CN117025635A - 一种葡萄果实颜色调控基因VvMYB308及其应用 - Google Patents
一种葡萄果实颜色调控基因VvMYB308及其应用 Download PDFInfo
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Abstract
本申请提供了一种葡萄果实颜色调控基因VvMYB308及其在调控葡萄品种色泽中的应用，所述葡萄果实颜色调控基因VvMYB308的核苷酸序列为SEQ ID N0.3。本申请克隆了一个与抑制葡萄花色苷合成相关的VvMYB308转录因子基因，对该基因在葡萄愈伤组织中的表达进行了分析，获得了其抑制花色苷作用的关键基序C2。该基因的发现和功能确认对培育不同色泽的葡萄品种具有重要意义。
Description
技术领域
本申请属于遗传学和分子生物学育种技术领域，具体地，本申请提供了一种葡萄果实颜色调控基因VvMYB308及其应用。
背景技术
葡萄属于葡萄科(Vitaceae)葡萄属(Vitis L.)植物，具有重要的经济价值。果实颜色是影响葡萄经济价值的重要性状，葡萄的果实颜色是由果皮积累的花色苷决定的。MYBA1和MYBA2激活花色苷合成途径基因，导致葡萄果皮积累花色苷最终形成有色葡萄，而这些有色葡萄果实颜色由浅到深变异类型极为丰富，呈现典型的数量性状特点。为了更深入地阐释葡萄果实颜色形成的分子机制，我们从前期的转录组数据中挑选出了一个潜在的花色苷合成抑制子VvMYB308，进行了转基因验证，分析了其抑制花色苷合成机制。
发明内容
一方面，本申请提供了一种葡萄果实颜色调控基因VvMYB308，其核苷酸序列为SEQID NO.3。
进一步地，所述基因编码的氨基酸序列为SEQ ID NO.4。
另一方面，本申请提供了葡萄果实颜色调控基因VvMYB308在调控葡萄品种色泽中的应用。
进一步地，所述应用中过表达所述葡萄果实颜色调控基因VvMYB308以降低葡萄果实中的花色苷含量。
进一步地，使用pCXSN过表达载体来过表达所述葡萄果实颜色调控基因VvMYB308。
进一步地，所述葡萄果实颜色调控基因VvMYB308的核苷酸序列与SEQ ID NO.3具有90％以上的序列同一性，且包括SEQ ID NO.10所示的C2基序。
进一步地，所述葡萄果实颜色调控基因VvMYB308的核苷酸序列为SEQ ID NO.3。
另一方面，本申请提供了上述方法在葡萄育种中的应用。
另一方面，本申请提供了一种葡萄育种用试剂盒，其含有过表达载体，所述过表达载体中插入有上述葡萄果实颜色调控基因VvMYB308。
进一步地，所述试剂盒用于降低葡萄果实中的花色苷含量。
本发明提供了一种抑制葡萄花色苷合成相关MYB转录因子及其编码基因与应用。抑制葡萄花色苷合成相关MYB转录因子来源于葡萄品种玫瑰香，命名为VvMYB308。VvMYB308的编码序列由873对碱基组成；编码290个氨基酸，含有R2和R3结构域，属于典型的R2R3-MYB转录因子。
利用任何一种可以引导外源基因在植物中表达的载体，将本发明所提供的VvMYB308的编码基因导入植物细胞，使植物花色苷生物合成受到抑制。使用本发明的基因构建植物表达载体时，可以在其转录起始核苷酸前加上任何一种增强启动子或诱导性启动子。为了便于对转基因植物细胞或植物进行鉴定及筛选，可对所使用的载体进行加工，如加入植物可选择性标记(GUS基因、荧光素酶基因等)或具有抗性的抗生素标记物(庆大霉素、卡那毒素等)。携带有本发明VvMYB308的表达载体可通过使用Ti质粒、Ri质粒、植物病毒载体、直接DNA转化、微注射、电导、农杆菌介导等常规生物学方法转化植物细胞或组织，并将转化的植物组织培育成植株。被转化的宿主既可以是单子叶植物，也可以是双子叶植物。
