CN113957086A - SlBIN2基因在调控番茄果实成熟和类胡萝卜素合成中的应用 - Google Patents

SlBIN2基因在调控番茄果实成熟和类胡萝卜素合成中的应用 Download PDF

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CN113957086A
CN113957086A CN202111510196.5A CN202111510196A CN113957086A CN 113957086 A CN113957086 A CN 113957086A CN 202111510196 A CN202111510196 A CN 202111510196A CN 113957086 A CN113957086 A CN 113957086A
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汪俏梅
孟凡亮
胡松申
刘丽红
郑积荣
王同林
李松文
李园园
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Zhejiang University ZJU
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Abstract

本发明涉及生物技术领域，具体公开了一种SlBIN2基因在调控番茄果实成熟和类胡萝卜素合成中的应用，该SlBIN2基因的核苷酸序列如SEQ ID No：1所示。SlBIN2基因敲除促进番茄果实类胡萝卜素的积累。

Description

SlBIN2基因在调控番茄果实成熟和类胡萝卜素合成中的应用
技术领域
本发明涉及生物技术领域，尤其是涉及番茄SlBIN2基因在调控果实成熟和类胡萝卜素合成中的应用。
背景技术
番茄(Solanum lycopersicum)起源于南美洲，明朝万历年间传入中国，现已成为中国乃至世界范围内栽培最广泛的蔬菜作物。据联合国粮农组织数据显示，2019年全球番茄产量高达1.8亿吨，总产值约1千亿美元，均位列果蔬首位，其中，我国番茄总产量约占世界总产量的34.78％，是世界上最大的番茄生产国。
随着国民消费水平的提高，消费者对番茄的需求已从单一的产量需求上升到品质需求。番茄具有良好的感官品质、营养品质和风味品质，是有益于人体健康的重要营养来源，其含有多种生物活性成分，如番茄红素、β-胡萝卜素和叶黄素等多种类胡萝卜素。类胡萝卜素是一类具有独特理化性质的萜类化合物，广泛存在于动植物和微生物中。在植物光合组织和非光和组织中，通过异戊二烯途径产生橙色、黄色和红色等色素，参与花卉、果实和蔬菜的着色，并在光合作用和光保护中发挥重要作用。同时，类胡萝卜素是人类饮食的必需成分，是维生素A的合成前体，对人体健康有着重要作用。除了作为色素和营养成分之外，类胡萝卜素也是植物激素脱落酸和独脚金内酯以及挥发性风味化合物的合成前体，参与番茄的生长发育和风味品质的形成。
番茄果实类胡萝卜素的代谢与果实成熟密切相关。番茄作为典型的呼吸跃变型果实，在果实成熟阶段，果实的颜色、质地、风味和口感等感官属性以及糖、酸、维生素和类胡萝卜素等营养物质方面都发生显著的变化。植物激素在番茄果实成熟和类胡萝卜素的代谢中发挥着重要作用，存在着不同激素既独立又互作调控的网络机制。目前普遍认为番茄果实成熟是乙烯主导、多种植物激素参与的调控过程。
除了乙烯参与番茄果实的成熟和类胡萝卜素积累之外，BR等植物激素也参与了番茄果实的成熟和类胡萝卜素的合成，但其信号转导途径的基因功能并没有完全验证。SlBIN2(Solyc07g055200)与拟南芥AtBIN2基因高度同源，是BR信号转导过程中的负调控蛋白激酶，能够磷酸化BZR1和BES1负向调控BR信号(Vert and Chory 2006)。拟南芥BIN2基因的已知功能大多与植物非生物胁迫、细胞分裂、细胞伸长和气孔发育等方面相关，SlBIN2基因目前已知的用途仅仅是诱导茎的分枝。
发明内容
本发明要解决的技术问题是提供一种参与番茄果实成熟和类胡萝卜素合成的基因、蛋白质以及相应的用途。
为了解决上述技术问题，本发明提供一种SlBIN2基因在调控番茄果实成熟和类胡萝卜素合成中的应用，SlBIN2基因的核苷酸序列如SEQ ID No：1所示。
作为本发明的应用的改进：SlBIN2负调控番茄果实成熟和类胡萝卜素合成。
作为本发明的应用的进一步改进：SlBIN2基因敲除促进番茄果实类胡萝卜素的积累。
在本发明中：基因SlBIN2编码的蛋白质具有如SEQ ID NO：2所示的氨基酸序列。
本发明还同时提供了含有上述基因的质粒以及含有上述基因的植物表达载体，为过表达载体pGWB17-35S::SlBIN2。
本发明还同时提供了一种含有上述基因的宿主细胞，宿主细胞为大肠杆菌细胞、农杆菌细胞。
本发明还同时提供了番茄中敲除SlBIN2基因的方法，包括以下步骤：
1)利用CRISPR/Cas9技术设计基因编辑的靶点sgRNA序列：5'-CTGGGACCTCAGCACCATAA-3’；
