CN109679956A - 基于BnaCnng52950D基因的启动子序列、重组载体及应用 - Google Patents
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Abstract
本发明属于基因工程及分子生物学领域,具体涉及一种甘蓝型油菜BnaCnng52950D基因的启动子序列,及其所构建的重组载体及应用。本发明通过生物信息学软件预测了该序列所包含的启动子元件,并通过构建植物表达载体对其启动子活性进行定性检测,结果表明所克隆序列可驱动GUS报告基因在角果柄基部和叶片中表达。该启动子的获得,为油菜转基因研究提供有力工具,并给油菜和它的近缘物种的研究奠定一定的基础。
Description
技术领域
本发明属于基因工程及分子生物学领域,具体涉及一种甘蓝型油菜BnaCnng52950D基因的启动子序列,及其所构建的重组载体与转基因植株。
背景技术
甘蓝型油菜作为世界四大油料作物之一,是世界上唯一的冬季油料作物,也是我国主要的油料、饲料和能源作物,常年播种面积和总产量居世界首位。但油菜现在仍面临产量和含油量较低、抗性较弱且不适宜机械化操作等问题。
启动子位于基因上游5'端,在基因表达调控中起着至关重要的作用,可被RNA聚合酶识别并与其结合,并准确起始转录一段特异DNA序列。转录水平的调控是基因表达调控方式中最经济有效也是最常用的调节方式。启动子作为重要的顺势作用元件不但决定着转录的方向、效率和所使用的RNA聚合酶的类型,而且通过与转录因子结合,还精确的控制基因表达的时间、空间以及强度。因此利用植物基因工程技术有望解决人类面临的能源、环境和粮食危机等问题。
启动子又分为上游启动子与核心启动子2个区域。核心启动子区域包含了一些非常重要的功能元件,如TATA-box、起始因子Inr、转录因子TFIIB识别位点BRE及下游启动子元件DPE等,能准确定位转录起点和方向,是基因调控的重要基础。上游启动子区包含了与下游基因表达调控相关的调控元件,例如GC-box、CAAT-box等。同时,根据启动子的作用方式和功能可以分为3类:组成型启动子,组织特异性启动子和诱导型启动子。由于组成型启动子在不用时间和不同组织中都能驱动下游基因的表达,浪费能量并且易影响植株正常的生理代谢,产生一些不良的多效性反应,因此有效利用特异表达的启动子,是解决转基因作物需求的关键。
综上所述,本发明的启示在于通过基因工程技术分离出具有较高应用价值的启动子,可控制基因,实现对油菜角果柄发育的影响,提高油菜光合效率,对有效解决我国植物油问题具有重要意义,并给油菜和它的近缘物种的研究奠定了一定的基础。
发明内容
有鉴于此,本发明的目的在于提供一个来源于甘蓝型油菜BnaCnng52950D基因的启动子序列,及其所构建的重组载体及应用。
为实现上诉目的,本发明的技术方案为:
一段基于BnaCnng52950D基因启动子的核苷酸片段,该片段由BnaCnng52950D基因启动子克隆而来,核苷酸序列如SEQ ID NO:1所示。
一种上述序列SEQ ID NO:1的核苷酸片段的克隆方法,具体步骤如下:
1)根据甘蓝型油菜基因组序列设计其上游1500bp启动子特异性扩增引物一对,序列为SEQ ID NO:2和SEQ ID NO:3。
2)以甘蓝型油菜ZS11的基因组DNA为模板,使用美国NEB公司的Q5高保真酶扩增进行PCR扩增。扩增程序为98℃预变性30s;98℃变性10s,56℃退火20s,72℃延伸1min,35个循环;最后72℃保温2min。
3)扩增引物中加入pENTR/D-TOPO载体,混匀后室温放置15min后全部转化大肠杆菌TOP10感受态细胞。
4)在含有卡那霉素的LB平板上进行筛选,挑选单克隆于含Kan(50mg/L)的LB液体中进行摇菌,设计两个引物对组合,对菌液进行PCR检测,挑选扩增片段大小与预测相一致的阳性克隆子送测序,测序正确的菌液摇菌提取质粒,命名为pEntry-52950。
