CN116855517A - 一种葡萄VvFLS3基因及其在抗高温中的应用 - Google Patents
一种葡萄VvFLS3基因及其在抗高温中的应用 Download PDFInfo
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Abstract
本发明涉及一种葡萄VvFLS3基因及其在抗高温中的应用,属于植物基因工程技术领域。在本发明中一种葡萄VvFLS3基因其CDS序列如SEQ ID NO.1所示。本发明发现过表达葡萄VvFLS3具有提高其抗高温的功能,其过表达植物可以通过促进体内槲皮素含量积累、提高黄酮醇合酶FLS(Flavonol synthase)的活性,降低葡萄叶片细胞膜透性和丙二醛(Malondialdehyde,MDA)含量,提高葡萄叶片PSII反应中心的光化学效率以及抗氧化酶活性来增强其抗高温能力。因此,本发明在提高植物抗高温胁迫方面具有重要意义。
Description
技术领域
本发明涉及植物基因工程技术领域,特别涉及一种葡萄VvFLS3基因及其在抗高温中的应用。
背景技术
葡萄(Vitis vinifera L.)是主要的经济果树之一。然而在葡萄的生长过程中,会受到各种各样环境因素的影响。其中,温度是制约葡萄生长和果实发育的主要调节因子。目前,全球变暖形势加剧,极端高温事件也越发频繁。高温会造成葡萄叶片枯黄焦灼、果实缩果及病害猝发,严重制约葡萄产业的发展。因此,鉴定葡萄高温响应功能物质,挖掘其合成关键基因并解析其调控机理,对培育耐热葡萄品种具有重要的科学意义和潜在的应用价值。
在高温胁迫下植物体内的活性氧含量迅速提高。过度合成的活性氧如果不能及时地进行清除,会产生严重的氧化应激,加剧膜脂的过氧化,甚至导致植物细胞的程序性死亡。植物在长期的进化过程中,逐渐形成了一套完整的抗氧化***来清除过量的活性氧,以减轻对植物细胞的伤害,这套保护***主要由酶促清除***和非酶促清除***组成。
植物有能力产生各种各样的代谢物。其中大多数是次级代谢物,发挥着广泛的生理和生态作用。类黄酮是一类高度多样的多酚次生代谢物,黄酮类化合物在花、果实、绿色组织和根中组成性地积累或在激发时被诱导。其中槲皮素是一种重要的黄酮醇物质,是清除活性氧的关键抗氧化物质,具有心脏保护、抗氧化、抗血管生成、抗炎或神经保护特性,甚至可能具有抗癌潜力。它合成的关键限速酶是黄酮醇合酶FLS。可直接催化二氢黄酮醇合成槲皮素,目前已分别从苹果、葡萄和花生等植物中克隆得到FLS基因。苹果中含有3个FLS家族基因,过表达MdFLS1促进黄酮醇合成,抑制苹果愈伤组织花青苷积累,提高耐盐性;葡萄中5个FLS基因在花芽和花中均有表达,但只有VvFLS4和VvFLS5在幼果和成熟果实中表达,其中VvFLS4的表达受光照调控。花生中已克隆出8个FLS基因,AhFLS3受低温诱导,AhFLS4参与抵御紫外胁迫及种子成熟过程。此外,FLS在其他植物抵御非生物胁迫中也具有重要作用,如盐胁迫处理可以显著诱导银杏中FLS1基因的表达,提高槲皮素含量;铁皮石斛DoFLS1定位在细胞质中,且干旱和低温处理均诱导其表达,但FLSs基因在植物抵御高温胁迫过程中的具体生物学功能尚不清楚。因此如能将FLSs基因在植物抵御高温胁迫中的应用研究并推广将对植物抵御高温胁迫的遗传改良具有重要意义。
发明内容
