CN110079537A - 葡萄细胞分裂素响应调节因子VvRR基因及其编码蛋白和应用 - Google Patents
葡萄细胞分裂素响应调节因子VvRR基因及其编码蛋白和应用 Download PDFInfo
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Abstract
本发明涉及葡萄细胞分裂素响应调节因子VvRR基因及其编码蛋白和应用，属于植物基因工程技术领域。本发明利用强启动子驱动原理的转基因技术，将VvRR基因的超量表达载体转入番茄中，从而获得转基因番茄植株。实验证明，相对于野生型番茄植株，超量表达VvRR基因的转基因番茄植株的果实坐果率显著提高。说明VvRR基因与植物果实坐果率有密切的联系，因此VvRR基因可以用于提高植物的果实坐果率。
Description
技术领域
本发明涉及葡萄细胞分裂素响应调节因子VvRR基因及其编码蛋白和应用，属于植物基因工程技术领域。
背景技术
葡萄和葡萄酒中富含有益于人体的矿物质、维生素和多种必需氨基酸等物质。其中，白藜芦醇具有防癌抗炎、延缓衰老和提高免疫力等功效而引起了全世界的关注，也促进了世界葡萄产业的发展。美洲葡萄或欧美葡萄杂交种的果实品质优良且抗逆性强，在鲜食葡萄产业中占据主导地位。但是，美洲葡萄或欧美杂交种葡萄表现出坐果率低的特点，在生产过程中需要大量使用植物生长调节剂来促进葡萄果实坐果。植物生长调节剂的使用一方面增加了果农的生产成本和劳动时间，同时也降低了葡萄果实品质，且增加了环境污染的风险。因此，通过生物技术培育坐果率高、品质优良且抗逆性强的转基因葡萄对于生产实践具有重要意义。
果实坐果是指胚珠受精后，子房发育成为幼小果实的关键过程，包括盛花后的细胞分裂和细胞延伸。该过程直接决定果实后期的生长和发育，主要包括果实的数量和大小、种子发育状态，最终决定果实产量和品质。葡萄主要通过子房特异细胞的分裂来起始幼果生长，一般在开始开花到盛花后5d；随着细胞的分裂膨大，子房转变为幼果，坐果过程发生在盛花后6-12d。大部分葡萄在盛花后9-10d落花严重，为第一个落果高峰期。美洲或欧美杂交种葡萄在这个时期落果更为严重，如巨峰等。葡萄生产中主要在盛花后5d左右使用细胞分裂素或配合赤霉素来促进葡萄坐果。果实坐果可依赖于、也可不依赖于授粉受精，其中后者也称为专性单性结实。外源激素处理在这两种坐果类型中都具有重要的调节作用。
细胞分裂素、赤霉素和生长素是参与果实坐果与幼果发育的主要激素，其中赤霉素和生长素调控果实坐果与幼果发育的分子机制的研究取得了一定的进展。GA3ox和GA20ox家族的大部分基因在果实坐果和幼果发育过程中表达量快速增加，基因定位表明这些上调表达基因主要定位在授粉后的子房中。GID1s(GA Insensitive Dwarf1s)是赤霉素受体基因，其在拟南芥果荚坐果与幼果发育中起重要作用。GID1A主要在拟南芥的雌蕊中表达，GID1B和GID1C分别在胚珠和子房壁中表达。GID1s突变后影响果实坐果和幼果发育，gid1a果实坐果率显著降低，gid1b和gid1c果实大小发生改变、种子发育不正常。图位克隆发现fwf位点包含一个ARF8基因，该基因导致不能启动果实和种子发育信号的产生，作为一个抑制子阻止心皮的发育，进而降低果实的坐果率。细胞分裂素在葡萄生产中的应用非常广泛，可以诱导花序由雄性转变为雌性、诱导卷须转变为其同源器官花序、促进果实坐果、果粒膨大和单性结实、延迟果实成熟和增加果实硬度等，其中以促进葡萄坐果的应用最具有代表性。到目前为止，细胞分裂素促进葡萄果实坐果的分子机制或关键基因还不清楚。
发明内容
本发明的目的是提供葡萄细胞分裂素响应调节因子VvRR基因，该基因能够提高植物果实的坐果率。
本发明还提供了葡萄细胞分裂素响应调节因子VvRR，该蛋白能够提高植物果实的坐果率。