5)利用PlantCARE数据库对扩增序列中的顺式作用元件进行分析,结果表明甘蓝型油菜BnaCnng52950D基因启动子含有大量核心元件TATA-box和CAAT-box及许多其他重要的顺式作用元件,包括许多光响应相关调节元件,如Box 4、GT1-motif和MRE等,也有与昼夜节律控制有关的顺式调节元件circadian和参与胚乳表达的顺式调节元件GCN4_motif,以及厌氧诱导的顺式调节因子等。
进一步,上诉克隆方法中使用的两个引物对组合为:上游引物对:M13R23+SEQ IDNO:2;下游引物对:M13F26+SEQ ID NO:3。
一种重组表达载体,包含序列为SEQ ID NO:1的核苷酸片段,以及植物表达载体pEarly502。
一种上诉重组表达载体的构建方法,其特征在于,具体步骤如下:
1)用限制性内切酶组合MluI酶切质粒pEntry-52950,将环形质粒变为线性,有利于提高LR反应的效率。
2)将线性化的pEntry-52950质粒与植物表达载体pEarly502进行LR重组反应,将BnaCnng52950D启动子序列重组至pEarly502载体中。
3)将重组载体全部转入大肠杆菌TOP10感受态细胞,在含有卡那霉素的LB平板上进行筛选,挑选克隆子进行摇菌培养。
4)设计两个引物对组合,对培养产物进行PCR检测,挑选扩增片段大小与预测相一致的阳性克隆子送测序,测序正确的菌液摇菌提取质粒,命名为pEarly502-52950。
进一步,步骤(4)中使用的引物对组合为SEQ ID NO:2+SEQ ID NO:4;SEQ ID NO:5+SEQ ID NO:6。
进一步,步骤(1)中的酶切体系为:ddH2O,14μl;10×NEB buffer 3.1,5μl;pEntry-52950质粒,30μl;MluI酶,1μl。
进一步,步骤(2)中的LR重组体系为:酶切后的pEntry-52950,3μl;植物表达载体pEarly502,2μl;5×LR Clonase Enzyme,1μl。
含有如SEQ ID NO:1所述启动子在培育短角果柄或长角果柄植物中的应用。
进一步,含有如SEQ ID NO:1所述启动子在包括培育短角果柄或长角果柄植物中的应用。
基于BnaCnng52950D基因启动子PCR扩增的引物一对,所述引物对序列如SEQ IDNO:2和SEQ ID NO:3所示。
基于重组表达载体pEarly502-52950PCR扩增的引物对组合,所述引物对组合为SEQ ID NO:2+SEQ ID NO:4;SEQ ID NO:5+SEQ ID NO:6。
本发明的有益效果在于:
1)本发明提供了一个优先在角果柄基部、叶片中表达的组织特异性启动子,及其所构建的载体与转基因植株。本发明增加了甘蓝型油菜组织特异性启动子的多样性,对解决我国菜籽油问题具有一定的意义。
2)本发明前期使用甘蓝型油菜转录组数据筛选具有组织特异性表达的基因BnaCnng52950D,克隆了该基因的启动子序列,利用Gateway技术构建了该启动子载体,将其与GUS报告基因相连接,采用农杆菌介导法在拟南芥中进行转化,检测分析转基因拟南芥中的GUS表达模式,通过在转基因植株中根、茎、叶、花、种子、荚果等进行GUS染色分析,结果发现其可驱动GUS报告基因在角果柄基部和叶片中表达。并通过生物信息学软件预测了该序列所包含的启动子元件。该启动子的获得,为油菜转基因研究提供有力工具,并给油菜和它的近缘物种的研究奠定一定的基础。
附图说明
图1BnaCnng52950D基因的启动子PCR扩增产物电泳图(左起第一栏为DNA分子量标准:5000bp,3000bp,2000bp,1000bp,750bp,500bp,200bp,100bp,第二栏为PCR产物)。
图2:BnaCnng52950D的启动子片段连接pENTR-D-TOPO载体(左起第一栏为DNA分子量标准:5000bp,3000bp,2000bp,1000bp,750bp,500bp,200bp,100bp,第二栏起为PCR产物)
图3BnaCnng52950D的启动子片段连接pEarly502载体(左起第一栏为DNA分子量标准:8000bp,5000bp,3000bp,2000bp,1000bp,750bp,500bp,200bp,100bp,第二栏起为PCR产物。)
