CN114517207A - 一种草莓MYB转录因子FaMYB5基因的应用 - Google Patents
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Abstract
本发明涉及植物分子基因工程技术领域，具体涉及一种草莓MYB转录因子FaMYB5基因的应用，通过克隆出草莓MYB转录因子FaMYB5基因以及含有上述FaMYB5基因的表达载体、宿主菌，这种FaMYB5基因能够应用于调控草莓果实中柠檬酸积累、表达CS1和GAD基因，在草莓中过量表达FaMYB5促进草莓果实柠檬酸代谢，同时柠檬酸合成主要基因CS1表达量上升和降解基因GAD表达量明显下降。
Description
技术领域
本发明涉及植物分子基因工程技术领域，具体涉及一种草莓MYB转录因子FaMYB5基因的用。
背景技术
有机酸是果实主要的品质组成因子，直接影响草莓果实的酸度和风味。适量的酸可以使水果更可口，果实酸度的调控具有很强的经济相关性，因为它与消费者的感知有关，因此是水果产业的重要制约因素。
大多数水果中主要有机酸为柠檬酸或苹果酸，柠檬酸是草莓果实中主要有机酸类型，占总有机酸的80％以上，其含量由合成、分解代谢、运输和液泡储存之间的平衡决定。不同品种之间的柠檬酸含量存在差异，并受遗传、成熟度和栽培管理措施等因素的影响。
果实有机酸代谢是一个复杂的生物学过程，在果实发育过程中，有机酸积累主要发生在果实膨大期，而随着果实成熟进程其含量逐渐下降。此外，柠檬酸不仅响应果实风味，也参与了果实的光合作用、呼吸作用以及氨基酸、芳香物质、酯类及酚类物质的合成、衰老等众多生物学过程。因此，草莓果实柠檬酸代谢研究是果实品质生物学的重要研究内容。
鉴于上述缺陷，本发明创作者经过长时间的研究和实践终于获得了本发明。
发明内容
本发明的目的在于解决如何提高果实中的柠檬酸含量的问题，提供了一种草莓MYB转录因子FaMYB5基因的应用。
为了实现上述目的，本发明公开了一种草莓MYB转录因子FaMYB5基因在表达FaCS1和FaGAD基因中的应用，所述草莓MYB转录因子FaMYB5基因的核苷酸序列如SEQ ID NO.1所示。
本发明还公开了一种草莓MYB转录因子FaMYB5基因调控草莓果实中柠檬酸代谢中的应用，所述草莓MYB转录因子FaMYB5基因的核苷酸序列如SEQ ID NO.1所示。
一种草莓MYB转录因子FaMYB5基因调控草莓果实中柠檬酸代谢中的应用，采用注射法侵染果实。
与现有技术比较本发明的有益效果在于：本发明克隆出草莓MYB转录因子FaMYB5基因，并通过过表达转基因草莓试验发现，FaMYB5与果实柠檬酸代谢相关，将该基因过量表达后，其表达量增高，FaCS1和FaGAD主要基因表达量明显增加。可见，该FaMYB5基因在果实柠檬酸代谢中将具有广泛的应用。
附图说明
图1为草莓果实中克隆得到FaMYB5；
图2为实时定量PCR检测草莓果实注射后转录因子MYB5、合成关键基因CS2和降解基因GAD表达图；
图3为过表达注射草莓果实后柠檬酸含量的变化图。
具体实施方式
以下结合附图，对本发明上述的和另外的技术特征和优点作更详细的说明。
实施例1
以NCBI中搜索到的草莓MYB5基因序列为模板，进一步设计引物；以红颜草莓大绿期果实的cDNA为模版，以MYB5-F为上游引物，以MYB5-R为下游引物，进行PCR反应，克隆得到MYB5基因，转化pMD19-T载体，提取质粒备用。本实施例使用的红颜草莓为安徽农业大学种质资源圃采集。
一、转化步骤如下：
(1)连接产物转化大肠杆菌感受态：重组DNA粘附在细菌细胞表面，经过42℃的热击处理90s，促进吸收DNA，然后在37℃非选择LB液体(未添加抗生素)培养基中震荡培养45min，均匀地涂布在添加氨苄青霉素(Amp)抗生素的固体培养基中37℃过夜培养；