本发明还提供了包含葡萄细胞分裂素响应调节因子VvRR基因的重组表达载体，该载体携带葡萄细胞分裂素响应调节因子VvRR基因，因此能够超表达VvRR基因，进而能够提高植物果实的坐果率。
本发明还提供了上述的包含葡萄细胞分裂素响应调节因子VvRR基因的重组表达载体的制备方法，能够制得该载体。
本发明还提供了上述的葡萄细胞分裂素响应调节因子VvRR基因和重组表达载体在植物品种育种中的应用，能够获得坐果率高的植物品种。
为了实现上述目的，本发明所采用的技术方案是：
葡萄细胞分裂素响应调节因子VvRR基因，其编码的氨基酸序列如SEQ ID NO.2所示。
本发明中利用强启动子(花椰菜花叶病毒35S启动子)驱动原理的转基因技术，将VvRR基因的超量表达载体转入番茄中，从而获得转基因番茄植株。实验证明，相对于野生型番茄植株，超量表达VvRR基因的转基因番茄植株的果实坐果率显著提高。说明VvRR基因与植物果实坐果率有密切的联系。
优选的，葡萄细胞分裂素响应调节因子VvRR基因的核苷酸序列如SEQ ID NO.1所示。
上述的核苷酸序列为葡萄中天然存在的序列，也可以根据该序列进行密码子优化，得到的优化序列也具有同样的效果。
葡萄细胞分裂素响应调节因子VvRR，其氨基酸序列如SEQ ID NO.2所示。
葡萄细胞分裂素响应调节因子VvRR是一个含375个氨基酸的蛋白，该蛋白能够提高植物果实的坐果率。
重组表达载体，所述重组表达载体包含葡萄细胞分裂素响应调节因子VvRR基因，所述葡萄细胞分裂素响应调节因子VvRR基因的核苷酸序列如SEQ ID NO.1所示。
本发明中的重组表达载体为植物过量表达载体，能够在植物中超表达目的基因。
重组表达载体的制备方法，包括：根据如SEQ ID NO.1所示的序列设计引物，克隆所述葡萄细胞分裂素响应调节因子VvRR基因，然后将所述葡萄细胞分裂素响应调节因子VvRR基因连接到pCAMBIA2300植物表达载体上，即得。
本发明中将VvRR基因开放阅读框连接至植物过量表达载体pCAMBIA2300上，能够形成重组表达载体pCAMBIA2300-VvRR。
上述的葡萄细胞分裂素响应调节因子VvRR基因在植物品种育种中的应用；具体的，在提高植物果实坐果率育种中的应用；更为具体的，在提高番茄果实坐果率育种中的应用。上述的的重组表达载体在植物品种育种中的应用；具体的，在提高植物果实坐果率育种中的应用；更为具体的，在提高番茄果实坐果率育种中的应用。
本发明中通过植物基因工程技术，从‘京秀’葡萄幼果中分离克隆出与果实坐果相关基因完整编码区段的DNA片段，并验证了VvRR基因的功能，发现超量表达之后转基因番茄果实坐果率显著提高。因此该基因可以用于提高植物的果实坐果率。
附图说明
图1为本发明中VvRR基因的5'-RACE扩增结果图；
图2为本发明中VvRR基因的全长扩增结果图；
图3为本发明中VvRR基因的ORF扩增结果图；
图4为本发明中农杆菌介导的子叶转化方法获得抗性番茄植株的过程图；
图5为本发明中转基因检测引物设计位点展示图；
图6为本发明中PCR检测转基因番茄植株结果图；
图7为本发明中野生型和转基因番茄植株的坐果率对比图；
图8为本发明中野生型与转基因番茄植株的坐果率统计分析图。
具体实施方式
下面结合具体实施例对本发明做进一步的详细说明。除特殊说明的之外，各实施例及试验例中所用的设备和试剂均可从商业途径得到。
葡萄细胞分裂素响应调节因子VvRR基因的实施例1
本实施例中葡萄细胞分裂素响应调节因子VvRR基因，其核苷酸序列如SEQ IDNO.1所示。
本实施例中葡萄细胞分裂素响应调节因子VvRR基因的克隆，包括如下步骤：
(1)5’RACE引物的设计：根据转录组测序获得的部分序列设计5’RACE引物VvRR5’RACE-R，其中：
VvNAC5’RACE-R：5'-TTCTTCTGCTCCTTCTTCTAAGC-3'(如SEQ ID NO.3所示)。
