactgattgaaaagattcttccagtttacaaggaagtcattgctgaactgaaggcagctggtgctagttggattcagtttgatgagcctactcttgttaaggatcttgattctcatcaattgcaagcattttctcatgcctactcagaattagagtcaccgctttccggattaaatgtcctcattgagacatattttgctgatgttcctgctgaagctttcaaaacagtgacttctttaaaatgtgttactgcactggggtttgatctagttcgtggatcaaagaatcttgatttgatcaagagtggttttccttcagaaaagtatctatttgctggagtagttgatgggaggaatatttgggctaatgatcttgctgcttctctcagtaccttgcaagctcttgagaacgtggtcggaaaagacaagcttgtggtctccacctcttgctcgcttctccacactgcagttgatttagtgaatgaaactaagttggatgaagaaattaagtcatggcttgcatttgctgcacaaaaattggttgaagttaacgcgttggcaaaggcgttggctggacaaaaagacgaggcattcttctctgctaatgctgcagctcgtacgtccagaaaatcctctcccagagtgaccaacggggctgtgcagaaggctgctgctgcgttgaagggctctgatcatcgcagagcgacaactgtaagtgcgaggttggaagctcaacagaagaagctgagtcttccatctcttccaaccactaccatcggttctttccctcagacattggagcttagaaaagttcgacgagagtacaaggctaacaagatctcggaggaagattatgtcaaatatattacggaggaaatcagcaaagtagtcaaactccaggaggatctagacattgatgttcttgtgcacggagagccagagagaaacgatatggtcgagtattttggggagcaattatctggttttgcttttacagccaatggatgggttcaatcttatggatctcgctgtgttaagccaccaataatctacggtgatgtcagtcgcccaaaaccaatgactgtcttctggtcttcacgagcacagagcatgagcaagcgtccaatgaagggaatgcttacaggacctgttaccattttaaattggtcttttgttagagatgaccagccaagatttgagacttgctaccaaatagctttggctattaaggatgaggttgaagatctcgagaaggctggcattaatgtcattcagattgatgaagctgctttaagagagggtttacctcttagaaaatccgaggaagctttctacttgaactgggctgtacattcattcaggattaccaactgtggtgttcaagacactacccagattcacactcacatgtgctattcaaacttcaacgacatcatccattcaattatcgacatggatgctgacgttatcaccatcgagaactccaggtctgacgagaaacttctttctgtgttccgcgagggagtgaagtatggtgctggcattggccccggagtatacgacatccattcacaaaggattccatcaacagaagaaatagctgacagaatcagcaagatgcttgcagtccttgataccaacatcctctgggttaaccccgactgtggcctcaaaacgcgcaagtatactgaagttaagcctgcactcagcaacatggtagcagctgctaagcttctccgcaaccagttggccagcacgaagtga。
使用上述SEQ ID NO.1所示基因编码的蛋白质,其特征在于:其氨基酸序列如SEQID NO.2所示。