(2)挑取单克隆，重新进行活化，即在新的含Amp抗生素的固体培养基上重新划线，每个挑取12个单克隆进行重新划线活化，37℃过夜培养；
(3)挑取菌落，按照上述PCR反应体系进行菌落PCR反应检测；
(4)根据菌落PCR结果，挑取菌落，置于添加Amp的液体LB培养基中震荡培养12h，送去上海生工生物工程有限公司测序；
(5)选取测序正确的单菌落，放入(20mL LB+20uL Amp)的LB液体培养基，37℃摇床摇12～16h，提质粒。
二、提质粒步骤如下：
(1)取1～4mL在LB培养基中培养过夜的菌液，12000×g离心1min，弃尽上清；
(2)加250μL Buffer SH1悬浮细胞沉淀，悬浮需均匀，不应留有小的菌块(确认Buffer S1中已加入RNaseA)；
(3)加入250μL Buffer SH2，温和并充分地上下翻转4～6次混合均匀使菌体充分裂解，直至形成透亮的溶液；
(4)加入350μL Buffer SH3，温和并充分地上下翻转4～6次，12000g离心10min；
吸取步骤(4)中的离心上清并转移到制备管(试剂盒内提供)，12000g离心1min，弃滤液；将制备管置回离心管，加500μL Buffer W1，12000g离心1min，弃滤液；将制备管置回离心管，加700μL Buffer W2，12000g离心1min，弃滤液，重复一次；将制备管移入新的1.5mL中，在制备管膜中央加50～80μL Eluent或去离子水，室温静置3～5min，12000g离心1min。
MYB5-F：ATGAGGAACCCATCTTCGTCTTCAT，
MYB5-R：CTATTGCGTCTGATTGACATTAACC。
PCR反应程序为：94℃变性1min；94℃变性30sec，55℃退火30sec，72℃延伸45sec，35个循环；72℃10min，4℃保温。
如图1所示，草莓果实中克隆得到FaMYB5基因序列全长1041bp，编码347个氨基酸。其碱基序列如SEQ ID NO.1所示，表达的蛋白的氨基酸序列如SEQ ID NO.2所示。
实施例2
将实施例1得到的FaMYB5基因，转化pMD19-T载体，提取质粒；以FaMYB5-pCXSN-F和FaMYB5-pCXSN-R为扩增引物，以pMD19-T-FaMYB5质粒为模板进行PCR反应，回收目的基因产物。将目的基因与pCXSN-FLAG载体进行连接。16℃连接12h后热激法转化大肠杆菌感受态。提取验证正确的菌株质粒，冻融法转化农杆菌GV3101。菌落PCR鉴定。
具体如下：
采用pCXSN-FLAG过量表达载体进行重组构建，在FaMYB5的5’端选取20bp左右的片段设计引物，并在特异引物前加入一个碱基A，作为目标片段的上游引物；在FaMYB5的3’端选取20bp左右的片段设计引物，作为目标片段的下游引物。引物信息如下：以MYB5-pMD19-T为模板扩增得到目标片段，PCR产物经回收后，经Soultion1连接至pCXSN-FLAG载体，构建成目的载体。选取菌落PCR反应后正确的单克隆菌株，提取质粒并以pCXSN-FLAG载体自身上游引物pCXSN-FLAG-F和MYB5下游引物FaMYB5-pCXSN-R进行PCR验证。
引物如下：
FaMYB5-pCXSN-F：ACGGGGGACTCTTGACCATGGCATGAGGAACCCATCTTCGTCTTC，
FaMYB5-pCXSN-R：TCTCCTTTACTAGTCAGATCTTTGCGTCTGATTGACATTAACCC，
pCXSN-FLAG-F：GATTACAAGGATGATGATGAT。