本发明克隆了一个与抑制葡萄花色苷合成相关的VvMYB308转录因子基因，对该基因在葡萄愈伤组织中的表达进行了分析，对培育不同色泽的葡萄品种具有重要意义。
附图说明
图1为VvMYB308的系统发育分析和序列分析；
图2为VvMYB308负向调节葡萄愈伤组织中花色苷生物合成相关实验结果；
图3为VvMYB308负向调节花色苷生物合成机制相关实验结果。
具体实施方式
材料：“赤霞珠”葡萄果实愈伤组织。
实施例1葡萄VvMYB308基因的克隆
提取葡萄总RNA
Quick-RNA提取试剂盒(华跃洋，中国北京)提取健壮无病虫害的葡萄果皮总RNA，并反转录成cDNA。根据VvMYB308基因特征，设计了特征性上下游引物，(F：ATGGGTAGATCTCCCTGCTGTG(SEQ ID NO.1)/R：TCATGAATCCAAGGCCGTGT(SEQ ID NO.2))。
以葡萄果皮cDNA为模板，进行VvMYB308基因克隆。PCR反应体系为50uL，其中包括模板2uL，F和R引物各1.5uL，PCR Mix 25uL，dd H2O 20uL。反应程序为95℃预变性3min；94℃变性30s，56℃退火15s，72℃延伸1min，32个循环；72℃延伸5min；使用胶回收试剂盒对目的片段进行胶回收。
测序验证葡萄VvMYB308基因
将胶回收得到的目的基因连接到pCE2-TA Blunt-Zero载体，将连接产物转化到大肠杆菌感受态细胞，挑取大肠杆菌感受态单克隆分别用通用引物M13F和M13R进行一代测序验证，最终获得VvMYB308基因相关序列：
实施例2 VvMYB308的系统进化分析和序列分析
葡萄和其他物种中与VvMYB308相关的R2R3-MYB TF的系统发生树使用Mega软件(v11.0.10)构建的最大似然系统发生树，有1000次引导重复。VvMYB308以黑色实心圆圈标出。
VvMYB308与其他已知黄酮类相关R2R3-MYB抑制因子氨基酸序列的比对。保留残基以黑色突出显示，部分保留残基以粉色或蓝色显示。C末端的C1和C2基序用方框标出。使用了以下GenBank编号(括号内)：拟南芥AtMYB4(AT4G38620)、AtMYB6(AT4G09460)、AtMYB7(AT2G16720)、AtMYB32(AT4G34990)；葡萄VvMYBA1(AB097923)、VvMYBPA2(EU919682)、VvMYBPA1(AM259485)、VvMYBC2-L1(JX050227)、VvMYBC2-L3(KM046932)、VvMYB4a(EF113078)、VvMYB4b(FJ792820)、VvMYB4-like(XP_002273328)、VvMYB308(XM_019220875)；桃PpMYB18(KT159234)，PpMYB10.1(XM_007216468)；苹果MdMYB10(DQ267897)，MdMYB16(HM122617)；草莓FaMYB1(AF401220)；柿子DkMYB4(AB503701)；金鱼草AmMYB308(P81393)and玉米ZmMYB31(NP_001105949)。
序列信息：VvMYB308的编码序列包含870bp碱基，编码290个氨基酸。在与MYBTFs的系统进化分析中，VvMYB308与AmMYB308、MdMYB16、VvMYB4a、VvMYB4b等R2R3-MYB抑制因子密切相关(图1a)。VvMYB308的结构与其他R2R3-MYB抑制因子相似。它们在N端含有一个bHLH结合结构域，在C端发现了一个保守的C1基序和一个C2基序(图1b)。这些结果表明，VvMYB308可能是一种候选的黄酮类化合物生物合成抑制因子，它可以与其他MYB因子一起调节浆果中黄酮类化合物的积累。
ELI INLHSFFGNKWSLIAGR(bHLH结合基序氨基酸序列，SEQ ID NO.5)；