本发明通过分析高温处理对葡萄叶片中FLS酶活及槲皮素关键合成家族基因VvFLSs表达特性分析发现VvFLS3表达量最高,有提高葡萄叶片抗高温性能的功能。因此,本发明提出如下技术方案:
一种葡萄VvFLS3基因,所述葡萄VvFLS3基因的CDS序列如SEQ ID NO.1所示。
在上述技术方案基础上,所述葡萄VvFLS3基因的编码氨基酸序列如SEQ ID NO.2所示。
在上述技术方案基础上,所述葡萄VvFLS3基因的编码氨基酸可作为疏水性蛋白发挥作用,三级结构多为α螺旋和无规卷曲,磷酸化位点主要包括丝氨酸位点12个、苏氨酸位点4个和络氨酸位点7个,无跨膜结构域。
在上述技术方案基础上,可将葡萄VvFLS3基因制备成重组表达载体、表达盒、转基因细胞系、重组菌或重组病毒,以应用于提高植物抗高温性能。
在上述技术方案基础上,葡萄VvFLS3基因在抗高温中的用途,所述葡萄VvFLS3基因在高温下,
降低葡萄叶片表型的皱缩、枯黄现象;和/或
增加葡萄叶片的槲皮素含量、提高FLS酶活、Fv/Fm值、ΦPSII值、超氧化物歧化酶(Superoxide dismutase,SOD)活性、过氧化物酶(Peroxidase,POD)活性;和/或
降低葡萄叶片的相对电导率、MDA含量;
通过促进葡萄叶片中槲皮素积累、降低葡萄叶片细胞膜透性和MDA含量,提高葡萄叶片PSII反应中心的光化学效率以及抗氧化酶活性来增强其抗高温能力。
在上述技术方案基础上,所述高温为35℃以上。
在上述技术方案基础上,所述目标植物为葡萄、拟南芥或烟草。
优选的,所述目标植物为葡萄,所述葡萄为品种‘左优红’和‘赤霞珠’。
基于同一个发明创造,本发明还提供了一种提高植物抗高温性能的方法,是将葡萄VvFLS3基因的CDS序列SEQ ID NO.1构建到表达载体中形成重组表达载体,然后将重组表达载体转化菌株,获得携带有葡萄VvFLS3基因的重组菌株,然后将重组菌株通过侵染植物叶片,使植物携带有葡萄VvFLS3基因,并最终通过葡萄VvFLS3基因的表达,调控植物的抗高温性能。
优选的,所述转化菌株为农杆菌GV3101。
本发明具有如下优点:
1、本发明发现瞬时转化葡萄VvFLS3具有提高葡萄抗高温的功能,其过表达植物可以通过降低葡萄叶片细胞膜透性和MDA含量,提高葡萄叶片PSII反应中心的光化学效率以及抗氧化酶活性来增强其抗高温能力。
2、降低葡萄叶片表型的皱缩、枯黄现象;提高葡萄叶片的Fv/Fm值、ΦPSII值、SOD酶活、POD酶活;降低葡萄叶片的相对电导率、MDA含量;
3、瞬时过表达VvFLS3可以提高葡萄叶片中FLS酶的活性,通过促进黄酮醇的积累尤其是槲皮素的积累来提高葡萄叶片的抗高温能力。
4、本发明葡萄VvFLS3可应用于异源过表达,调控植物的抗高温性能。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。显而易见地,下面描述中的附图仅仅是本发明的一种实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图引伸获得其它的实施附图。
图1:实施例一,高温胁迫对不同葡萄品种叶片FLS酶活影响;
图2:实施例一,高温胁迫对不同葡萄品种VvFLSs的相对表达量影响;
图3:实施例二,A:VvFLS3 PCR扩增产物;B:pMD19-T-VvFLS3转化大肠杆菌DH5α菌落PCR验证;C:pMD19-T-VvFLS3重组质粒酶切验证;