SEQ ID NO.2为:
MLTVAKKLSSIYMRLVLPTPSSSTFLSFGSSVSVFSPPRGTQFLRLNFRFRTMASHVVGYPRMGPKRELKFALESFWDGKSNSEDLEKVAADLRLSIWKQMADAGIKYIPSNTFSYYDQVLDTTAMLGAVPPRYGWNGGEIGFDVYFPMARGNASVPAMEMTKWFDTNYHYIVPELGPDVKFSYASHKAVSEYKEAKSLGIDTVPVLVGPVSFLLLSKAAKGVEKSFPLLSLIEKILPVYKEVIAELKAAGASWIQFDEPTLVKDLDSHQLQAFSHAYSELESPLSGLNVLIETYFADVPAEAFKTVTSLKCVTALGFDLVRGSKNLDLIKSGFPSEKYLFAGVVDGRNIWANDLAASLSTLQALENVVGKDKLVVSTSCSLLHTAVDLVNETKLDEEIKSWLAFAAQKLVEVNALAKALAGQKDEAFFSANAAARTSRKSSPRVTNGAVQKAAAALKGSDHRRATTVSARLEAQQKKLSLPSLPTTTIGSFPQTLELRKVRREYKANKISEEDYVKYITEEISKVVKLQEDLDIDVLVHGEPERNDMVEYFGEQLSGFAFTANGWVQSYGSRCVKPPIIYGDVSRPKPMTVFWSSRAQSMSKRPMKGMLTGPVTILNWSFVRDDQPRFETCYQIALAIKDEVEDLEKAGINVIQIDEAALREGLPLRKSEEAFYLNWAVHSFRITNCGVQDTTQIHTHMCYSNFNDIIHSIIDMDADVITIENSRSDEKLLSVFREGVKYGAGIGPGVYDIHSQRIPSTEEIADRISKMLAVLDTNILWVNPDCGLKTRKYTEVKPALSNMVAAAKLLRNQLASTK。
用于扩增番茄SlMETS1基因全长的引物为:
上游引物5‘-ATGTTAACTGTTGCTAAGAAATTGA -3’;
下游引物5‘-AGCTCACTTCGTGCTGGC -3’。
一种番茄SlMETS1基因敲除突变体获得方法,其特征在于,包括如下步骤:
步骤1,SlMETS1CRISPR/Cas9载体的构建,以pCBC-DT1T2质粒为模板进行四引物PCR扩增,引物序列如下:
F0 5‘- TGCACCATTCCAACCGTATCTGTTTTAGAGCTAGAAATAGC -3’;
R0 5‘- AACATGGCTAGGGGAAACGCCTCAATCTCTTAGTCGACTCTAC-3’;
BsF 5‘- ATATATGGTCTCGATTGCACCATTCCAACCGTATCTGTT -3’;
BsR 5‘- ATTATTGGTCTCGAAACATGGCTAGGGGAAACGCCTCAA -3’;
扩增后对目的条带切胶以后进行胶回收处理;将胶回收产物与pHSE401质粒进行酶切-连接;
步骤2,利用步骤1得到的酶切-连接产物对番茄进行遗传转化;
步骤3,通过Bar试纸条对步骤2得到的遗传转化外植体进行遴选,得到SlMETS1基因敲除植株,即为番茄SlMETS1基因敲除突变体。
本发明所述的一种SlMETS1基因及其在调控番茄生长发育中的应用,其有益效果为:
SlMETS1基因具有调控番茄生长发育的功能,使用CRISPR/Cas9基因编辑技术获得SlMETS1基因敲除番茄,其种子单粒重显著上升、发芽势显著下降、子叶长度显著变短、育性显著下降、花柱长度显著缩短、花粉活力显著下降;另外,由于mets突变体花柱长度变短且花粉活力下降,因此mets突变体很难进行天然自花授粉,可以在杂交育种时免去去雄步骤。上述SlMETS1的功能具有较好的应用潜力,为研究植物生长发育提供了基因资源和新思路,具有广泛的应用前景和较高的使用价值。
附图说明
本发明有如下附图:
图1为SlMETS1基因克隆示意图;
图2为SlMETS1氨基酸进化树分析图;
图3为CRISPR带靶点片段构建过程图;
图4为Bar试纸条检测结果(A)与凝胶电泳检测结果(B);