(2)提取‘京秀’葡萄坐果前后幼果中的总RNA，按照TaKaRa公司 RACE5’Kit的说明书进行反转录；
(3)5’RACE技术克隆VvRR基因全长：按照TaKaRa公司 RACE 5’Kit的说明书进行PCR反应，其中正向引物为UPM，反向引物为VvRR5’RACE-R，反应体系和反应程序按照试剂盒说明书进行。其中，UPM的序列为：
5'-TAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3'(如SEQ ID NO.4所示)。
(4)PCR产物进行回收、载体连接、转化、测序，得到VvRR基因的开放阅读框(openreading frame,ORF)为1128bp，编码375个氨基酸。
更为具体的，葡萄细胞分裂素响应调节因子VvRR基因的克隆包括：
1、葡萄幼果总RNA的提取与纯化
按照SDS/酚法进行RNA的提取及纯化。具体操作如下：
(1)取0.2g幼果置于研钵中，在液氮中充分研磨后，加入装有800μL提取缓冲液[140mM LiCl，10mM EDTA，10mM Tris，5％(w/v)SDS，2％(w/v)PVP]的2mL离心管中涡旋混匀；
(2)加入等体积的氯仿-异戊醇(24∶1)，涡旋混匀，4℃，12000rpm离心15min；
(3)转移上清至一新的2mL离心管中，加入1/3体积5M，pH4.8的KAc，涡旋混匀，4℃，12000rpm离心10min；
(4)转移上清至一新的2mL离心管中，加入等体积氯仿-异戊醇(24∶1)，涡旋混匀，4℃，12000rpm离心10min；
(5)转移上清至一新的2mL离心管中，加入1/3体积8M LiCl，-20℃放置1h以上，4℃，12000rpm离心15min；
(6)弃上清，用75％乙醇洗涤沉淀两次；
(7)倾去75％乙醇，待乙醇挥发干净后，加入30μL DEPC-H2O溶解RNA；
(8)按下述体系于1.5mL离心管中加入各组分：
表1
(9)37℃温育30min；
(10)加入等体积的酚-氯仿-异戊醇(25∶24∶1)，涡旋混匀，4℃，12000rpm离心10min；
(11)转移上清至一新的1.5mL离心管中，加入等体积氯仿-异戊醇(24∶1)，4℃，12000rpm离心10min；
(12)转移上清至一新的1.5mL离心管中，加入1/10体积3M NaAC(pH5.2)，2.5倍体积无水乙醇，-80℃过夜；
(13)4℃，12000rpm离心15min后；弃上清，75％乙醇洗涤两次，室温干燥；
(14)沉淀溶于20μL DEPC-H2O中，-80℃冻存。
2、葡萄细胞分裂素响应调节因子VvRR基因的5'-RACE扩增
(1)First-Strand cDNA的合成
a)在一0.2mL PCR薄壁管中加入如下试剂：
表2
b)混匀并离心后，于70℃温育2min，然后于冰上冷却2min；
c)在上述反应体系中加入如下物质：
表3
d)混匀后在PCR仪上于42℃反应1.5h；
e)将反应产物用100μL TE[10mM Tris-Cl(pH8.0)，1mM EDTA(pH8.0)]溶液稀释；
f)将稀释后的反应产物在70℃加热7min；
g)最终的反应产物于-20℃保存备用。
(2)反转录产物的PCR扩增
a)在一0.2mL PCR薄壁管中加入如下试剂：
表4
b)将上述试剂混匀离心后，加2滴矿物油于表面，置于PCR仪上反应，PCR程序为：5cycles：94℃30s，72℃3min→5cycles：94℃30s，70℃30s，72℃，3min→25cycles：94℃30s，68℃30s，72℃3min；
c)将PCR产物进行琼脂糖凝胶电泳，结果如图1所示，其中M为Maker，1-2号泳道为目的条带。将目的条带所在的胶块切下后，用凝胶回收试剂盒回收目的条带，然后将回收产物与pGEM-T easy载体连接并倒入DH5α感受态细胞中进行克隆，通过蓝白斑筛选后选择阳性克隆进行测序。
