CN117467636A - 一个参与番茄黄酮醇3-o-葡萄糖苷形成的基因及其应用 - Google Patents
一个参与番茄黄酮醇3-o-葡萄糖苷形成的基因及其应用 Download PDFInfo
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- CN117467636A CN117467636A CN202311345922.1A CN202311345922A CN117467636A CN 117467636 A CN117467636 A CN 117467636A CN 202311345922 A CN202311345922 A CN 202311345922A CN 117467636 A CN117467636 A CN 117467636A
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Abstract
本发明提供一个参与番茄黄酮醇3‑O‑葡萄糖苷形成的基因及其应用,所述基因为糖基转移酶。经验证,当UDP‑葡萄糖作为糖供体时,该蛋白可将槲皮素和山奈酚糖基化并形成对应黄酮醇3‑O‑葡萄糖苷。本发明利用CRISPR/Cas9技术敲除这个糖基转移酶基因,获得纯合突变的番茄果实,其黄酮醇糖苷显著低于野生型。本发明所述的糖基转移酶具有调控番茄果实黄酮醇糖苷积累的作用,对于改善果实的营养价值和经济价值,促进番茄新品种培育具有重要应用价值,可在调节植物应对低温和UV‑B胁迫过程中积累黄酮醇糖苷中应用。
Description
技术领域
本发明属于植物分子生物技术和基因工程领域,涉及一个参与番茄黄酮醇3-O-葡萄糖苷形成的基因及其应用。
背景技术
类黄酮是植物中重要的次生代谢产物,其中黄酮醇是其重要组成部分。黄酮醇以糖苷的形式贮藏在植物体内,番茄中的黄酮醇糖苷主要以槲皮素和山奈酚糖苷为主。研究表明,黄酮醇糖苷在植物生长发育和抵御逆境胁迫等方面发挥重要作用。在UV-B照射条件下,黄酮醇糖苷大量积累,影响植物的药用价值和经济价值。此外,黄酮醇糖苷具有活性氧清除能力,在抗氧化、抗病毒及预防心血管疾病等方面具有积极作用,有利于人体健康。
糖基转移酶(UGT)是一种可以催化小分子苷元与糖供体相连形成糖苷的蛋白酶,是黄酮醇糖苷形成的必需酶。糖基化反应在植物发育以及抗逆过程中的重要作用,使其受到广泛关注。越来越多的UGT家族成员被报道,但功能被充分验证的成员尚少。目前在黑莓、苹果、梨、甜橙等果实中发现了与黄酮醇糖基化相关的糖基转移酶。番茄作为重要的经济作物,调控黄酮醇糖苷积累的糖基转移酶尚未见报道。
发明内容
本发明的目的在于提供一个参与番茄黄酮醇3-O-葡萄糖苷形成的基因,属于糖基转移酶家族成员。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一个参与番茄黄酮醇3-O-葡萄糖苷形成的基因,其核苷酸序列如SEQ ID NO.1所示。PCR扩增的上游引物的核苷酸序列如SEQ ID No.2所示,PCR扩增的下游引物的核苷酸序列如SEQ ID No.3所示。
所述糖基转移酶编码的氨基酸序列如SEQ ID NO.16所示。
所述糖基转移酶基因可以糖基化槲皮素和山奈酚,形成对应的黄酮醇3-O-葡萄糖苷。
本发明的另一个目的是提供所述的糖基转移酶基因在调节番茄果实应对低温和UV-B胁迫过程中积累黄酮醇糖苷中应用。所述糖基转移酶作为候选基因在改善番茄果实的营养价值和经济价值方面具有应用前景。
本发明所述糖基转移酶基因以UDP-葡萄糖为糖供体,将槲皮素和山奈酚糖基化形成对应黄酮醇3-O-葡萄糖苷。
本发明所述糖基转移酶基因可以影响番茄果实在低温贮藏期间的黄酮醇糖苷积累。