图5为T0代和T1代mets突变体敲除类型;
图6为mets突变体T1代总蛋白凝胶跑胶检测结果(A)与含量测定结果(B);
图7为mets突变体T1代甲硫氨酸、色氨酸、丝氨酸、苏氨酸含量测定结果;
图8为mets突变体T0-2果实大小表型;
图9为mets突变体T1代种子单粒重(A)和发芽势(B)比较;
图10为mets突变体T1代子叶长度比较(A)与表型(B);
图11为mets突变体T1代花解剖图(A)和花柱比较(B);
图12为mets突变体T1代花柱长度比较(A)和花粉活力比较(B);
图13为mets突变体T1代花粉活力染色观察图。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1:番茄培养方法
将‘moneymaker’番茄于28℃的恒温培养箱中培养至四叶一心时期,培养箱光照强度:600 μmolm-2s-1,光周期16 h光照8 h黑暗,温度白天25℃/夜晚18℃。
实施例2:植物RNA提取
取适量植物于2 mL无酶离心管,加入火烧灭菌后的钢珠,用植物磨样器研磨成粉末;
TRizol 法提取 RNA:
(1)向样品管中加入1 mL TRizol 提取液,涡旋震荡混匀,室温静置5 min;
(2)将离心机4℃预冷,12,000 rpm离心5 min,取上清加入无RNase酶的1.5 mL离心管中;
(3)向1.5 mL离心管中加入200 μL氯仿,颠倒混匀,室温静置5 min;
(4)4℃,12,000 rpm离心15 min,取上清液于新的1.5 mL离心管;
(5)重复步骤(3)、(4);
(6)向上清液中加入750 μL异丙醇充分颠倒混匀,室温静置10 min;
(7)4℃,12,000 rpm离心15 min,弃上清;
(8)用1.5 mL 80%乙醇(-20℃预冷)清洗RNA沉淀,7500 rpm 4℃离心 5 min后弃上清;
(9)离心管开盖室温干燥约5 min使剩余乙醇蒸发干燥;
(10)向离心管中加入40-60 μL RNA-free水溶解RNA沉淀,用NanoDrop 测定RNA浓度后放入-80℃冰箱保存。
实施例3:RNA反转录为cDNA
按诺唯赞生物HiScript® II Q RT SuperMix for qPCR(+gDNA wiper)(R223-01)试剂盒步骤进行。
基因组DNA去除:
在无RNA酶的离心管中进行溶液配制如下:
模板RNA 1 μg
4×gDNA wiper Mix 4 μL
RNase-free ddH2O up to 16 μL
用移液器轻轻吹打混匀。42℃ 2 min。
进行逆转录反应体系配制如下:
在上一步的反应管中直接加入5×HiScript Ⅱ qRT SuperMix Ⅱ
5×HiScript Ⅱ qRT SuperMix Ⅱ 4 μL
第1步的反应液 16 μL
用移液器吸打混匀。50℃ 15 min,85℃ 5 s。
实施例4:SlMETS1基因全长克隆
根据番茄SlMETS1基因片段设计克隆引物。
设计的引物序列如下:
上游引物5‘-ATGTTAACTGTTGCTAAGAAATTGA -3’
下游引物5‘-AGCTCACTTCGTGCTGGC -3’
上述引物均由北京擎科新业生物技术有限公司合成。
PCR反应体系:PrimeSTAR®Max DNA Polymerase(TaKaRa公司)如下表1
表1
以番茄cDNA为模板PCR反应程序如下表2
表2
PCR产物检测:根据目标片段大小制作1%琼脂糖凝胶,加入核酸染料(万分之一),0.1%TAE电泳缓冲液,110-120 v电压电泳约25 min,紫外灯下检测PCR产物片段大小,可得条带(图1)。对目的条带切胶回收处理。
实施例5:SlMETS1氨基酸进化树分析
