CN117625640A - 广金钱草转录因子DsMYB在调控黄酮生物合成中的应用 - Google Patents
广金钱草转录因子DsMYB在调控黄酮生物合成中的应用 Download PDFInfo
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Abstract
本发明公开了广金钱草转录因子DsMYB在调控黄酮生物合成中的应用。所述DsMYB60基因的核苷酸序列如SEQ ID NO.1所示。本发明首次披露了广金钱草MYB转录因子DsMYB60基因序列,并通过过表达DsMYB60基因,证实了该基因能够促进烟草总黄酮的积累。本发明提供的广金钱草MYB转录因子DsMYB60基因可以作为一个优良的基因资源,广泛应用于植物遗传育种领域,对改良植物黄酮类物质代谢积累,特别是培育高黄酮水平的植物品种具有重要意义。
Description
技术领域
本发明属于基因工程技术领域,具体涉及广金钱草转录因子DsMYB在调控黄酮生物合成中的应用。
背景技术
转录因子(transcription factor)是一群能与基因5'端上游特定序列专一性结合,从而保证目的基因以特定强度在特定时间与空间表达的蛋白质分子。根据转录因子的作用特点可分为两类:第一类为普遍转录因子;第二类转录因子为组织细胞特异性转录因子。人们在植物中已经找到了很多调控黄酮类物质次生代谢的特异性转录因子基因,主要包括编码MYB、MYC、bZIP、WD40蛋白和锌指蛋白等基因家族。MYB基因家族广泛存在于植物中,是植物中最大的转录因子基因家族之一。在黄酮生物合成过程中,MYB转录因子扮演着重要角色,可调控与黄酮类物质合成相关的酶基因的表达,从而有效地调控黄酮类物质的生物合成。
广金钱草属于豆科山蚂蝗属草本植物,收录于《中国药典》,主产于我国广东、广西等地,含有黄酮、生物碱、多糖和挥发油类有效成分。具有利湿退黄和利尿通淋的功效,主治黄疸尿赤、热淋、石淋和小便涩痛,是两广地区重要的药用植物。黄酮类化合物是广金钱草主要的药效成分,具有广泛的药理活性和重要的应用价值。然而,目前关于广金钱草药用活性成分黄酮类化合物的调控机制仍不清晰,较大制约了该类植物的开发利用,因此本研究拟通过基因克隆等分子生物学手段来高效挖掘调控广金钱草黄酮类物质的关键基因,解析其代谢途径的遗传机制和调控网络,这对广金钱草的基因工程和创新利用具有重要意义。
发明内容
本发明的第一个目的是提供一种新的广金钱草MYB转录因子DsMYB60基因,该基因的核苷酸序列如SEQ ID NO.1所示。
本发明的第二个目的是提供含有上述的广金钱草MYB转录因子DsMYB60基因的生物材料。
优选的,所述生物材料为表达盒、重组载体或重组菌。
优选的,所述重组载体的表达载体为pBI121-eGFP。
优选的,所述重组菌的宿主菌为农杆菌GV3101。
本发明的第三个目的是提供上述的广金钱草MYB转录因子DsMYB60基因或含有该基因的生物材料在促进植物黄酮类物质生物合成的应用。
优选的,所述植物包括烟草和广金钱草。
本发明的第四个目的是提供一种促进植物黄酮类物质生物合成的方法,包括将广金钱草MYB转录因子DsMYB60基因转入植物细胞或组织中的步骤。
优选的,所述将广金钱草MYB转录因子DsMYB60基因转入植物细胞或组织中的方法包括农杆菌介导法、植物病毒载体法或直接DNA转化法。
本发明首次披露了广金钱草MYB转录因子DsMYB60基因序列,并通过过表达DsMYB60基因,证实了该基因能够促进烟草总黄酮的积累。本发明提供的广金钱草MYB转录因子DsMYB60基因可以作为一个优良的基因资源,广泛应用于植物遗传育种领域,对改良植物黄酮类物质代谢积累,特别是培育高黄酮水平的植物品种具有重要意义。
附图说明
图1为目的基因DsMYB60的琼脂糖凝胶电泳分析。