农杆菌转化步骤如下：
(1)从-80℃取出保存的感受态农杆菌于冰上融化；
(2)每100μL感受态加1μg质粒DNA混匀，依次于冰上静置5min、液氮5min、37℃5min、冰浴5min；
(3)加入700μL无抗生素的LB液体培养基，于28℃振荡培养2～3h；
(4)5000rpm离心三分钟收集菌，留取50μL左右的上清轻轻吸打重悬菌块涂布于含相应抗生素的LB平板上，倒置放于28℃培养箱培养2～3天
(5)挑单菌落培养并鉴定，鉴定正确的菌液每300μL加入50％甘油700μL置于-80℃保存。
实施例3
瞬时转MYB转录因子FaMYB5基因草莓的获得及该基因在草莓果实的表达模式
(1)挑选实施例2鉴定正确的单菌落进行草莓果实侵染；选择大绿果时期的八倍体果实为试材，采用注射法进行果实侵染，具体方法如下：
①挑去含pCXSN-FLAG-MYB5质粒的阳性克隆，接种至15mL LB培养基中(含有50mgL-1的卡那和50mg L-1利福平)，28℃、200rpm摇菌至OD600≈2.0。
②取20mL新鲜的LB液体培养基(含有50mg L-1的卡那和50mg L-1利福平)，接入1mL步骤1的农杆菌，28℃、200rpm摇至OD600≈0.6。
③收集菌液，室温、6000rpm离心3min。
④用侵染液重新悬浮菌液，室温条件下，6000r/s离心3min。侵染液配置：分别配置浓度为1.0mol L-1的MES、1.0mol L-1的MgCl2以及浓度为1.0mol L-1的乙酰丁香酮，取200μL的MES、200μL MgCl2、20μL乙酰丁香酮，灭菌水定容至20mL，即为侵染液，备用。
⑤重复步骤④一次。
⑥最后用20mL侵染液重新悬浮菌株，室温静止2h，注射法侵染草莓果实。
数据测定：将pCXSN-FLAG-MYB5农杆菌注射果实做RT-qPCR，分析该基因在草莓果实的表达模式。
(2)PCR程序为：
(3)PCR反应程序为：95℃，2min；95℃，15sec；60℃，1min(50个循环)；溶解曲线。
(4)柠檬酸含量的测定方法为：
将草莓样品超低温研磨成粉末，-80℃冰箱存放，使用时称取0.50g定容到5mL，放至超声波中低温超声20min，用低温高速，4℃，10000r/min，8分钟，再吸取管子中的中清液2mL至2mL离心管中，再低温10000r/min，10min，吸取样品溶液过0.22μm MCE膜，上样。HPLC程序：乙腈-(NH4)2HPO4缓冲系统，流动相A：色谱级乙腈，流动相B：0.5％的磷酸氢二铵缓冲液，pH＝2.6，pH用磷酸调节，柱温：25℃，流速：1.0mL/min，洗脱方式：A：1％，B：99％等度洗脱，检测波长：210nm，进样体积：20μL。
以上所述仅为本发明的较佳实施例，对本发明而言仅仅是说明性的，而非限制性的。本专业技术人员理解，在本发明权利要求所限定的精神和范围内可对其进行许多改变，修改，甚至等效，但都将落入本发明的保护范围内。
Claims (3)
1.一种草莓MYB转录因子FaMYB5基因在表达FaCS1和FaGAD基因中的应用，其特征在于，所述草莓MYB转录因子FaMYB5基因的核苷酸序列如SEQ ID NO.1所示。
2.一种草莓MYB转录因子FaMYB5基因调控草莓果实中柠檬酸代谢中的应用，其特征在于，所述草莓MYB转录因子FaMYB5基因的核苷酸序列如SEQ ID NO.1所示。
3.如权利要求2所述的一种草莓MYB转录因子FaMYB5基因在调控草莓果实中柠檬酸代谢中的应用，其特征在于，采用注射法侵染果实。
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