将SlMETS1氨基酸序列在http://www.ncbi.nlm.nih.gov网站上提供的软件中进行同源性分析,并在TAIR(https://www.arabidopsis.org/)、Sol Genomics Network(https://solgenomics.net/)、CuGenDB(http://www.cucurbitgenomics.org/)、Phytozome(https://phytozome-next.jgi.doe.gov/)等数据库对搜索结果进一步鉴定;将整理好的物种氨基酸数据用MEGA-X等软件进行蛋白序列比对分析和进化树绘制分析。其结果如图2所示,发现Solyc01g009180.2和PGSC0003DMP400015309序列进化距离最近,同样是茄科作物,Capana00g004772反而遗传变异的程度更大,除此之外葫芦科、十字花科和禾本科的进化距离逐渐增大。
实施例6:SlMETS1CRISPR/Cas9载体的构建
在番茄数据库(Sol Genomics Network)中查找目的基因的外显子,在适合外显子上选择2个靶点,设计4对引物DT1-BsF/R、DT1-F0/R0。以pCBC-DT1T2质粒为模板进行四引物PCR扩增。
设计的引物序列如下:
F0 5‘-TGCACCATTCCAACCGTATCTGTTTTAGAGCTAGAAATAGC -3’
R0 5‘-AACATGGCTAGGGGAAACGCCTCAATCTCTTAGTCGACTCTAC-3’
BsF 5‘-ATATATGGTCTCGATTGCACCATTCCAACCGTATCTGTT -3’
BsR 5‘-ATTATTGGTCTCGAAACATGGCTAGGGGAAACGCCTCAA -3’
扩增体系(50 μL)如下表3:
表3
混合好体系后离心,进行PCR,PCR程序如下表4:
表4
将PCR产物使用1%的琼脂糖凝胶电泳检测,如图3所示,对目的条带切胶以后进行胶回收处理。将胶回收产物进行酶切-连接处理如下表5:
表5酶切-连接体系(20 μL):
混合好体系后离心,进行PCR,PCR程序如下表6:
表6
将PCR产物纯化回收。
实施例7:番茄遗传转化
播种和萌发T0
取一定数量的‘moneymaker’番茄种子,加入2.5%的NaClO,混合摇匀8 min。消毒后用灭菌水洗涤7-8次,将种子倒入种子萌发T0培养基,每瓶30-40粒。将培养基放入黑暗培养室中6天,光下培养2天后进行组织培养。
预培养阶段T1
将生长8-9天的番茄小苗剪去根部和子叶叶尖,将其余子叶和下胚轴去除生长点剪成小段。将处理好的外植体置于预培养培养基上,培养基放置事先灭菌干燥后的滤纸,子叶背面朝上放置。光下培养2天。
共培养阶段T1
使用MS溶液重悬农杆菌至OD600值0.15-0.2。将重悬液倒入灭菌烘干的100ml烧杯中,将外植体浸泡在浸染液中,浸染5 min后将外植体捞出置于滤纸上,吸干浸染液。将外植体置于预培养培养基上,叶背面朝上,暗培养2天。
芽诱导阶段T21
将共培养2天后的外植体从黑暗中取出,全部置于芽诱导培养基T21,叶正面朝上。光下培养7天后转入新的T21培养基中继续继代培养,之后每14天进行下一次继代,直到外植体分化为有正常生长点的一簇芽。
芽伸长期T22
待外植体芽长到约2 cm时转入芽伸长培养基T22中,培养2周。
生根期Tr
当芽长到4-5 cm时,通过试纸条鉴定阳性苗,剪掉愈伤组织后将芽转移到生根培养基Tr,培养1个月左右。
驯化期
将生长到一定高度的苗取出,用纸擦去根上的培养基,种到基质中正常培养。
实施例8:SlMETS1敲除植株的鉴定
使用Bar试纸条对实施例7得到的外植体进行检测,得到3株阳性番茄植株(SlMETS1敲除植株),试纸条检测与凝胶电泳检测结果如图4(A)、图4(B)所示,3个阳性株系分别命名为T0-2、T0-9、T0-18。
通过测序结果如图5(A)发现,这三株突变体番茄均为不同敲除方式的纯合体。其中T0-2有8种不同的敲除方式,T0-9和T0-18各有4种不同的敲除方式。这三株T0代突变体只有T0-2结果获得22粒种子,经测序鉴定发现,只有T1-⑧是相同敲除方式的纯合体,其余都是不同敲除方式的纯合体,敲除位点序列如图5(B)所示。其中T1-⑧在靶点1和靶点2位置上各敲除了1 bp的核苷酸,其余5个植株的敲除类型为三种混杂,分别是:靶点1和靶点2各敲除1bp,同T1-⑧一样;靶点1敲除22 bp,靶点2敲除1 bp;靶点1敲除2 bp,靶点2敲除83 bp。
实施例9:mets突变体番茄总蛋白提取及含量检测
1.总蛋白提取
(1)取200 mg植物材料于2 mL离心管中(加入火烧灭菌后的钢珠),用植物磨样器研磨成粉末;