图2为目的基因DsMYB60和克隆载体重组质粒的双酶切图。
图3为目的基因DsMYB60过表达载体转化农杆菌后的菌落PCR图。
图4为目的基因DsMYB60烟草异源转化。
具体实施方式
以下实施例是对本发明的进一步说明,而不是对本发明的限制。
实施例1.编码转录因子DsMYB60的基因的克隆
1、广金钱草基因组总RNA的提取
根据多糖多酚植物总RNA提取试剂盒操作说明提取广金钱草总RNA,1%琼脂糖凝胶电泳检测RNA的完整性,用Nano核酸分析仪检测RNA的OD260/280值和OD260/230值。使用PrimeScriptTM RT reagent Kit with gDNA Eraser将合格的总RNA逆转录为cDNA,-20℃保存备用。
2、编码转录因子DsMYB60的基因的克隆
上游引物DsMYB60-F:5’-CCCCCGGGGGATGGGAAGACCACCATGTT-3’(SEQ ID NO.2);
下游引物DsMYB60-R:5’-CGAGCTCGTTAGAAAAACTTGGCATC-3’(SEQ ID NO.3);
以反转录cDNA为模板,添加上下游引物,进行PCR扩增。PCR反应体系及程序如下:
PCR反应程序:94℃,5min;94℃,30s;55℃,30s;72℃,1min;30个循环;72℃,10min;4℃保存。用凝胶回收试剂盒(Quick Gel ExtractionKit)回收纯化PCR产物(图1),将回收的目的片段与/>-Blunt Simple Cloning Vector按照载体与目的片段摩尔比为1:7的条件配成3-5μL的连接体系。在25℃连接10min。将连接产物转化Trans1-T1感受态细胞,筛选阳性克隆,得到/>-Blunt Simple-DsMYB60质粒(图2)。经PCR扩增得到的DsMYB60基因的核苷酸序列如SEQ ID NO.1所示,具体序列为:ATGGGAAGACCACCATGTTGTGACAAAGAAGGTGTCAAGAAAGGGCCTTGGACTCCTGAAGAAGACATCATATTGGTGTCTTATATACAGGAACATGGCCCTGGAAATTGGAGGGCAGTTCCTACCAAAACAGGGTTGTCAAGGTGCAGCAAGAGTTGCAGACTTAGATGGACTAATTACCTGAGGCCAGGAATCAAACGTGGTAACTTCACAGAACAAGAGGAGAAGATGATAATCCATCTTCAAGATCTTTTAGGAAACAGATGGGCTGCAATAGCTTCATACCTTCCACAGAGAACAGACAATGACATAAAGAACTATTGGAACACTCACTTGAGAAAGAAACTGAAAAAGATGCAAACAGGTTGTGAAAGTGGTTTGGGAGAAGGGTTTTCTGCTTCAAGGCAAATCCCTAGAGGCCAGTGGGAAAGAAGGCTCCAAACTGATATTCAAATGGCAAAGAAAGCTCTCAGTGAAGCTCTTTCACAAGAGAATAATAAAAATAATAAGCCTACTTCTTTGTTATCTGCATCAAACTCAAACCCTTCTGATACTAGCAGCTCTTTCTCTTCCACAAAACCAACACATTCTTTGTGTTATGCATCAAGTGCTGAGAATATAGCACGCATGCTGAAGGGTTGGATGAAAAACCCACCAAAGTCTTCAAGGACTAACTCATCTGTGACTCAAAATTCCTTCAATAACTTGGCTGGTGCTGATACTGCTTCTAGTGGAGCAAATGGATCTGATCTGTCTGAGAATTTTGAATCTTTGTTGTATTTTGACCAGTCTTTGGAGTCTTCAAACTCTGAACAAGTTTCTCAGTCTTTGTCTCCTGAGACCACTGTTTTGCAAGATGAAAGCAAACCTAATATTGGTGCAGAAATAATGCCCTTTTCTTTGCTTGAGAAGTGGCTTCTTGATGAGGCTGGTTCTCTAGATAAAATTGGTTTTGGTGATGCCAAGTTTTTCTAA。