2)利用步骤1)所得的序列合成引物，并构建到CRISPR/Cas9载体中；
3)将步骤2)所得的载体遗传转化野生型番茄品种，从而获得相应的转基因植株；从所述转基因番茄植株中鉴定出敲除SlBIN2基因的植株。
本发明还同时提供了上述基因的用途：用于构建转基因番茄，所述转基因番茄能够调控番茄果实成熟和提高类胡萝卜素的含量。
本发明首次构建了番茄SlBIN2基因过表达转基因植株和基因编辑植株，并进行功能研究。通过测定果实乙烯释放量和类胡萝卜素的含量，发现SlBIN2基因在番茄果实乙烯释放和类胡萝卜素积累中起到负向调控作用。通过对番茄SlBIN2基因的编辑，培育果实成熟提前和类胡萝卜素含量更高的番茄材料，在提高番茄感官和营养品质方面具有良好的应用前景。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细说明。
图1是SlBIN2基因过表达载体pGWB17-35S::SlBIN2载体图谱。
图2是SlBIN2基因过表达的株系中SlBIN2基因的表达量。
图3是番茄SlBIN2基因编辑株系的CRISPR/Cas9靶点位置及测序结果。
图4是SlBIN2基因过表达和基因编辑株系与野生型番茄果实乙烯释放量。
图5是SlBIN2基因过表达和基因编辑株系与野生型番茄果实成熟进程对比图。绿熟期(Mature green，MG)，破色期(Breaker，B)，粉色期(Pink，B+3)，红熟期(Red ripe，B+7)；
B+3代表番茄果实粉色期(Pink，B+3)，B+7代表番茄果实红熟期(Red ripe，B+7)。
图6是SlBIN2基因过表达和基因编辑株系与野生型番茄果实类胡萝卜素含量。abcd代表株系与对照具有显著差异(p<0.05)。
具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
一、获得番茄SlBIN2基因以及该基因过表达载体的构建：
用Primer Premier 6.0设计该基因双向扩增引物，取种植在浙江大学紫金港校区温室的野生型番茄AC(Ailsa Craig)叶片，提取RNA反转录成的cDNA为模版(cDNA的提取方式为常规方法，可参照专利CN104561025A)，上游特异性引物为SlBIN2-F，下游引物为SlBIN2-R，用PrimerSTAR高保真酶扩增SlBIN2基因全长，即SEQ ID No：1。
引物序列为：SlBIN2-F：5’-ATGGCCTCGATACCGCTGGGAC-3’
SlBIN2-R：5’-TTACGTCGCACCAGGAAATG-3’
PCR扩增反应体系：2xPrimerSTAR buffer 25ul、dNTP Mixture 5ul、PrimerSTARDNA polymerase 1ul、ddH2O 14ul、cDNA2ul、上下游引物各1.5ul，共50ul。PCR反应程序为：98℃预变性5分钟；98℃变性10秒，60℃退火10秒，72℃延伸90秒，35个循环；最后72℃终延伸5分钟，扩增出的基因全长连接到pQB-V3载体上，将重组质粒送至擎科公司测序确认序列是否正确，然后通过同源重组的方法将目的片段转移到pGWB17终载体上，构建好的过表达载体命名为pGWB17-35S::SlBIN2(图1)。
二、番茄SlBIN2基因敲除的CRISPR/Cas9载体构建
利用在线专业软件(http://crispr.mit.edu/)，在SlBIN2基因的编码序列(SEQID NO:1)中设计CRISPR/Cas9编辑的靶点sgRNA序列：5'-CTGGGACCTCAGCACCATAA-3’；并在生物技术公司合成相应的靶点引物序列：5'-ATTGTTATGGTGCTGAGGTCCCAG-3’与5'-AAACCTGGG ACCTCAGCACCATAA-3’。将靶点引物退火，连接至中间载体AtU6-26-sgRNA-SK，将PCR验证阳性且测序正确的质粒提取，用Nhe I和Spe I双酶切，电泳后将片段大小约642bp的片段切胶回收，回收片段即sgRNA cassette。然后将sgRNA cassette连接至Spe I酶切后的pCAMBIA1300-pYAO:Cas9质粒，利用双元载体上的引物序列进行菌落PCR鉴定，挑取鉴定正确的单克隆扩繁并提取质粒，利用Sal I和Kpn I进行酶切验证，酶切片段长度约为670bp的质粒为SlBIN2的CRISPR/Cas9基因编辑载体。
三、转基因材料的构建与检测：
将过表达载体pGWB17::SlBIN2和CRISPR/Cas9基因编辑载体分别转化农杆菌LBA4404菌株，用番茄子叶作为外植体，与菌液进行共培养，获得愈伤组织，愈伤组织经过分化培养基和生根培养基获得转基因阳性苗，利用PCR和RT-PCR验证阳性转基因植株。
选取过表达T2代符合3：1分离比且表达量较高(满足种子在卡那霉素(50mg/L)的培养基上长侧根与未长侧根的分离比符合3：1，且基因表达量升高两倍以上条件的属于过表达)的株系作为研究对象(图2)。图2中，WT代表野生型番茄AC(Ailsa Craig)，OE-1、OE2、OE4分别代表SlBIN2的三个过表达株系。