3、葡萄细胞分裂素响应调节因子VvRR基因全长的扩增
反转录按照TaKaRa PrimeScript 1st Strand cDNA Synthesis Kit说明书进行。具体操作步骤如下：在PCR管中加入：Random 6mers(50μM)1μL，dNTP Mixture(10mM each)1μL，Total RNA2μg，RNase free dH2O补齐至10μL，充分混匀，瞬时离心使溶液至PCR管底部。在PCR仪上65℃反应5min，冰上急冷。在PCR管中加入： Buffer 4μL，RNaseInhibitor(40U/μL)0.5μL， RTase(200U/μL)1μL，RNase Free dH2O补齐至20μL。在PCR仪上进行下述反应：30℃，10min；42℃，60min；95℃，5min；4℃，保存。
反转录产物的PCR扩增。cDNA模板1μL，全长正向引物2μL，全长反向引物2μL，PCRBuffer 5μL，dNTP Mix 2.5μL，DNA Polymerase 1.0μL，PCR-Grade Water补齐至50μL。PCR反应程序为：94℃30sec；94℃30sec，56℃30sec，72℃3min，30cycles；72℃10min；4℃Forever。
全长正向引物：5'-CCAGAGATGCAGAACGGGAT-3'(如SEQ ID NO.5所示)；
全长反向引物：5'-GCCGCAATACGTAGCGATAGC-3'(如SEQ ID NO.6所示)。
PCR产物经1.2％琼脂糖凝胶电泳，结果如图2所示，其中M为Maker，3-4号泳道为目的条带。将目的条带所在的胶块切下后，用凝胶回收试剂盒回收目的条带，然后将回收产物与pGEM-T easy载体连接并倒入DH5α感受态细胞中进行克隆，通过蓝白斑筛选后选择阳性克隆进行测序，获得pGEM-T easy-VvRR质粒。葡萄果实成熟相关基因VvRR的全长序列如SEQID NO.7所示，全长为1437个核苷酸；通过分析发现该基因的编码区共1128bp，如SEQ IDNO.1所示，为ORF片段。
葡萄细胞分裂素响应调节因子VvRR的实施例1
本实施例中葡萄细胞分裂素响应调节因子VvRR，其氨基酸序列如SEQ ID NO.2所示。
重组表达载体的实施例1
本实施例中重组表达载体包含葡萄细胞分裂素响应调节因子VvRR基因，所述葡萄细胞分裂素响应调节因子VvRR基因的核苷酸序列如SEQ ID NO.1所示。
重组表达载体的制备方法的实施例1
葡萄细胞分裂素响应调节因子VvRR基因过量表达载体的构建
为研究葡萄细胞分裂素响应调节因子VvRR基因的功能，将包含有VvRR基因编码区在内的共1128bp的ORF片段正确插入植物过量表达载体pCAMBIA2300上。
根据葡萄细胞分裂素响应调节因子VvRR基因的实施例1中克隆到的VvRR基因ORF序列，设计可以扩增VvRR基因ORF的上下游引物VvRR-ORF-F和VvRR-ORF-R：
VvRR-ORF-F：5'-CACATGGCCATGAAGGGCTAC-3'(如SEQ ID NO.8所示)；
VvRR-ORF-R：5'-TCAACCAGTCAATGTCTCGTCAG-3'(如SEQ ID NO.9所示)。
根据pCAMBIA2300载体上的酶切位点，在引物VvRR-ORF-F的5’端加上酶切位点XbaI，在引物VvRR-ORF-R的5’端加上酶切位点KpnI，具体序列如下所示：
VvRR-ORF-XbaI-F：5'-GGGTCTAGACACATGGCCATGAAGGGCTAC-3'(如SEQ ID NO.10所示)；
VvRR-ORF-KpnI-R：5'-GGGAAGCTTTCAACCAGTCAATGTCTCGTCAG-3'(如SEQ ID NO.11所示).