实施例2.过表达载体pBI121-eGFP-DsMYB60的构建
(1)对基因克隆为阳性,并且测序正确的菌液进行质粒提取,获得-BluntSimple-DsMYB60质粒。具体操作步骤如下:
1)加入250μL的Solution I(含RNaseA),使用振荡器剧烈振荡充分悬浮细菌沉淀;
2)加入250μL的Solution II轻轻上下翻转混合5~6次,使菌体充分裂解;
3)加入350μL的4℃预冷的Solution III轻轻上下翻转混合5~6次,直至形成紧实凝集块,然后室温静置2分钟;
4)室温12000rpm离心10分钟,取上清;
5)上清液转移至Spin Column中,12000rpm离心1分钟,弃滤液;
6)加入500μL Buffer WA,12,000rpm离心30sec,弃滤液;
7)加入700μL Buffer WB(已加入乙醇),12,000rpm离心30sec,弃滤液;
8)重复步骤7);
9)12,000rpm离心1分钟,除尽残留洗液;
10)将Spin Column安置于新的1.5mL的离心管上,在Spin Column膜的中央加入50μL的灭菌水,室温静置1分钟;
11)12,000rpm离心1分钟洗脱DNA。
(2)取pBI121-eGFP载体和-Blunt Simple-DsMYB60质粒用Sma I和SacⅡ双酶切,酶切体系为:
组分 | 体积 |
10×Quickcut buffer | 5μL |
Sma I | 2μL |
SacⅡ | 2μL |
质粒/pBI121-eGFP | 41μL |
以上体系在37℃酶切反应30分钟。反应完毕后进行1%琼脂糖凝胶电泳,并回收目的片段与载体大片段。将目的片段与pBI121-eGFP载体按照摩尔比为1:6的比例加入0.2mL的EP管中,再加入与DNA溶液等量的DNA Ligation mix,混匀后在16℃连接30分钟。连接产物转化Trans1-T1感受态细胞,转化步骤同目的基因连接克隆载体转化大肠杆菌,在含氨苄青霉素与卡那霉素的固体培养基倒置过夜培养。挑取阳性单克隆扩大培养,提取质粒pBI121-eGFP-DsMYB60,电泳和测序验证含SEQ ID NO.1所示的DsMYB60基因序列。
(3)GV3101农杆菌的转化与鉴定:
1)按照上述质粒提取的方法提取大肠杆菌中的重组质粒;
2)吸取约500ng的重组质粒加入刚融化的50μL GV3101农杆菌感受态细胞中;
3)依次于冰上静置5分钟,液氮5分钟,37℃水浴5分钟,冰浴5分钟;
4)加入1mL无抗液体LB培养基,28℃,180rpm避光培养2-3个小时;
5)取200μL菌液均匀涂布在含卡那霉素和利福平的固体培养基上,28℃倒置、避光培养36个小时;
6)挑取阳性菌进行PCR扩增,吸取1μL农杆菌进行菌体PCR,将菌体PCR产物经1%凝胶电泳检测,可以看到菌体PCR产物DNA有明显的单一条带,表明转化的农杆菌含有目的基因片段,可以用于瞬时转化烟草的实验并且证明植物表达载体pBI121-eGFP-DsMYB60的构建成功(图3)。
实施例3.植物表达载体pBI121-eGFP-DsMYB60通过农杆菌介导的方法转化到异源的烟草
1、本氏烟草的瞬时转化
(1)烟草的培养:本氏烟草种子播种于灭菌处理的营养土中,铺保鲜膜保湿4-5天。一周后,将长出3片真叶的烟草移植于小盆中,转到光照培养箱中生长。培养条件:光照/黑暗为16h/8h,温度22℃,湿度80%,以生长期5-6周的烟草作为实验材料。