合成SlBIN2基因PCR扩增的上游引物5’-ATGGCCTCGATACCGCTGGGAC-3’和下游引物5’-TTACGTCGCACCAGGAAATG-3’，以基因编辑番茄植株及其对照品种AC的基因组DNA为模板，用2×Taq PCR Master Mix(TIANGEN公司)对SlBIN2基因进行PCR扩增，PCR扩增体系为20μl，含有2×Taq PCR Master Mix 10μl，上、下游引物(10μM)各1μl，模板DNA1μl(<1μg)，无菌水7μl；PCR扩增程序为：94℃预变性5min；94℃变性30sec，55℃退火30sec，72℃延伸30sec，35个循环；72℃延伸10min。
PCR产物测序分析后，鉴定了两个成功敲除SlBIN2基因的株系bin2-5和bin2-10，这两个株系的植株中SlBIN2基因编码区分别缺失10个和2个碱基(图3)，使SlBIN2基因发生移码突变，导致该基因功能缺失。bin2-5和bin2-10株系植株中SlBIN2基因的核苷酸序列如图3所述。
四、转基因株系果实成熟和乙烯释放量研究
SlBIN2基因过表达转基因株系和基因编辑株系与野生型番茄花期标记后30天果实到达绿熟期(MG)，果实顶端变红时到达破色期(B)，通过表型的观察和统计，野生型番茄从花期标记到果实破色期平均天数约34天，OE-1、OE-2、OE-4三个SlBIN2基因过表达株系从花期标记到果实破色期平均天数分别约35、35和36天，KO-5和KO-10两个SlBIN2基因编辑株系从花期标记到果实破色期平均天数分别约32和33天。结果表明，SlBIN2基因过表达延缓番茄果实成熟，SlBIN2基因敲除促进番茄果实成熟。
利用1050mL圆形保鲜盒(安立格，中国)和PM6白色气动隔板直通接头外螺纹接头(fssto，中国)制作测定乙烯释放速率容器，打孔放置接头，并套上橡胶细管同时用止水夹夹住。每个盒子作为一个生物学重复，每次试验包含三个生物学重复；每个盒子抽气两次，每次为一次技术性重复。称重两到三个果实并记录重量放置于一个盒子中。将果实放入保鲜盒后扣紧并关闭止水夹，放置两小时环境条件与外源处理环境相同，抽取1mL气体注入气相。
采用气相色谱仪系统包括GC 6890N(Agilent Technologies，美国)、空气发生器(KF-2L，杭州德克尔，中国)及FID检测器。色谱柱填料为Porapak Q 50/80。载气为氮气，氢气流量为40.0mL/min空气流量为400.0mL/min。采用恒定尾吹流量模式。柱温(加热炉)为100度。前进样口温度为140度，检测器温度为230度。采用乙烯标准品(10-6mol/mol，载气为N2，南京特种气体厂，中国)进行外标法计算。
SlBIN2基因过表达转基因株系和基因编辑株系与野生型番茄花期标记后30天果实到达绿熟期，依次对花期标记30天到43天的番茄果实进行取样并检测乙烯释放量，乙烯释放量的结果如图4所述，据图4，可获得以下总结性结论：两个SlBIN2基因编辑株系乙烯释放高峰最早出现且乙烯释放量最高，野生型番茄果实次之，三个SlBIN2基因过表达株系果实的乙烯高峰最晚出现且释放量最低，说明SlBIN2基因敲除促进番茄果实乙烯释放进而促进番茄果实成熟，SlBIN2基因过表达延缓乙烯释放进而延缓番茄果实成熟进程。
五、转基因株系果实类胡萝卜素含量研究
SlBIN2基因过表达转基因株系和基因编辑株系与野生型番茄果实生长至绿熟期(MG)，开始取果实样品(外果皮)，果实顶端变红时取破色期(B)果实样品，破色期后三天取粉色期(B+3)果实样品，破色期后7天取红熟期(B+7)果实样品，一共4个时期的样品(图5)。
取一定量果实组织进行研磨，取0.5g果实粉末在50mL箔纸包裹的离心管中称重。然后立即加入30mL提取液(乙烷:丙酮:乙醇＝1:1:1体积比)，然后150r/min摇动30min。然后在混合物中加入15mL双蒸水，4度1500g离心10min。上清液用0.22μm有机相过滤头过滤，再用氮吹仪浓缩至液体几乎不存在。加入0.5mL溶解液(四氢呋喃:乙腈:甲醇＝3:10:11，体积比)三次，共1.5mL。此步溶液用于进行上样。
取20μl的样品，使用岛津HPLC系统(Shimadzu,Kyoto,Japan)进行测定，包括C18色谱柱(5μm particle size,4.6mm x 250mm,Elite analytical instruments Co.,Ltd.,大连，中国)、自动进样器和SPD-M20A二极阵列检测器。流动相为(甲醇:乙腈＝90:10体积比，同时加入0.05％三乙胺)，流速为1.2mL/min。检测波长为475nm。采用番茄红素、β-胡萝卜素和叶黄素标准品(Sigma,St Louis,MO,USA)进行外标法计算含量。结果以μg g-1FW(freshweight，鲜重)为单位表示。