以pGEM-T easy-VvRR质粒为模板，用VvRR-ORF-XbaI-F与VvRR-ORF-KpnI-R进行扩增，结果如图3所示，其中M为Maker，5-6号泳道为目的条带。回收目的条带后连接到pMD19-T克隆载体，转化TOP10感受态细胞，在附加Amp的LB培养基上进行蓝白斑筛选，分别经过菌液PCR与质粒酶切检测，pMD19-T-VvRR阳性克隆送公司测序。用KpnI、XhoI双酶切重组克隆载体pMD19-T-VvRR与植物表达载体pCAMBIA2300，回收线性化载体与目标片段，连接并转化TOP10，经Kan抗生素筛选，挑取单克隆摇菌，菌液检测后提质粒酶切检测，形成植物表达载体pCAMBIA2300-VvRR。
葡萄细胞分裂素响应调节因子VvRR基因及重组表达载体的应用的实施例1
1、抗性番茄植株的获得
将植物表达载体pCAMBIA2300-VvRR转化入农杆菌中。将含有目的基因(pCAMBIA2300-VvRR载体)的农杆菌菌液于超低温冰箱取出后，在冰上溶化，取200μL接种于液体LB培养基中(含60mg·L-1Kan和60mg·L-1Gent)，28℃180rpm培养20h后取30μL该菌液接种于20mL液体LB培养基中，相同条件下，进行二次活化，培养20h左右至菌液浑浊。将菌液转移到灭菌的50mL离心管中，6000rpm 8min，去上清，再用液体MS培养基(含200μM AS和3％的蔗糖)重悬菌液，28℃、180rpm培养3-4h，在紫外可见分光光度计上检测菌液浓度，可用重悬液稀释，使菌液浓度达到试验所确定的最佳浓度(OD600＝0.1-0.2)，备用。
以Micro-Tom番茄为材料，选取饱满的种子，用70％的乙醇消毒10s，再用有效氯1％的次氯酸钠溶液消毒15min，用无菌水冲洗干净，置于MS培养基中，光照培养至种子萌发长出子叶。将无菌番茄子叶切去叶尖和基端，切成0.5cm2的小块，近轴面向下置于MS培养中预培养3d，用准备好的菌液侵染10min后，用无菌滤纸吸干表面菌液，接种于共培养培养基(MS+IAA0.2mg·L-1+ZT 2.0mg·L-1+AS 200μM)，黑暗培养48h后脱菌，用浓度600mg·L-1的头孢霉素溶液、羧苄青霉素溶液各清洗2遍，在用无菌水冲洗3次，接种于分化培养基(MS+IAA0.2mg·L-1+ZT 2.0g·L-1+Carb 400mg·L-1)中10d后转至筛选培养基(MS+IAA0.2mg·L-1+ZT 2.0mg·L-1+Carb 400mg·L-1+Kan 20mg·L-1)，每20d继代一次，至长出抗性芽，抗性芽接入生根培养基(MS+IAA0.2mg·L-1+Carb 200mg·L-1)中，发育成完整植株(抗性番茄植株)，移栽至温室中(如图4所示，其中A为萌发7d后的番茄无菌苗；B为农杆菌侵染后的子叶形成愈伤组织；C为愈伤组织再生形成幼苗；D为转基因幼苗生根培养)。
2、转基因番茄植株的检测
用无液氮DNA快速提取法提取抗性番茄叶片DNA，作为模板，设计特异引物一对。
其序列如下所示：
转基因检测-F：5'-CCTAACAGAACTCGCCGTAAAG-3'(如SEQ ID NO.12所示)；
转基因检测-R：5'-GCCGGTGGTGCAGATGAAC-3'(如SEQ ID NO.13所示)。该引物为根据pCAMBIA2300载体上报告基因GFP和CaMV 35S启动子的序列来设计的，通过检测选载体和目的基因是否存在来说明转基因是否成功(如图5所示)。
检测体系：模板：2μL；正向引物1μL；反向引物1μL；rTaq酶0.25μL；dNTP2μL；Buffer2.5μL；水16.25μL。PCR扩增程序：94℃，3min；94℃，30s；57℃，60s；72℃，2min，29个循环；72℃，5min；4℃保存。PCR产物进行0.8％琼脂糖凝胶电泳，以DNA Mark DL 2000为标样，检测PCR产物。分别以pCAMBIA2300-VvRR质粒DNA和未转化的植株DNA PCR产物为阳性和阴性对照。检测结果如图6所示，M为Maker，N为阴性对照，P为阳性对照，其中3、7、8三个株系含有载体和目的基因的电泳条带，为阳性转基因植株，其他1、2、4、5、6五个株系只含有载体(空载体)的电泳条带，没有目的基因的电泳条带，为假阳性植株。
3、番茄植株的果实坐果率统计
花后35d，观察野生型番茄植株和转基因番茄植株的坐果情况，结果如图7所示，A为野生型番茄植株，B为转基因番茄植株；可以明显看出，转基因植株的的果实较多。统计野生型番茄植株和转基因番茄植株的坐果率，坐果率统计方法：坐果率＝坐果数/开花总数×100％。统计结果如图8所示，可以明显看出与野生型番茄植株相比，转基因型番茄植株#3、#7、#8的坐果率大大提高。
<110> 河南科技大学
<120> 葡萄细胞分裂素响应调节因子VvRR基因及其编码蛋白和应用
<160> 13
<170> SIPOSequenceListing 1.0
<211> 1128
<212> DNA
<213> 葡萄
<221> VvRR基因
<400> 1
atggccatga agggctactt tttcgtccgg gcggccgtct attcgcggcg cgctatcagc 60
gcaaagtacg gcgaagcagt ggaccatgag gcgtggctaa agccggtcat gatccggggt 120