GAGCTCATCATCAACCTCCACAGCTTCTTTGGAAACAAATGGTCTCTCATTGCGGGGAGATTACCG(bHLH结合基序核苷酸序列，SEQ ID NO.6)；
LYSRGIDPQTHRPLS(C1基序氨基酸序列，SEQ ID NO.7)；
CTCTACAGCCGGGGAATCGACCCCCAAACTCACCGCCCCCTCAGC(C1基序核苷酸序列，SEQ IDNO.8)；
PQLNLELSIGLP(C2基序氨基酸序列，SEQ ID NO.9)；
CCACAACTTAACCTTGAGCTCTCCATAGGCCTTCCT(C2基序核苷酸序列，SEQ ID NO.10)。
实施例3葡萄VvMYB308基因功能验证
通过overlap PCR方法去除VvMYB308的bHLH结合序列、C1基序和C2基序，并分别命名为MYBRbH、MYBRC1和MYBRC2。将这些序列插入pCXSN载体。将连接产物转化到大肠杆菌感受态细胞，挑取单克隆进行一代测序。测序正确的载体用冻融法将表达载体分别导入农杆菌菌株GV3101。选择适量的愈伤组织进行遗传转化。
遗产转化成功的阳性愈伤组织在继代培养基上继代培养，在花色苷诱导培养基上诱导花色苷积累。继代培养基为固体B5培养基，添加30g/L蔗糖、2.5g/L酸水解酪蛋白、3.0g/L植胶、0.1mg/Lα-萘乙酸和0.2mg/L激动素，pH值为5.9-6.0。培养条件为24小时黑暗培养，温度为25℃，每25天对愈伤组织进行一次继代。花色苷诱导培养基由固体缺氮培养基(含3mM硝酸盐的B5培养基)组成，添加80g/L蔗糖、2.5g/L酸水解酪蛋白、3.0g/L植物凝胶、0.01mg/Lα-萘乙酸和0.2mg/L激动素，pH值为5.9-6.0。花色苷诱导培养条件为25℃，16/8-h光/暗循环。
使用Micro Plant Anthocyanin Assay Kit(Solarbio，中国北京)提取总花色苷。用提取液从0.1克样品中提取花色苷，然后在pH值为1.0和4.5的缓冲液中分别在530纳米和700纳米处测量花色苷提取物的吸光度。pH值为1时，530nm和700nm处的吸光度值记作A1和A1’；pH值为4.5时，530nm和700nm处的吸光度值记作A2和A2’，ΔA＝(A1-A1’)-(A2-A2’)花色苷总含量＝[ΔA÷(ε×d)×103×F]×V÷W。(F：稀释倍数，10；d：比色皿光学直径，1cm；W：样品质量，g；ε：花色苷摩尔消光系数，2.69×104mL/mmol/cm；V：提取液总体积，1mL；103：单位换算系数，1mmol＝103μmol；)。
使用Quick-RNA提取试剂盒(华跃洋，中国北京)提取总RNA。qRT-PCR采用Taq ProUniversal SYBR qPCR Master Mix(Vazyme，南京，中国)，扩增程序如下：95℃30s，然后95℃10s和60℃30s，40个循环。以UBIQUITIN1为内参基因进行归一化处理。采用2(-ΔΔCt)法计算基因的相对表达水平。
使用MatchmakerTMGold酵母单杂交系统(Clontech)进行酵母双杂交(Y2H)检测。VvMYC1基因被插入pGBKT7载体。将VvMYB308插入pGADT7载体。然后，将两种质粒共转到Y2H酵母菌菌株中，酵母细胞在缺乏Leu和Trp的SD培养基(SD/-Leu/-Trp，DDO)上生长。阳性克隆通过PCR鉴定，并在缺乏Ade、His、Leu、Trp和Leu的SD培养基上进一步生长。pGADT7-T+pGBKT7-p53作为阳性对照，pGADT7-T7+pGBKT7-Lam作为阴性对照。