(2)从-80℃超低温冰箱中取出含DsMYB60的GV3101菌种平板划线活化,待长出菌后,挑单菌落于2mL含卡那霉素和利福平的培养基中摇菌培养18-24小时。
(3)取1mL菌液接种于35mL液体LB培养基中,振荡培养,使菌液浓度OD600值达到0.8-1.0之间。5000rpm离心10分钟收集菌体,用含10mM MgCl2和10mM MES的重悬液洗涤菌体2次,菌体沉淀用终浓度为100μM MAS的等体积重悬液重悬,28℃避光放置2-3个小时。
(4)使用1mL注射器吸取含有菌体的重悬液,去掉针头,从烟草背面注射,使菌液在烟草叶片扩散开。注射后的烟草放回培养箱暗培养24h,然后转弱光培养,2天后收取材料进行后续分析。
2、烟草总黄酮含量的测定
(1)样品的前处理:烟草叶片置于80℃烘箱烘干至恒重,烘干至恒重的叶片粉碎后过三号筛。
(2)供试溶液的制备:精密称取芦丁适量,加入70%乙醇溶解,容量瓶定容,配成0.5mg/mL的供试品溶液。
(3)标准曲线的制作:取6支试管按照表1加入标准溶液,依次加入5%亚硝酸钠、10%硝酸铝和4%氢氧化钠,分别静置6min、6min、15min后,在510nm测定吸光值。得到芦丁的线性方程为y=1.8223x-0.0121,R2=0.9999(n=5),可见相关性较好。线性范围为0.1mg/mL-0.5mg/mL。
(4)样品总黄酮含量测定:称取0.1g粉末加入8mL 70%乙醇,超声提取30min,将提取液于25℃,5000r/min转速下离心10min。取1mL提取液加入试管,按表1加入各试剂,并按标准曲线制作方法反应。发现转化pBI121-eGFP空载对照的烟草中总黄酮含量为6.87mg/g,转化pBI121-eGFP-DsMYB60后烟草的总黄酮含量相比空载对照提高了23.8%。说明DsMYB60的超表达促进烟草总黄酮的生成(图4)。
表1芦丁标准曲线测定的试剂添加表
综上所述,本发明构建了含有MYB转录因子DsMYB60的植物表达载体pBI121-eGFP-DsMYB60,其中DsMYB60为首次报道。所构建的载体可导入烟草中,促进烟草总黄酮的积累。
以上仅是本发明的优选实施方式,应当指出的是,上述优选实施方式不应视为对本发明的限制,本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说,在不脱离本发明的精神和范围内,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.广金钱草MYB转录因子DsMYB60基因,其特征在于,其核苷酸序列如SEQ ID NO.1所示。
2.含有权利要求1所述的广金钱草MYB转录因子DsMYB60基因的生物材料。
3.根据权利要求2所述的生物材料,其特征在于,所述生物材料为表达盒、重组载体或重组菌。
4.根据权利要求3所述的生物材料,其特征在于,所述重组载体的表达载体为
pBI121-eGFP。
5.根据权利要求3所述的生物材料,其特征在于,所述重组菌的宿主菌为农杆菌GV3101。
6.权利要求1所述的广金钱草MYB转录因子DsMYB60基因或权利要求2所述的生物材料在促进植物黄酮类物质生物合成的应用。
7.根据权利要求6所述的应用,其特征在于,所述植物包括烟草和广金钱草。
8.一种促进植物黄酮类物质生物合成的方法,其特征在于,包括将权利要求1所述的广金钱草MYB转录因子DsMYB60基因转入植物细胞或组织中的步骤。
9.根据权利要求8所述的方法,其特征在于,所述将广金钱草MYB转录因子DsMYB60基因转入植物细胞或组织中的方法包括农杆菌介导法、植物病毒载体法或直接DNA转化法。
10.根据权利要求8所述的方法,其特征在于,所述植物包括烟草和广金钱草。
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