(2)加入0.5 mL提取试剂,混匀后冰上放置20分钟,期间数次颠倒混匀,以便蛋白溶解;
(3)12000 rpm离心15分钟,弃去沉淀。将上清液转移至新的离心管中,直接使用或在-80℃中保存。
2.总蛋白含量检测
采用SDS-PAGE蛋白质凝胶电泳和考马斯亮蓝染色的方法进行总蛋白含量检测,胶图及含量如图6(A)和图6(B)所示。mets突变体的蛋白含量相对于对照并无显著变化。
实施例10:mets突变体番茄氨基酸含量检测
(1)取200 mg植物材料于2 mL离心管中(加入火烧灭菌后的钢珠),用植物磨样器研磨成粉末;
(2)氨基酸含量测定由北京博云华康基因科技有限公司进行。
氨基酸含量检测结果如图7所示。突变体相对于WT植株色氨酸、苏氨酸、丝氨酸含量显著降低,但甲硫氨酸含量相对来说无明显变化,证明除SlMETS1外,还有其他蛋白或途径可以合成甲硫氨酸。
实施例11:mets突变体番茄其他生理性状检测
1.T0代果实和T1代种子发芽势比较
取突变体T0-2与对照第一花序所结第一穗果相同位置的果实进行对比,如图8所示,发现T0-2果实与对照相比大小更小。
T0-2果实与对照果实中种子含量对比如图8所示,对照果实内种子比T0-2果实种子数多。
对mets和对照种子进行称量,估算单粒重,发现mets突变体相对于对照种子单粒重显著提升。对种子进行催芽,拍摄催芽3天发芽势情况,比较发芽势和发芽率,发现种子发芽势显著下降,发芽率略微下降,如图9和所示。
2.mets突变体与WT子叶长度比较
经观察发现,mets和对照相比,子叶长度显著变短,如图10所示,表明SlMETS1基因影响番茄子叶的生长。
3.mets突变体与WT花柱长度比较
经田间试验观察发现,mets突变体出现了显著的育性下降,为了解mets突变体育性显著下降的类型和原因,解剖并比较观察mets突变体T1代5株植株和对照植株的花,发现mets突变体相对于对照花柱显著变短,如图11所示。花柱长度统计柱形图如图12(A)所示。表明mets突变体雌蕊的发育受到抑制。
4.mets突变体与WT花粉活力比较
花粉活力检测:
(1)在中午12时左右取新鲜花朵,小心去除花瓣和雌蕊;
(2)将花粉置于载玻片,滴加2-3滴亚历山大染色液(北京酷来搏科技有限公司SL7660);
(3)充分混合,立即盖上盖玻片染色5-10 h;
(4)吸去多余液体,显微镜下观察。
经比较和观察发现,mets突变体花粉活力相对于对照显著下降,活力花粉数显著减少,如图12(B)和13所示。表明mets突变体雄蕊的发育受到抑制。
由于mets突变体花柱头缩短,且花粉活力显著下降,导致mets突变体无法正常自花授粉,因此使用mets突变体作为母本进行杂交育种可以免除去雄步骤。
本说明书中未作详细描述的内容属于本领域专业技术人员公知的现有技术。
Claims (1)
1.一种雄蕊的发育受到抑制的番茄SlMETS1基因敲除突变体获得方法,其特征在于,包括如下步骤:
步骤1,SlMETS1 CRISPR/Cas9载体的构建,以pCBC-DT1T2质粒为模板进行四引物PCR扩增:
扩增后对目的条带切胶以后进行胶回收处理;将胶回收产物与pHSE401质粒进行酶切-连接;
步骤2,利用步骤1得到的酶切-连接产物对番茄进行遗传转化;
步骤3,通过Bar试纸条对步骤2得到的遗传转化外植体进行遴选,得到SlMETS1基因敲除植株,即为番茄SlMETS1基因敲除突变体;
步骤1中所述四引物为:
F0 5‘- TGCACCATTCCAACCGTATCTGTTTTAGAGCTAGAAATAGC -3’;
R0 5‘- AACATGGCTAGGGGAAACGCCTCAATCTCTTAGTCGACTCTAC-3’;
BsF 5‘- ATATATGGTCTCGATTGCACCATTCCAACCGTATCTGTT -3’;
BsR 5‘- ATTATTGGTCTCGAAACATGGCTAGGGGAAACGCCTCAA -3’;
其中,所述番茄SlMETS1基因的核苷酸序列如SEQ ID NO.1所示。
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