本发明所述糖基转移酶可以影响番茄果实在UV-B照射后的黄酮醇糖苷积累。
本发明所述低温贮藏方法为,果实放置于4℃冰柜,贮藏7d和21d。
本发明所述UV-B照射条件为,UV-B强度为180μw/cm2,环境温度为20℃,UV-B照射时长为7d。
本发明的有益效果:本发明提供了一个可以糖基化槲皮素和山奈酚的糖基转移酶,属于植物分子生物技术和基因工程技术领域,所述基因具有SEQ ID NO.1所示的核苷酸序列。本发明中,所述糖基转移酶基因能够糖基化槲皮素和山奈酚。该基因重组蛋白能够在体外糖基化槲皮素和山奈酚,形成对应黄酮醇3-O-葡萄糖苷。利用CRISPR/Cas9技术获得两个纯合突变体株系。在4℃低温处理7d和21d(C7 d,C21 d)以及UV-B照射7d后,突变体番茄果实中的黄酮醇糖苷积累显著低于野生型。由此表明,所述基因可以糖基化槲皮素和山奈酚,并在果实低温和UV-B照射条件下影响体内黄酮醇糖苷积累,从而影响果实的营养价值和经济价值。该发明在番茄新品种培育方面具有指导意义和应用价值。
附图说明
图1为重组蛋白SlUGT75C1的SDS-PAGE分析。
图2为SlUGT75C1蛋白对槲皮素的体外酶活测定结果。
图3为SlUGT75C1蛋白对山奈酚的体外酶活测定结果。
图4为SlUGT75C1基因纯合突变体slugt75c1#1和slugt75c1#2的蛋白质突变结果。
图5为突变体番茄果实低温处理后的芦丁含量(不同字母表示显著性差异,P<0.05)。
图6为突变体番茄果实低温处理后的山奈酚芸香糖苷含量(不同字母表示显著性差异,P<0.05)。
图7为突变体番茄果实UV-B照射7d后的芦丁含量(不同字母表示显著性差异,P<0.05)。
图8为突变体番茄果实UV-B照射7d后的山奈酚芸香糖苷含量(不同字母表示显著性差异,P<0.05)。
具体实施方式
本发明结合附图和实施例,作进一步的说明。
本发明提供了上述方案所述糖基转移酶基因SlUGT75C1编码的蛋白质,所述蛋白质的氨基酸序列如SEQ ID NO.16所示;所述蛋白质含有470个氨基酸。
本发明中,所述双酶切体系优选为:Cutsmart buffer 5μL、载体1μg、内切酶各1μL、水补足至50μL;所述双酶切的体系优选为:37℃酶切3h。
本发明中,所述回收的试剂盒优选采用TAKARA凝胶回收试剂盒进行;所述连接的试剂盒优选采用Vazyme的一步克隆试剂盒进行。
本发明还提供了上述方案所述的糖基转移酶基因SlUGT75C1在番茄育种中的应用;所述应用优选为糖基转移酶基因SlUGT75C1在番茄果实营养价值改善的基因工程育种中的应用。
下面结合实施例对本发明提供的一个参与番茄黄酮醇3-O-葡萄糖苷形成的基因及其应用SlUGT75C1及其在黄酮醇糖苷积累上的应用进行详细说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1:重组蛋白SlUGT75C1的获得
(一)实验方法
1.重组载体构建和大肠杆菌转化
根据SlUGT75C1在番茄基因组数据库Solanum lycopersicum ITAG3.2中的参考序列,利用PCR技术,结合引物对SEQ ID NO.2和SEQ ID NO.3扩增获得SlUGT75C1全长SEQ IDNO.1。PCR反应体系为50μL,成分分别为:25μL PrimeSTAR Max Premix(2x)酶(TAKARA),上下游引物(10μM)各2μL,2μL桃果实cDNA,19μL H2O。PCR反应程序为:98℃10s;60℃15s,72℃40s,35个循环。PCR产物与用限制性内切酶BamH I和EcoR I酶切后的目标载体(pGEX-4T-1)连接,用热激法转化大肠杆菌感受态DH5α,挑取阳性菌落后测序验证。将序列确认正确的质粒用热激法转化大肠杆菌感受态BL21(DE3)pLysS(Promega),挑取阳性菌落PCR检测验证。