通过overlap PCR方法去除了VvMYB308的bHLH结合结构域、C1基序和C2基序，并分别命名为MYBRbH、MYBRC1和MYBRC2(图2a)。将VvMYB308的四种编码序列插入pCXSN载体并转化到葡萄愈伤组织中(图2c)。这些愈伤组织在花色苷诱导培养基上培养14天，光周期为16/8小时。WT愈伤组织呈现红色并积累了大量花色苷，而过表达VvMYB308的转基因愈伤组织颜色为白色，表明VvMYB308抑制了葡萄愈伤组织中花色苷的生物合成。过表达MYBRbH、MYBRC1和MYBRC2的转基因愈伤组织颜色为红色，但其花色苷含量低于WT，过表达MYBRC2的转基因愈伤组织花色苷含量在四种转基因愈伤组织中最高(图2b，2d)。这些结果表明，VvMYB308的抑制活性来自于bHLH结合域、C1基序和C2基序，而C2基序在赋予VvMYB308抑制活性中起着重要作用。
双荧光素酶报告试验，从玫瑰香葡萄中扩增VvUFGT(1.8kb)起始密码子上游的启动子区域，并将其插入载体pGreenII 0800-LUC的多克隆位点中。用冻融法将转化到含有pSoup辅助质粒的农杆菌菌株GV3101中，并在28℃下培养2d。将混合的菌体重悬于10ml含有10mM MgCl2、200μM乙酰丁香酮和10mM MES(pH＝5.7)的缓冲液中，注射前在28℃下静置培养2h。将含有基因表达载体的农杆菌缓冲液按比例混合。将混合后的缓冲液注入4周大的烟草幼苗的嫩叶中。注射三天后，使用双荧光素酶试剂盒(Vazyme，中国南京)测量LUC与Ren活性的比值。
烟草叶片中的瞬时过表达实验表明，VvMYB308抑制了VvMYBA1和VvMYC1对花色苷生物合成的激活(图3a)。为了验证VvMYB308是否通过影响VvMYC1来抑制花色苷的生物合成，使用双荧光素酶试验测定了VvMYB308在不同VvMYC1剂量下对VvUFGT的抑制作用。当VvMYB308、VvMYBA1和VvMYC1的剂量比为6∶6∶0.1和6∶6∶0.2时，VvUFGT启动子的活性受到抑制；而当VvMYC1的剂量增加到6∶6∶0.3和6∶6∶0.4时，VvUFGT的启动子活性不再受VvMYB308的影响(图3d)。因此，在VvMYC1水平较低时，VvMYB308可以通过竞争性结合VvMYC1来抑制VvUFGT的表达。此外，VvMYB308的不同突变体对VvUFGT的启动子活性有不同的抑制作用。没有C2基序的MYB308RC2不能抑制VvUFGT启动子的相对活性，说明VvMYB308的抑制活性主要来自C2基序(图3e)。
Claims (10)
1.一种葡萄果实颜色调控基因VvMYB308，其特征在于，所述葡萄果实颜色调控基因VvMYB308的核苷酸序列为SEQ ID N0.3。
2.根据权利要求1所述的葡萄果实颜色调控基因VvMYB308，所述葡萄果实颜色调控基因VvMYB308编码的氨基酸序列为SEQ ID N0.4。
3.葡萄果实颜色调控基因VvMYB308在调控葡萄品种色泽中的应用。
4.根据权利要求3所述的应用，其中过表达所述葡萄果实颜色调控基因VvMYB308以降低葡萄果实中的花色苷含量。
5.根据权利要求4所述的应用，其中使用pCXSN过表达载体来过表达所述葡萄果实颜色调控基因VvMYB308。
6.根据权利要求3或4所述的应用，其中所述葡萄果实颜色调控基因VvMYB308的核苷酸序列与SEQ ID N0.3具有90％以上的序列同一性，且包括SEQ ID NO.10所示的C2基序。
7.根据权利要求6所述的应用，其中所述葡萄果实颜色调控基因VvMYB308的核苷酸序列为SEQ ID N0.3。
8.根据权利要求3-7任一项所述的方法在葡萄育种中的应用。
9.一种葡萄育种用试剂盒，其特征在于，所述试剂盒含有过表达载体，所述过表达载体中插入有上述葡萄果实颜色调控基因VvMYB308。
10.根据权利要求9所述的试剂盒，所述试剂盒用于降低葡萄果实中的花色苷含量。
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