SlBIN2基因过表达和基因编辑株系与野生型番茄果实类胡萝卜素含量如图6所述，从上到下依次是番茄红素含量、β-胡萝卜素含量、叶黄素含量和总类胡萝卜素含量。根据图6，可获得以下总结性结论：两个SlBIN2基因编辑株系果实的番茄红素和总类胡萝卜素含量在果实发育各个时期均显著高于野生型，同时三个SlBIN2基因过表达株系果实的番茄红素和总类胡萝卜素含量在果实发育各个时期均显著低于野生型。说明SlBIN2基因敲除促进番茄果实类胡萝卜素的积累。
最后，还需要注意的是，以上列举的仅是本发明的若干个具体实施例。显然，本发明不限于以上实施例，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。
序列表
<110> 浙江大学
<120> SlBIN2基因在调控番茄果实成熟和类胡萝卜素合成中的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1314
<212> DNA
<213> 番茄(Solanum lycopersicum)
<400> 1
atggcctcga taccgctggg acctcagcac cataatccgc cggaaaatca ccatcaccac 60
caccttcagc cgccgccgca gctcgtgcag cagcagcagc aacagcctgt gaatcgtgaa 120
ctaggaggag gaggaggagc tgcagtaagg aacgccggag cacgaccgga aatggaatcc 180
gaaaaggaaa tgtcagctgc tgttgttgag ggtaatggtg ccgtcactgg tcacataatt 240
tccaccacca ttggaggcaa gaatggagaa ccaaaaagga ccatcagtta tatggcagag 300
cgagttgtcg gtacagggtc ctttgggata gtgtttcagg caaaatgctt ggaaactgga 360
gagactgtgg ccataaagaa ggttttacag gacaaacggt ataaaaatcg tgaactacaa 420
ctgatgcgct tgatggatca cccaaatgtc attactctaa agcactgctt cttttccacg 480
actagtagag atgagctttt tcttaatttg gtcatggatt atgtccctga aagtttatac 540
aaggttttaa agcactatag caattcaaat caaaggatgc cactcatata tgtcaaactt 600
tacatgtatc agatattcag agggctggct tacattcata atgttccaag gatttgccat 660
agagatgtga aacctcaaaa tcttttggtt gatcctctga cccatcaagt caagctgtgt 720
gattttggaa gtgcaaaagt cctggtaaat ggtgaagcaa atatttcata catttgctct 780
cgctactaca gagctccaga actcatattt ggtgccacag agtatacaac atcaattgat 840
atttggtcag caggctgtgt ccttgctgag cttcttctgg ggcagccgct ctttcctggc 900
gaaaatgcag tagatcaact ggtggagatc atcaaggtcc ttggtactcc tactcgggaa 960
gaaattcgat gtatgaaccc aaactacaca gatttcagat tcccacagat aaaagctcat 1020
ccttggcaca aggtattcca taaaagaatg cctcctgaag caattgatct tgcctcacgg 1080
cttcttcaat attcaccaag tcttcgctgt actgcactag aagcatgtgc acattcgttc 1140
tttgatgagc ttcgtgagcc caatgcccgt ctccctaatg gacgtccatt tccacctctt 1200
ttcaacttta aacaagagtt aactggagct tcacctgatt tggtcaacaa gctgatccct 1260
gagcatgtgt ggaggcaact tggtttgaat ttcccatttc ctggtgcgac gtaa 1314
<210> 2
<211> 437
<212> PRT
<213> 番茄(Solanum lycopersicum)
<400> 2
Met Ala Ser Ile Pro Leu Gly Pro Gln His His Asn Pro Pro Glu Asn
1 5 10 15
His His His His His Leu Gln Pro Pro Pro Gln Leu Val Gln Gln Gln
20 25 30