2.诱导表达
挑选转化后的大肠杆菌单菌落,加入20mL含有氨苄抗生素的LB中,37℃过夜培养。过夜培养后的菌液按照1:50的比例转入300mL含有氨苄抗生素的LB中,培养至OD600=0.5-0.6。加入终浓度为1mM的IPTG于16℃过夜诱导至0D600=1.8-2.0。4000g,20min离心获得沉淀,以约15mL的1×PBS溶液重新悬浮,于-80℃超低温冰箱冻存24h后转至30℃水浴融解破碎。
3.蛋白纯化
破碎后的菌液进行离心(4℃,10000rpm,30min),上清液以HV除菌膜(0.45μm,diameter 33mm,Millipore USA)过滤后,使用GST标签蛋白纯化试剂盒纯化蛋白。蛋白液用脱盐柱PD-10(GE Healthcare UK)进行脱盐,并将蛋白置换到Tris-HCI缓冲液(100mMTris,2mM DTT,pH 7.5),加入10%甘油将蛋白储存于-80℃的超低温冰箱。
(二)实验结果
纯化后的蛋白进行SDS-PAGE电泳,结果如图1所示,SlUGT75C1-GST蛋白为78kDa,条带大小正确,纯化蛋白确认为SlUGT75C1蛋白,可用于后续研究。
实施例2:重组蛋白SlUGT75C1的体外酶活试验
(一)实验方法
1.体外酶活反应
反应在Tris-HCl缓冲液(100mM Tris,pH 7.5)中进行,50μL反应体系中加入1.2μg纯化蛋白,1mM底物苷元及1mM UDP-葡萄糖,底物分别为槲皮素和山奈酚。在30℃下孵育1h后,通过加入50μL甲醇停止反应。12000g,离心15min后的上清溶液进行LC-MS分析。
2.LC-MS液相色谱检测
使用仪器为Agilent 1290Infinity LC System(Agilent Technologies,USA),色谱柱为SunFire C18分析柱(5μm,4.6×250mm;Waters,USA),检测波长为370nm。色谱条件以0.1%甲酸作为溶剂A和100%乙腈(加入0.1%甲酸)作为溶剂B,1mL/min的流速进行检测。质谱由装有ESI源的Agilent6460三重四极杆质谱仪(Agilent Technologies,USA),以负离子模式分析。扫描范围为100-1000m/z,雾化器压力设定为45psi。黄酮醇糖苷的检测波长为370nm,槲皮素3-O-葡萄糖苷的m/z值为465[M+H]+,山奈酚3-O-葡萄糖苷的m/z值为449[M+H]+。实验结果参见图2和图3,图2为SlUGT75C1蛋白对槲皮素的体外酶活测定结果,图3为SlUGT75C1蛋白对山奈酚的体外酶活测定结果。
(二)实验结果
在UDP-葡萄糖存在的情况下,大肠杆菌诱导的SlUGT75C1蛋白可以将槲皮素和山奈酚糖基化形成对应的黄酮醇3-O-葡萄糖苷。
实施例3:突变体材料的获得
1.pYLCRISPR/Cas9-SlUGT75C1基因编辑载体的构建
靶序列的选择:通过http://skl.scau.edu.cn/网址,进行靶序列预测,最终选择两个靶序列分别为T1和T2。
启动子的选择:使用拟南芥来源的AtU3d启动T1靶位点,AtU3b启动T2靶位点。