图4pEarly502-52950D的农杆菌鉴定(左起第一栏为DNA分子量标准:8000bp,5000bp,3000bp,2000bp,1000bp,750bp,500bp,200bp,100bp,第二栏起为PCR产物)。
图5pEarly502-52950D的拟南芥阳性植株鉴定(左起第一栏为DNA分子量标准:5000bp,3000bp,2000bp,1000bp,750bp,500bp,200bp,100bp,第二栏起为PCR产物)。
图6利用SnapGene绘制的pEarly502-52950D的载体图
图7BnaCnng52950D的启动子元件分析
具体实施方式
以下将参照附图,对本发明的优选实施例进行详细描述。优选实施例中未注明具体条件的实验方法,通常按照常规条件,所举实施例是为了更好地对本发明的内容进行说明,但并不是本发明的内容仅限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整,仍属于本发明的保护范围。
实施例1
启动子序列克隆
1.前期使用甘蓝型油菜转录组数据筛选具有组织特异性表达的基因BnaCnng52950D,根据甘蓝型油菜基因组序列设计其上游1500bp启动子扩增引物PR和PF。
2.以甘蓝型油菜ZS11的基因组DNA为模板,使用美国NEB公司的Q5高保真酶扩增,PCR程序为:98℃预变性30s;98℃变性10s,56℃退火20s,72℃延伸1min,35个循环;最后72℃保温2min。扩增产物进行电泳检测,在凝胶成像系统中观察电泳结果,条带大小正确片段利用胶回收试剂盒方法回收目的片段。
3.扩增后PCR产物4μl与Salt Solution 1μl混合后,取2.5μl到一个新的离心管,加入0.5μl的pENTR/D-TOPO载体,混匀后室温放置15min。全部转化大肠杆菌TOP10感受态。在含有卡那霉素的LB平板上进行筛选,挑选单克隆于800μl含Kan(50mg/L)的LB液体中进行摇菌,利用引物对M13F26和基因反向引物(PR),M13R23和基因正向引物(PF)对摇至浑浊的菌液进行PCR检测,挑选扩增片段大小与预测相一致的阳性克隆子送测序,测序正确的菌液摇菌提取质粒,命名为pEntry-52950。
实施例2
启动子顺势作用元件分析
利用PlantCARE数据库对BnaCnng52950D基因启动子扩增序列中的顺式作用元件进行分析,结果表明甘蓝型油菜BnaCnng52950D基因启动子含有大量核心元件TATA-box和CAAT-box及许多其他重要的顺式作用元件,包括许多光响应相关调节元件,如Box 4、GT1-motif和MRE等,也有与昼夜节律控制有关的顺式调节元件circadian和参与胚乳表达的顺式调节元件GCN4_motif,以及厌氧诱导的顺式调节因子等。
表1 BnaCnng52950D启动子中的顺式作用元件
实施例3
构建重组表达载体
1.用限制性内切酶组合MluI酶切质粒pEntry-52950,将环形质粒变为线性,有利于提高LR反应的效率。酶切体系(50μl):ddH2O,14μl;10×NEB buffer 3.1,5μl;pEntry-52950质粒,30μl;MluI酶,1μl。
2.酶切30min后,将线性化的pEntry-52950质粒与植物表达载体pEarly502进行LR重组反应,将BnaCnng52950D启动子序列重组至pEarly502载体中。LR重组反应体系(6μl):酶切后的pEntry-52950,3μl;植物表达载体pEarly502,2μl;5×LR Clonase Enzyme,1μl。
3.将连接体系混匀后室温放置4min,加入0.5μl蛋白酶K,37℃放置10min,全部转入大肠杆菌TOP10感受态,在含有卡那霉素的LB平板上进行筛选,挑选克隆子进行摇菌,利用引物对PF+RGUS502,以及FBAR+RBAR进行PCR检测,挑选扩增片段大小与预测相一致的阳性克隆子送测序,测序正确的菌液摇菌提取质粒,分别命名为pEarly502-52950。