atgtatgtat tccgatatcc tattgaatca cggagcggat accctagccg acaggtcact 180
atcgcttctt cacagcagtc agaatatgtg ctagctgttg atgacagcct tatcgataga 240
aaattgattg agaagctcct caagaactca tcctatcaag taactacagt tgattctggt 300
agcaaggccc ttgaatttct gggtttgcat gaaaatgacc caaacacacc ttccgtttct 360
ccaaacagtc atcaggaagt ggaggtgaat cttataatta ctgactactg tatgcctgga 420
atgacaggct atgatttact caaaaaaatc aaggaatctt catctttgag aaatatacca 480
gtagtgatca tgtcatctga gaatgtgcct tcaaggatca ccagatgctt agaagaagga 540
gcagaagaat tttttctgaa accagttcag atatcagatg tgaatcggct taaacctcat 600
atgatgggat ggcgcactaa aatattggct aaagtgcaaa tagcctggaa gccgggcatg 660
cccaatgcga tgggctctat gccacatact gccagccggg agctcctgca ttatctaatt 720
gacggaatat gcgtttgtct actctggttg cgaacctcaa tagggatcga ttacgggcat 780
ggtattttac atgagacggt atacgtgaga ggtctcccgt tggaggggca ggccgccgtc 840
aaagatctaa ttcgcgtgcc ggagttaaag gagaaggtga tacccgaaat gcccctaagc 900
ctatcgacgg gcctcccggg gacatactcc aaagagattc tcgcgacgtg gccgactggt 960
ccgccggctc ttcgcggcga ggtaaaaaga caagagtatg cagtcaggta ccggggactg 1020
caggaaccgt ggctcccgac ctgttatgcc aactatgcat accagaagga cgacatgacg 1080
aggctaatcc gtctgataca agaccctgac gagacattga ctggttga 1128
<211> 375
<212> PRT
<213> 葡萄
<221> VvRR蛋白
<400> 2
MET Ala MET Lys Gly Tyr Phe Phe Val Arg Ala Ala Val Tyr Ser
1 5 10 15
Arg Arg Ala Ile Ser Ala Lys Tyr Gly Glu Ala Val Asp His Glu
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Ala Trp Leu Lys Pro Val MET Ile Arg Gly MET Tyr Val Phe Arg
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Tyr Pro Ile Glu Ser Arg Ser Gly Tyr Pro Ser Arg Gln Val Thr
50 55 60
Ile Ala Ser Ser Gln Gln Ser Glu Tyr Val Leu Ala Val Asp Asp
65 70 75
Ser Leu Ile Asp Arg Lys Leu Ile Glu Lys Leu Leu Lys Asn Ser
80 85 90
Ser Tyr Gln Val Thr Thr Val Asp Ser Gly Ser Lys Ala Leu Glu
95 100 105
Phe Leu Gly Leu His Glu Asn Asp Pro Asn Thr Pro Ser Val Ser
110 115 120
Pro Asn Ser His Gln Glu Val Glu Val Asn Leu Ile Ile Thr Asp
125 130 135
Tyr Cys MET Pro Gly MET Thr Gly Tyr Asp Leu Leu Lys Lys Ile
140 145 150
Lys Glu Ser Ser Ser Leu Arg Asn Ile Pro Val Val Ile MET Ser
155 160 165
Ser Glu Asn Val Pro Ser Arg Ile Thr Arg Cys Leu Glu Glu Gly
170 175 180
Ala Glu Glu Phe Phe Leu Lys Pro Val Gln Ile Ser Asp Val Asn
185 190 195
Arg Leu Lys Pro His MET MET Gly Trp Arg Thr Lys Ile Leu Ala