含有靶位点的sgRNA表达盒的制备:经过2轮PCR,分别获得AtU3d-T1-gRNA和AtU3b-T2-gRNA 2个DNA片段。AtU3d-T1-gRNA为包含靶向T1位点的sgRNA的表达盒,其表达由AtU3d启动子驱动;AtU3b-T2-gRNA为包含靶向T2位点的sgRNA的表达盒,其表达由AtU3b启动子驱动。制备方法如下:第一轮PCR反应体系为25μL,包含KOD酶(TOYOBO)0.5μL、10×KOD Plus Buffer(TOYOBO)2.5μL、dNTP 2.5μL、MgSO4 1.5μL、pYLgRNA-LacZ-AtU3d/AtU3b质粒1μL、U-F引物(10μmol/L)0.5μL、gR-R引物(10μmol/L)0.5μL、gR-SlUGT75C1-T1/T2引物0.25μL、AtU-SlUGT75C1-T1/T2引物0.25μL、ddH2O 15.5μL。pYLgRNA-LacZ-AtU3d质粒、PYLgRNA-AtU3b质粒均记载在文献(Ma et al.,A Robust CRISPR/Cas9 System forConvenient,High-Efficiency Multiplex Genome Editing in Monocot and DicotPlants[J].Molecular Plant,2015,8(8):1274-1284)中。所用引物序列分别为SEQ IDNO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8和SEQ ID NO.9所示。第一轮PCR反应程序为:94℃120s;94℃10s,58℃15s,68℃20s,28个循环;68℃7min。PCR反应结束后,获得2种PCR产物。第二轮PCR反应体系为20μL,包含KOD酶(TOYOBO)0.5μL、10×KOD PlusBuffer(TOYOBO)2.0μL、dNTP 2.0μL、MgSO4 1.2μL、第一轮PCR反应产物(稀释10倍)1.0μL、特异性引物对(Pps-GGL、Pgs-GG2或Pps-GG2、Pgs-GGR,10μmol/L)0.25μL、ddH2O 12.8μL。所用引物序列分别为SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13所示。第二轮PCR反应程序为:94℃120s;94℃10s,58℃15s,68℃20s,25个循环;68℃7min。第二轮PCR反应结束后,将2种PCR产物等量混合后进行纯化。
pYLCRISPR/Cas9-SlUGT75C1重组载体的制备:采用Golden Gate cloning方法(Maet al.,Robust CRISPR/Cas9 System for Convenient,High-Efficiency MultiplexGenome Editing in Monocot and Dicot Plants[J].Molecular Plant,2015,8(8):1274-1284)完成目的sgRNA表达盒与pYLCRISPR/Cas9-Pubi-H载体的酶切连接反应。反应体系为:10×CutSmart Buffer(NEB)1.5μL、pYLCRISPR/Cas9-Pubi-H载体2.0μL、经过纯化的AtU3d-T1-gRNA和AtU3b-T2-gRNA两个DNA片段的混合物1.0μL、BsaΙ-HF(NEB)0.5μL、T4 DNAligase(Promaga)1.0μL、10×T4DNA ligase Buffer(Promaga)1.5μL、ddH2O 7.5μL。反应条件为:37℃10min,10℃5min,20℃5min,3个循环;37℃3min,10℃5min,20℃5min,10个循环;37℃5min。将酶切连接产物用热激法转化大肠杆菌感受态DH5α,挑取阳性菌落后测序验证,将序列确认正确的质粒转入农杆菌GV3101感受态细胞,并挑取阳性克隆菌体保存。
2.突变体植株的获得