实施例4
重组表达载体转化根癌农杆菌GV3101
1.实验室购买农杆菌感受态。
2.取1管100μl-80℃保存的农杆菌GV3101感受态,冰水浴解冻,冻融后加入10μlpEarly502-52950表达载体质粒,用枪头轻轻混匀,先冰水浴5min,液氮浸没8min后迅速放入37℃水浴锅中热激5min,在超净工作台下加入800μl空白YEB液体培养基后于28℃、180r摇床复苏3h,复苏结束后于室温5000rpm离心5min。
3.吸取600μl上清,剩下菌液重悬,均匀涂布于添加抗生素Kan50mg/L+Gen 25mg/L+Rif 20mg/L+Str 25mg/L的YEB固体培养基上,放入28℃培养箱倒置培养2d,挑取单斑菌落。
4.将菌落接种于添加抗生素Kan 50mg/L+Gen 25mg/L+Rif 20mg/L+Str 25mg/L的YEB液体培养基,28℃、250rpm摇床至浑浊。利用引物RGUS502+基因前引物,FBAR+RBAR进行PCR检测农杆菌菌液,选取结果正确的菌液在超净工作台上加入等量50%的甘油后置于-80℃保存,用于后续拟南芥转化。
实施例5
浸花法转化拟南芥
1.将上述200μl保存的菌液添加至200ml YEB液体培养基中,添加抗生素Kan50mg/L+Gen 25mg/L+Rif 20mg/L+Str 25mg/L后摇菌,于28℃、250r摇床上培养48h,培养到OD600=1.0即可准备浸染。
2.将培养菌液转入50ml离心管中,4℃,5000rpm离心20min,弃上清,用重悬液(200ml ddH2O+10蔗糖+100μl silwet L-77+0.1g MES+0.05g乙酰丁香酮)均匀重悬至OD600=0.8左右。将拟南芥花序浸泡在浸染液中5s,取出放在塑料盘中用黑色塑料膜覆盖保湿,暗培养24h后解开薄膜,在正常条件下继续培养转化后的拟南芥植株,之后再重复浸染两次,直至收获。取T3代转基因阳性植株进行GUS染色试验。
实施例6
转基因植株GUS染色
1.配制GUS染液:5g/L X-gluc,0.1mol/L K3[Fe(CN)6],0.1mol/L K4[Fe(CN)6],0.5mol/L EDTE母液,0.2mol/L磷酸缓冲液溶解,调节pH为8.0。
2.取新鲜植物组织于90%丙酮中固定20min,弃丙酮,加入适量不含X-Gluc的GUS染色液润洗一遍,之后加入含X-Gluc的GUS染色液,真空处理20min。
3.置于37℃孵育24h,再用无水乙醇浸泡24h,除去叶绿素,最后观察结果并拍照。如果有GUS活性的植株就会发生蓝色反应。经过显色反应发现转基因拟南芥在角果柄基部和叶片出现明显的蓝色反应。
表2转基因拟南芥GUS活性定性检测结果
序号 | 植株 | 显色部位 | 显色程度 |
1 | pEarly502-52950D-1花蕾 | 叶片根部 | 明显 |
2 | pEarly502-52950D-2花蕾 | 叶片根部 | 较明显 |
3 | pEarly502-52950D-1荚果 | 角果柄基部 | 明显 |
4 | pEarly502-52950D-3花蕾 | 叶片 | 明显 |
5 | pEarly502-52950D-2荚果 | 角果柄基部 | 明显 |
6 | pEarly502-52950D-1幼苗 | 叶片 | 极明显 |
7 | pEarly502-52950-1早期种子 | 未显色 | ---- |
8 | pEarly502-52950-1晚期种子 | 未显色 | ---- |
9 | pEarly502-52950D-1叶片 | 未显色 | ---- |
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的宗旨和范围,其均应涵盖在本发明的权利要求范围当中。
序列表
<110> 西南大学
<120>基于BnaCnng52950D基因的启动子序列、重组载体及应用
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 1327