图4:实施例二,A:pCAMBIA2301-VvFLS3表达载体质粒扩增;B:pCAMBIA2301-VvFLS3重组质粒转GV3101菌落PCR验证;C:pCAMBIA2301-VvFLS3双酶切验证;
图5:实施例二,VvFLS3蛋白三级结构预测;
图6:实施例二,VvFLS3氨基酸序列的亲水性/疏水性预测;
图7:实施例二,VvFLS3氨基酸的磷酸化位点分析;
图8:实施例二,VvFLS3蛋白的跨膜结构域分析;
图9:实施例三,VvFLS3瞬时转化对葡萄叶片耐热表型的影响;
图10:实施例三,VvFLS3瞬时转化对葡萄叶片槲皮素含量的影响;
图11-图16:实施例三,分别为VvFLS3瞬时转化对葡萄叶片Fv/Fm值、ΦPSII值、SOD酶活、POD酶活、相对电导率、MDA含量的影响;
图17:实施例四,高温胁迫对VvFLS3异源过表达拟南芥表型分析;
图18:实施例四,高温胁迫对VvFLS3异源过表达拟南芥存活率的影响;
图19-图24:实施例四,高温胁迫对VvFLS3异源过表达拟南芥Fv/Fm值、ΦPSII值、SOD酶活、POD酶活、相对电导率、MDA含量的影响。
具体实施方式
下面结合附图和实施例对本发明作进一步说明:
实施例一:高温下葡萄叶片中FLS酶活及VvFLSs表达特性分析
1、实验材料
葡萄驯化苗:生长4月龄的‘左优红’(Zuoyouhong)和‘赤霞珠’(CabernetSauvignon)葡萄驯化苗,由青岛农业大学植物逆境生理与分子生物学实验室培养驯化;
2、实验方法
(1)材料培养:以生长3月龄的葡萄组培苗为材料,将其从组培瓶中移出,用清水冲洗干净根部培养基,注意不要划伤根部,后将其移栽至含有营养土的小盆中,放置在育苗托盘中,置于温度为25℃,湿度为55%,光周期为16/8h的温室中培养,注意打开顶部透气孔通气,1月后选取长势一致驯化苗进行后续处理。
(2)材料处理方法:以生长4月龄‘左优红’和‘赤霞珠’葡萄驯化苗为材料,使用恒温恒湿培养箱对其进行高温处理(温度:45℃,相对湿度:50%-60%),分别在0h,3h,6h,9h,12h取样,进行FLS酶活、RNA的提取和表达量的检测。
(3)FLS酶活测定方法:FLS活性的检测采用PBS提取粗酶液并参考植物黄酮醇合成酶(FLS)ELISA试剂盒(上海恒远生物科技有限公司)进行检测。
(4)葡萄RNA提取:使用CTAB试剂提取葡萄驯化苗RNA。超低温冰箱-80℃保存。
(5)实时荧光定量PCR检测
表1实时荧光定量PCR引物序列
表2实时荧光定量PCR反应体系
实时荧光定量PCR反应体系为:95℃预变性5min,(95℃变性10s,56℃退火30s,72℃延伸20s)40个循环,以ACTIN作为内参,每个样品进行3个重复,按2-△△CT的方法计算相关基因的表达量。
3、实验结果与分析
实验结果如图1和图2所示,以生长4月龄‘左优红’及‘赤霞珠’葡萄驯化苗为材料,对其进行高温处理(温度:45℃,相对湿度:50%-60%),分别在处理0、3、6、9、12h后在植株的相同叶位取样,后提取FLS酶液经试剂盒检测发现高温处理后在‘左优红’和‘赤霞珠’两个葡萄品种中FLS酶活都呈现上升的趋势(图1),同时提取RNA后,对5个葡萄VvFLSs家族基因表达量进行实时荧光定量PCR检测,结果如图2显示,5个基因表达量都有不同程度的上调,且VvFLS3表达量最高,有提高葡萄叶片抗高温性能的功能。