采用农杆菌介导的叶盘转化法将pYLCRISPR/Cas9-SlUGT75C1基因编辑载体转入野生型Ailsa Craig番茄。所有操作均在无菌工作台中进行,植物培养温度均为25℃,具体操作步骤如下:
播种及种子萌发:取适量Ailsa Craig番茄种子,50%次氯酸钠消毒10min后,无菌水清洗5-8次,将种子播种于1/2MS培养基上。播种后将其避光培养至出芽后转至光下,光周期为16h光照/8h黑暗。1L的1/2MS培养基包含2.2g MS盐、10g蔗糖、100mg肌醇,琼脂8g,pH为5.8。
预培养:种子在光下生长一周后,待两片子叶完全展开,真叶开始生长时,将子叶切成5×5mm的方块,背面朝上放置铺有滤纸的KCMS固体培养基上,黑暗条件下培养2d。1L的固体KCMS培养基包含4.44g MS盐、30g蔗糖、100mg肌醇,琼脂8g,pH为5.8,灭菌后加乙酰丁香酮至浓度为100mg/L。
侵染液的制备:切子叶前1d将保存的pYLCRISPR/Cas9-SlUGT75C1农杆菌划线,所用固体培养基为含有卡那霉素与庆大霉素的LB,28℃培养2d后,挑取单菌落于3mL相应抗生素的液体LB培养基中。次日吸取200-300μL菌液于20mL含有卡那霉素与庆大霉素的LB中,28℃培养至OD600=0.5-0.6。5000rpm,离心10min后,收集菌体,用30mL液体KCMS培养基重悬菌体。1L的液体KCMS培养基包含4.44g MS盐、30g蔗糖、100mg肌醇,pH为5.8,灭菌后加乙酰丁香酮至浓度为100mg/L。
侵染及共培养:预培养2d的子叶于侵染液中侵染2-3min,于滤纸上吸干菌液。菌液吸干后,将子叶背面朝上重新放回固体KCMS培养基上,避光条件下培养2d。
诱导愈伤:将共培养2d后的外植体背面朝下,转至2Z培养基中,于光下培养2-3周,光周期为16h光照/8h黑暗。每7-10d更换一次培养基。1L的2Z固体培养基包含4.44g MS盐、20g蔗糖、100mg肌醇,琼脂7.4g,pH为6.0,灭菌后加入潮霉素(10mg/L)、特美汀(200mg/L)和玉米素(2mg/L)。
诱导芽分化:待外植体诱导出愈伤后,将其转入0.2Z固体培养基中,直至长出1-2cm小芽。每7-10d更换一次新的培养基。1L的0.2Z固体培养基包含4.44g MS盐、20g蔗糖、100mg肌醇,琼脂7.4g,pH为6.0,灭菌后加入潮霉素(10mg/L)、特美汀(200mg/L)和玉米素(0.2mg/L)。
生根培养:待外植体诱导出芽后,将其转至R固体培养基中,待外植体生根后移入土中,并进行测序筛选阳性植株。1L的R固体培养基包含4.44g MS盐、20g蔗糖、琼脂8g,pH为6.0,灭菌后加入潮霉素(5mg/L)、特美汀(150mg/L)、IAA(1mg/L)。
阳性植株的筛选:提取叶片DNA,结合引物对SEQ ID NO.14和SEQ ID NO.15进行PCR扩增,扩增序列测序确认突变位点后,获得T1代植株进行纯和突变体筛选。获得的SlUGT75C1#1和SlUGT75C1#2分别在T2靶序列处突变,其中SlUGT75C1#1的第902-1241bp缺失,SlUGT75C1#2的第1012-1297bp缺失,均导致终止密码子提前。两种突变体的截短蛋白如图4所示。
实施例4:低温处理后,SlUGT75C1突变体植株的黄酮醇糖苷与野生型相比显著下降
(一)实验方法
1.突变体植株的种植及低温处理
经鉴定过的突变体与野生型番茄种植于浙江大学人工气候室(25℃,16h/8h光周期)。对成熟果实进行采收,并进行4℃低温处理。低温处理时间分别为7d和21d,处理后果实经过液氮速冻后贮藏于-80℃待用,采收部位为番茄外果皮。每个株系包含3个生物学重复,每个重复包含5个果实。
2.黄酮醇糖苷的提取及测定