200 205 210
Lys Val Gln Ile Ala Trp Lys Pro Gly MET Pro Asn Ala MET Gly
215 220 225
Ser MET Pro His Thr Ala Ser Arg Glu Leu Leu His Tyr Leu Ile
230 235 240
Asp Gly Ile Cys Val Cys Leu Leu Trp Leu Arg Thr Ser Ile Gly
245 250 255
Ile Asp Tyr Gly His Gly Ile Leu His Glu Thr Val Tyr Val Arg
260 265 270
Gly Leu Pro Leu Glu Gly Gln Ala Ala Val Lys Asp Leu Ile Arg
275 280 285
Val Pro Glu Leu Lys Glu Lys Val Ile Pro Glu MET Pro Leu Ser
290 295 300
Leu Ser Thr Gly Leu Pro Gly Thr Tyr Ser Lys Glu Ile Leu Ala
305 310 315
Thr Trp Pro Thr Gly Pro Pro Ala Leu Arg Gly Glu Val Lys Arg
320 325 330
Gln Glu Tyr Ala Val Arg Tyr Arg Gly Leu Gln Glu Pro Trp Leu
335 340 345
Pro Thr Cys Tyr Ala Asn Tyr Ala Tyr Gln Lys Asp Asp MET Thr
350 355 360
Arg Leu Ile Arg Leu Ile Gln Asp Pro Asp Glu Thr Leu Thr Gly
365 370 375
<211> 23
<212> DNA
<213> 人工序列
<221> VvNAC5’RACE-R
<400> 3
ttcttctgct ccttcttcta agc 23
<211> 44
<212> DNA
<213> 人工序列
<221> UPM
<400> 4
taatacgact cactataggg caagcagtgg tatcaacgca gagt 44
<211> 20
<212> DNA
<213> 人工序列
<221> 全长正向引物
<400> 5
ccagagatgc agaacgggat 20
<211> 21
<212> DNA
<213> 人工序列
<221> 全长反向引物
<400> 6
gccgcaatac gtagcgatag c 21
<211> 1437
<212> DNA
<213> 葡萄
<221> VvRR基因的全长序列
<400> 7
ccagagatgc agaacgggat cggacccgga ctacggcatc cctcgcactg atacctcccc 60
gcgcctgtcg gcaagcggtg tcacatacga tgccgtacgg aagcagatat atcgatttac 120
atctgcacta cgcgtccatt tctcaatgac cacatggcca tgaagggcta ctttttcgtc 180
cgggcggccg tctattcgcg gcgcgctatc agcgcaaagt acggcgaagc agtggaccat 240
gaggcgtggc taaagccggt catgatccgg ggtatgtatg tattccgata tcctattgaa 300
tcacggagcg gataccctag ccgacaggtc actatcgctt cttcacagca gtcagaatat 360
gtgctagctg ttgatgacag ccttatcgat agaaaattga ttgagaagct cctcaagaac 420
tcatcctatc aagtaactac agttgattct ggtagcaagg cccttgaatt tctgggtttg 480
catgaaaatg acccaaacac accttccgtt tctccaaaca gtcatcagga agtggaggtg 540
aatcttataa ttactgacta ctgtatgcct ggaatgacag gctatgattt actcaaaaaa 600
atcaaggaat cttcatcttt gagaaatata ccagtagtga tcatgtcatc tgagaatgtg 660
ccttcaagga tcaccagatg cttagaagaa ggagcagaag aattttttct gaaaccagtt 720
cagatatcag atgtgaatcg gcttaaacct catatgatgg gatggcgcac taaaatattg 780
gctaaagtgc aaatagcctg gaagccgggc atgcccaatg cgatgggctc tatgccacat 840
actgccagcc gggagctcct gcattatcta attgacggaa tatgcgtttg tctactctgg 900