实施例二:VvFLS3基因的克隆、载体构建及生物信息学分析
1、实验材料
菌株:大肠杆菌DH5α,农杆菌GV3101由本实验室保存。
主要试剂:Pfu-x7聚合酶,DL5000 DNA Marker,M5 Taq DNA聚合酶,M5 dNTPs(10mM),2×M5 Hiper SYBR Premix EsTaq,质粒提取、胶回收试剂盒(购自Mei5 Bio),BamHI、Pst I、限制性内切酶,T4 DNA连接酶,SolutionⅠ(购自Takara)。
2、实验方法
表3PCR引物序列
表4PCR反应体系
PCR反应程序:98℃预变性5min,(98℃变性30s,56℃退火30s,72℃延伸15s/kb)35个循环,72℃终延伸10min,16℃保存。PCR扩增产物的电泳、回收。
表5菌落PCR反应体系
PCR反应程序:98℃预变性5min,(98℃变性30s,56℃退火30s,72℃延伸15s/kb)35个循环,72℃终延伸10min,16℃保存。PCR扩增产物的电泳、摇菌,提质粒后送公司测序鉴定是否为阳性菌。
表6测序载体构建反应体系
16℃金属浴过夜连接。
表7VvFLS3的基本理化性质进行分析
3、实验结果与分析
为了进一步验证VvFLS3参与高温胁迫应答,我们对该基因进行克隆经PCR扩增得到目的基因VvFLS3,结果显示,在700bp左右处能看见一条清晰的条带,与在NCBI网站上公布的葡萄‘黑比诺’品种的序列长度基本一致(图3A)将扩增得到的VvFLS3目的片段切胶回收,与pMD19-T载体连接后转化到大肠杆菌DH5α感受态中,涂板过夜培养挑取单菌落后进行菌落PCR鉴定(图3B),将阳性克隆菌液保菌提取质粒,经BamHⅠ和PstⅠ双酶切鉴定(图3C)正确后测序,得到核苷酸序列如SEQ ID NO.1所示;所述核苷酸序列SEQ ID NO.1的编码氨基酸序列如SEQ ID NO.2所示。
实验结果如图4所示,以构建成功的pMD19-T-VvFLS3质粒为模板,经PCR扩增得到带有BamH I和Pst I酶切位点目的基因片段,将该目的基因片段胶回收后,与pCAMBIA2301空载体分别进行双酶切,连接后转化大肠杆菌DH5α,后进行菌落PCR鉴定,提取质粒后送测序。测序结果比对正确后,将pCAMBIA2301-VvFLS3重组质粒转化农杆菌GV3101后进行菌落PCR鉴定,选取阳性菌进行保菌用于后续实验。
如图5至图8所示,对VvFLS3氨基酸序列进行生物信息学分析发现,VvFLS3氨基酸多为疏水性,推测VvFLS3作为一个疏水性蛋白发挥作用;三级结构多为α螺旋和无规卷曲,磷酸化位点主要包括丝氨酸、苏氨酸和酪氨酸,其中丝氨酸位点12个,苏氨酸位点4个,酪氨酸位点7个,跨膜结构域分析发现VvFLS3无跨膜结构域,预测其可能在细胞核内发挥作用。
实施例三:VvFLS3瞬时转化葡萄叶片抗高温功能分析
1、实验材料
葡萄驯化苗:生长4月龄的‘左优红’(Zuoyouhong)和‘赤霞珠’(CabernetSauvignon)葡萄驯化苗,由青岛农业大学植物逆境生理与分子生物学实验室培养驯化;
菌株:大肠杆菌DH5α,农杆菌GV3101由本实验室保存。
2、实验方法
(1)瞬转葡萄叶片
1)农杆菌的活化:将保存于-80℃的pCAMBIA2301空载体质粒的GV3101农杆菌菌株及P19菌株于含抗生素的LB+Kana+Rif液体培养基中,28℃,250rpm恢复培养,待菌液摇起后转移少量菌液28℃、250rpm振荡二次扩大培养约10h,至OD600=0.6。