取0.1g研磨过的外果皮粉末,加入1mL的50%甲醇水溶液(含0.1%甲酸),涡旋混匀,超声30min后,12000rpm,离心15min。吸取上清重复离心15min后,取900μL上清液至新的2mL离心管中,使用真空浓缩仪器,30℃条件旋转蒸发至干。90μL甲醇将样品复溶后,12000rpm,离心15min,上清液进行HPLC分析。
黄酮醇糖苷HPLC检测采用Waters的高效液相色谱仪(e2695 pump,2998PDAdetector)并外接ODS C18色谱柱(SunFire,4.6×250mm,Waters)。流动相为0.1%甲酸水溶液(A相),以及含有0.1%甲酸的50%乙腈水溶液(B相)。梯度程序为:0-45min,23%-50%B;45-50min,50%-100% B;50-55min,100% B;55-56min,100%-23% B;56-60min,23%B。黄酮醇糖苷检测波长为370nm,柱温为25℃,流速为1mL/min,进样体积为10μL。黄酮醇糖苷的含量通过对应标准品的标准曲线进行相对定量。
实验结果参见图5和图6。图5为突变体番茄果实低温处理后的芦丁含量;图6为突变体番茄果实低温处理后的山奈酚芸香糖苷含量(不同字母表示显著性差异,P<0.05)。
(二)实验结果
如图5和图6所示,得出以下结论:a.番茄经过低温处理7d和21d后,突变体植株无法正常积累芦丁,突变体的芦丁含量显著低于WT。b.番茄经过低温处理21d后,突变体无法正常积累山奈酚芸香糖苷,突变体果实的山奈酚芸香糖苷显著低于WT。因此,SlUGT75C1对低温条件下的黄酮醇糖苷积累具有重要作用。
实施例5:UV-B处理后,SlUGT75C1突变体植株的黄酮醇糖苷与野生型相比显著下降
(一)实验方法
1.突变体植株的种植及UV-B处理
经鉴定过的突变体与野生型番茄种植于人工气候室(25℃,16h/8h光周期)。对破色果实进行采收,并进行UV-B照射处理。UV-B照射条件为,UV-B强度为180μw/cm2,贮藏温度为20℃,照射时间为7d。处理后果实经过液氮速冻后贮藏于-80℃待用,采收部位为番茄外果皮。每个株系包含3个生物学重复,每个重复包含5个果实。
2.黄酮醇糖苷的提取及测定
见实施例4。
实验结果参见图7和图8。图7为突变体番茄果实UV-B照射7d后的芦丁含量;图8为突变体番茄果实UV-B照射7d后的山奈酚芸香糖苷含量(不同字母表示显著性差异,P<0.05)。
(二)实验结果
如图7和图8所示,得出以下结论:a.番茄在UVB处理后,突变体果实的黄酮醇糖苷无法正常积累,导致突变体植株的黄酮醇糖苷显著下降,包括芦丁(图7)和山奈酚芸香糖苷(图8)。因此,SlUGT75C1对UV-B条件下的黄酮醇糖苷积累具有重要作用。
Claims (5)
1.一个参与番茄黄酮醇3-O-葡萄糖苷形成的基因,其特征在于,所述基因为糖基转移酶,该基因的核苷酸序列如SEQ ID NO.1所示,该基因编码的氨基酸序列如SEQ ID NO.16所示。
2.根据权利要求1所述的基因,其特征在于,所述基因以UDP-葡萄糖为糖供体,将槲皮素和山奈酚糖基化形成对应黄酮醇3-O-葡萄糖苷。
3.权利要求1所述的糖基转移酶基因在调节植物应对低温和UV-B胁迫过程中积累黄酮醇糖苷中应用,其特征在于,所述植物为番茄果实。
4.根据权利要求3所述的应用,其特征在于,所述低温为将果实放置于4℃冰柜,贮藏7d和21d。
5.根据权利要求3所述的应用,其特征在于,所述UV-B胁迫为UV-B照射,照射条件为:UV-B强度为180μw/cm2,环境温度为20℃,UV-B照射时长为7d。
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