ttgcgaacct caatagggat cgattacggg catggtattt tacatgagac ggtatacgtg 960
agaggtctcc cgttggaggg gcaggccgcc gtcaaagatc taattcgcgt gccggagtta 1020
aaggagaagg tgatacccga aatgccccta agcctatcga cgggcctccc ggggacatac 1080
tccaaagaga ttctcgcgac gtggccgact ggtccgccgg ctcttcgcgg cgaggtaaaa 1140
agacaagagt atgcagtcag gtaccgggga ctgcaggaac cgtggctccc gacctgttat 1200
gccaactatg cataccagaa ggacgacatg acgaggctaa tccgtctgat acaagaccct 1260
gacgagacat tgactggttg aagccatgcc tcctataact tccgtaaggg ttaatggaac 1320
gctacagccg gcgtgcaaaa gacaaggtat aagaccgttg ctcttagcgt ctattgacgt 1380
aactatttcg ggtcacttac gcgagacctt ggcgcagcta tcgctacgta ttgcggc 1437
<211> 21
<212> DNA
<213> 人工序列
<221> VvRR-ORF-F
<400> 8
cacatggcca tgaagggcta c 21
<211> 23
<212> DNA
<213> 人工序列
<221> VvRR-ORF-R
<400> 9
tcaaccagtc aatgtctcgt cag 23
<211> 30
<212> DNA
<213> 人工序列
<221> VvRR-ORF-XbaI-F
<400> 10
gggtctagac acatggccat gaagggctac 30
<211> 32
<212> DNA
<213> 人工序列
<221> VvRR-ORF-KpnI-R
<400> 11
gggaagcttt caaccagtca atgtctcgtc ag 32
<211> 22
<212> DNA
<213> 人工序列
<221> 转基因检测-F
<400> 12
cctaacagaa ctcgccgtaa ag 22
<211> 19
<212> DNA
<213> 人工序列
<221> 转基因检测-R
<400> 13
gccggtggtg cagatgaac 19
Claims (8)
1.葡萄细胞分裂素响应调节因子VvRR基因，其特征在于：其编码的氨基酸序列如SEQID NO.2所示。
2.根据权利要求1所述的葡萄细胞分裂素响应调节因子VvRR基因，其特征在于：其核苷酸序列如SEQ ID NO.1所示。
3.葡萄细胞分裂素响应调节因子VvRR，其特征在于：其氨基酸序列如SEQ ID NO.2所示。
4.重组表达载体，其特征在于：所述重组表达载体包含葡萄细胞分裂素响应调节因子VvRR基因，所述葡萄细胞分裂素响应调节因子VvRR基因的核苷酸序列如SEQ ID NO.1所示。
5.如权利要求4所述的重组表达载体的制备方法，其特征在于：包括：根据如SEQ IDNO.1所示的序列设计引物，克隆所述葡萄细胞分裂素响应调节因子VvRR基因，然后将所述葡萄细胞分裂素响应调节因子VvRR基因连接到pCAMBIA2300植物表达载体上，即得。
6.如权利要求1所述的葡萄细胞分裂素响应调节因子VvRR基因或者权利要求4所述的重组表达载体在植物品种育种中的应用。
7.根据权利要求6所述的葡萄细胞分裂素响应调节因子VvRR基因或者重组表达载体的应用，其特征在于：在提高植物果实坐果率育种中的应用。
8.根据权利要求7所述的葡萄细胞分裂素响应调节因子VvRR基因或者重组表达载体的应用，其特征在于：在提高番茄果实坐果率育种中的应用。
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| CN110862992A (zh) * | 2019-11-04 | 2020-03-06 | 河南科技大学 | 野葡萄锚定重复蛋白VdANK1的编码基因与应用 |
| CN116769793A (zh) * | 2023-07-18 | 2023-09-19 | 西北农林科技大学 | 葡萄细胞分裂素响应调节因子基因VvRR1及其应用 |
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| CN110452917A (zh) * | 2019-09-16 | 2019-11-15 | 河南科技大学 | 野葡萄VyGOLS基因及其编码蛋白在干旱胁迫中的应用 |
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| CN116769793A (zh) * | 2023-07-18 | 2023-09-19 | 西北农林科技大学 | 葡萄细胞分裂素响应调节因子基因VvRR1及其应用 |
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