2)将扩摇的菌液5000rpm离心15min集菌,去除上清液,将沉淀以等体积转化悬浮液(配方见表1)悬浮,置于28℃、250rpm振荡培养至OD600=0.6,等体积混匀两种菌液置于28℃摇床轻摇20min备用;
表8转化悬浮液
3)将表面扎好小孔的叶片置入装有50mL混合菌液的中,抽真空10min,重复3次,直至菌液完全进入,至叶片成透明状态。
4)用灭过菌的滤纸拭去叶片上残留的菌液,将叶片培养于瓷盘中,叶柄基部置于湿润的双层脱脂棉中,叶片正面朝上,瓷盘底部始终保持湿润,用保鲜膜封住瓷盘,以保持湿度,并扎小孔透气;
5)将瓷盘置于26℃恒温培养箱中黑暗培养12h之后,转移至光照培养箱培养2d,光照培养箱条件设置为:25℃,2000Lux光照14h/黑暗10h;
6)将瞬转叶片进行高温处理,方法:45℃,4小时,设置3组重复,观察表型变化,拍照记录,取样检测相关耐热指标。
7)耐热指标的测定
细胞膜相对透性采用电导法测定;丙二醛含量(MDA)使用硫代巴比妥酸(TBA)法测定;氮蓝四唑法和愈创木酚法分别测定超氧化物歧化酶SOD、过氧化物酶POD的活性。
叶绿素荧光参数(Fv/Fm、ΦPSII)采用便携式脉冲调制荧光仪仪器测定。
3、实验结果与分析
如图9-图16所示,以生长4月龄‘左优红’及‘赤霞珠’驯化苗叶片为材料,将pCAMBIA2301空质粒和2301-35S:VvFLS3重组质粒分别转化农杆菌GV3101中,通过抽真空浸入到葡萄叶片中,45℃高温处理6h后,观察表型并取样检测相对电导率及MDA含量的变化。结果显示,高温胁迫后,2301-35S:VvFLS3重组质粒瞬时转化葡萄叶片的相对电导率及MDA含量均低于2301空对照,槲皮素含量、FLS酶活、Fv/Fm值、ΦPSII值、SOD酶活、POD酶活均高于2301空对照说明瞬时转化2301-35S:VvFLS3能够通过促进槲皮素含量积累,提高FLS酶的活性,降低葡萄叶片细胞膜透性和MDA含量,增强葡萄叶片PSII反应中心的光化学效率以及抗氧化酶活性来提高其抗高温能力。
实施例四:VvFLS3异源过表达拟南芥耐热功能分析
1、实验材料
拟南芥(Arabidopsis thaliana)为Columbia(Col-0)生态型,由本实验室保存;VvFLS3异源过表达拟南芥阳性植株,由本实验室构建获得。
2、实验方法
(1)拟南芥培养方法:挑取饱满一致的拟南芥种子,用消毒液进行消毒10min后,无菌水冲洗5遍,后加入1mL无菌水放于4℃冰箱春化3天后进行点种,生长10天后,将其移至含草炭木营养土的育苗穴盘中,置于温度为23℃,湿度为55%,光周期为16/8h的温室中培养,培养至4周龄用于成苗表型实验及耐热指标检测,培养至5-6周龄用于侵染转化。
(2)拟南芥阳性植株的筛选:采用农杆菌蘸花法进行转化获得VvFLS3异源过表达拟南芥,待收种后经氯气灭菌消毒并均匀撒种于含有卡那霉素的MS筛选培养基上,选取子叶绿色的VvFLS3阳性苗后移入土中,取莲座叶提取RNA并反转为cDNA进行验证,最终获得了异源过表达VvFLS3的拟南芥阳性植株。
(3)MS固体培养基的配制:4.4g/L,蔗糖30g/L,琼脂10g/L,调节pH=5.8-5.85。