Gln Gln Gln Pro Val Asn Arg Glu Leu Gly Gly Gly Gly Gly Ala Ala
35 40 45
Val Arg Asn Ala Gly Ala Arg Pro Glu Met Glu Ser Glu Lys Glu Met
50 55 60
Ser Ala Ala Val Val Glu Gly Asn Gly Ala Val Thr Gly His Ile Ile
65 70 75 80
Ser Thr Thr Ile Gly Gly Lys Asn Gly Glu Pro Lys Arg Thr Ile Ser
85 90 95
Tyr Met Ala Glu Arg Val Val Gly Thr Gly Ser Phe Gly Ile Val Phe
100 105 110
Gln Ala Lys Cys Leu Glu Thr Gly Glu Thr Val Ala Ile Lys Lys Val
115 120 125
Leu Gln Asp Lys Arg Tyr Lys Asn Arg Glu Leu Gln Leu Met Arg Leu
130 135 140
Met Asp His Pro Asn Val Ile Thr Leu Lys His Cys Phe Phe Ser Thr
145 150 155 160
Thr Ser Arg Asp Glu Leu Phe Leu Asn Leu Val Met Asp Tyr Val Pro
165 170 175
Glu Ser Leu Tyr Lys Val Leu Lys His Tyr Ser Asn Ser Asn Gln Arg
180 185 190
Met Pro Leu Ile Tyr Val Lys Leu Tyr Met Tyr Gln Ile Phe Arg Gly
195 200 205
Leu Ala Tyr Ile His Asn Val Pro Arg Ile Cys His Arg Asp Val Lys
210 215 220
Pro Gln Asn Leu Leu Val Asp Pro Leu Thr His Gln Val Lys Leu Cys
225 230 235 240
Asp Phe Gly Ser Ala Lys Val Leu Val Asn Gly Glu Ala Asn Ile Ser
245 250 255
Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala Pro Glu Leu Ile Phe Gly Ala
260 265 270
Thr Glu Tyr Thr Thr Ser Ile Asp Ile Trp Ser Ala Gly Cys Val Leu
275 280 285
Ala Glu Leu Leu Leu Gly Gln Pro Leu Phe Pro Gly Glu Asn Ala Val
290 295 300
Asp Gln Leu Val Glu Ile Ile Lys Val Leu Gly Thr Pro Thr Arg Glu
305 310 315 320
Glu Ile Arg Cys Met Asn Pro Asn Tyr Thr Asp Phe Arg Phe Pro Gln
325 330 335
Ile Lys Ala His Pro Trp His Lys Val Phe His Lys Arg Met Pro Pro
340 345 350
Glu Ala Ile Asp Leu Ala Ser Arg Leu Leu Gln Tyr Ser Pro Ser Leu
355 360 365
Arg Cys Thr Ala Leu Glu Ala Cys Ala His Ser Phe Phe Asp Glu Leu
370 375 380
Arg Glu Pro Asn Ala Arg Leu Pro Asn Gly Arg Pro Phe Pro Pro Leu
385 390 395 400
Phe Asn Phe Lys Gln Glu Leu Thr Gly Ala Ser Pro Asp Leu Val Asn
405 410 415
Lys Leu Ile Pro Glu His Val Trp Arg Gln Leu Gly Leu Asn Phe Pro
420 425 430
Phe Pro Gly Ala Thr
435

Claims (3)

1.SlBIN2基因在调控番茄果实成熟和类胡萝卜素合成中的应用，其特征在于：SlBIN2基因的核苷酸序列如SEQ ID No：1所示。
2.根据权利要求1所述的应用，其特征在于：SlBIN2负调控番茄果实成熟和类胡萝卜素合成。
3.根据权利要求1或2所述的应用，其特征在于：SlBIN2基因敲除促进番茄果实类胡萝卜素的积累。
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