(4)拟南芥处理方法:选取生长4周龄长势一致的野生型和过表达拟南芥为材料,对其进行高温处理(温度:45℃,相对湿度:50%-60%)10h,后置于温室中(25℃)恢复24h后,观察表型,拍照,取样,进行相关耐热指标的检测。
3、实验结果与分析
以生长4周龄OE-VvFLS3和野生型拟南芥为材料,对其进行45℃高温处理后,进行表型观察,并统计存活率。结果显示,OE株系在高温处理10h后植株长势优于野生型株系,统计存活率结果也显示高于野生型,耐热指标检测发现过表达VvFLS3能够降低拟南芥叶片的相对电导率、MDA含量,提高拟南芥叶片暗反应下的最大光化学效率(Fv/Fm)和实际光化学效率(ΦPSII)、同时也提高了超氧化物歧化酶(SOD)、以及过氧化物酶(POD)等抗氧化酶的活性,以上结果表明异源过表达VvFLS3能够提高植物的抗高温能力。
上面以举例方式对本发明进行了说明,但本发明不限于上述具体实施例,凡基于本发明所做的任何改动或变型均属于本发明要求保护的范围。
Claims (10)
1.一种葡萄VvFLS3基因,其特征在于,所述葡萄VvFLS3基因的CDS序列如SEQ ID NO.1所示。
2.如权利要求1所述的葡萄VvFLS3基因,其特征在于,所述葡萄VvFLS3基因的编码氨基酸序列如SEQ ID NO.2所示。
3.根据权利要求2所述的葡萄VvFLS3基因,其特征在于,所述葡萄VvFLS3基因的编码氨基酸可作为疏水性蛋白发挥作用,三级结构多为α螺旋和无规卷曲,磷酸化位点主要包括丝氨酸位点12个、苏氨酸位点4个和酪氨酸位点7个,无跨膜结构域。
4.根据权利要求1-3任一项所述葡萄VvFLS3基因的应用,可将葡萄VvFLS3基因制备成重组表达载体、表达盒、转基因细胞系、重组菌或重组病毒,以应用于提高植物抗高温性能。
5.葡萄VvFLS3基因在抗高温中的用途,其特征在于,所述葡萄VvFLS3基因的CDS序列如SEQ ID NO.1所示;所述葡萄VvFLS3基因在高温下,
降低葡萄叶片表型的皱缩、枯黄现象;和/或
促进葡萄叶片中槲皮素含量的积累、提高葡萄叶片的FLS酶活性、Fv/Fm值、ΦPSII值、超氧化物歧化酶活性、过氧化物酶活性;和/或
降低葡萄叶片的相对导率、MDA含量;
通过促进葡萄叶片中槲皮素含量积累、提高FLS酶的活性,降低葡萄叶片细胞膜透性和MDA含量,提高葡萄叶片PSII反应中心的光化学效率以及抗氧化酶活性来增强其抗高温能力。
6.根据权利要求5所述的葡萄VvFLS3基因在抗高温中的用途,其特征在于,所述高温为35℃以上。
7.根据权利要求5所述的葡萄VvFLS3基因在抗高温中的用途,其特征在于,所述目标植物为葡萄、拟南芥或烟草。
8.根据权利要求7所述的葡萄VvFLS3基因在抗高温中的用途,其特征在于,所述目标植物为葡萄,所述葡萄为品种‘左优红’和‘赤霞珠’。
9.一种提高植物抗高温性能的方法,其特征在于,是将权利要求1中葡萄VvFLS3基因的CDS序列SEQ ID NO.1构建到表达载体中形成重组表达载体,然后将重组表达载体转化菌株,获得携带有葡萄VvFLS3基因的重组菌株,然后将重组菌株侵染植物叶片,使植物携带有葡萄VvFLS3基因,并最终通过葡萄VvFLS3基因的表达,调控植物的抗高温性能。
10.根据权利要求9所述的一种提高植物抗高温性能的方法,其特征在于,所述转化菌株为农杆菌GV3101。
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