<212> DNA
<213> Brassica napus
<220>
<221> gene
<400> 1
TTTTAGGTTT TAACCCCCTC CCAAAAAAAA TCATTACATG TGGTGCTAAA ATAACTTAAA 60
CTACATAAAT AATATTTATT TATGGGTATG TTAATATCCT TGCTAAACTA TATGGCATTA 120
CCATAAAGTT GATATATCTG TTTGTCTGAA AATAAATAAA ACAACATCTT TACCCAACTA 180
ATGTTGAAAT AAACTATTTA ATTTATTACA CATTTCACAT AGGAGATAAG AATGATGACA 240
ATTTTGATAC TTAATATGGG ATGATATCTA CTTTTTAAAC TATATTTCCG ATGATAAATC 300
TACTAACATA AGAGTAGAGA ATGGAAAATG TTAATAAGAT CAGAAAAAGT TACTTTCTGA 360
TCAAAAATAG TAATGGAAAC TAAAATCTAA ATTTAGTCAT TAAATAAAAA TATACTAGGT 420
GATTTTCCTG TGTACATGCA CATATATAAA TATTAAACTT TAAAAATCAT AATAATTGAT 480
CAACATTTTT GGCACTTTTG TTTATAAGTT ATAAGTGTTT TATAACTTTT ATTAAAGGTG 540
TCAAAATGGA TAATCCAACT CAAACCATCC TATATCCATA TGAACCCATT CTCATATGAA 600
CCATGAATAA ACCCAATCTA TTTAGGTAAT GGTTTGGATC AATCCATAAC TCATTTAAAT 660
TAATAGGTTG ATAGTTTCAA TGAGTTGACT CACTATTATT TATTATCAAT ATTAAAAATC 720
AATCAAAATA AGTTAATAAT TATGTAAATT TTAAATAAAA GTATTACTAA CACTGTCTAA 780
AATTTGATTA TGCAAATTTA AATGTAAAAA AAAAAAGCTA ACAAAATTAT TGAACATAAA 840
AAATAAAACA GAATATTTTC ATTAGACTAT TGCTTTAAAG TCTTTGTAGA ACATTCCAAC 900
ACTTTACAAA CAGATGAAAA CGAAACCATA CGAAAGGGAT CAAGCACAGC AACTCTGTCA 960
CCGTACTTAT CCGCAGATGA TCTCCATATA TCAGGAACAG CCTTTCCATT CATTGATTTA 1020
ATTTCTCTAT TATTTTGTTG ATTCAGAAGT TCGTATAAAT ATATATATAT AATATACTTT 1080
CCTAAATAAG AGAGATTCCT GTGAGATGGC AAAATAACAT GATAAACACA AATAATGTTA 1140
ATCATATCAA CGGCATTATA AAAAAAGGAA AGGTGGACTT GCTAAAAAAC GGGTGGAAAG 1200
TAATTCAGTC AAAGATATAT GGTGTGGACT ATTTTTTGAA CACCATATAA AGAAGTTAAC 1260
CAAACTATAA CATCACCACA TATAACAACT CTCATCATTT TAACTTCTTC ACCACACCAC 1320
ATCAAAA 1327
<210> 2
<211>31
<212> DNA
<213>人工序列
<220>
<223> 引物PF
<400> 2
CACCTTTTAG GTTTTAACCC CCTCCCAAAA A 31
<210> 3
<211>26
<212> DNA
<213>人工序列
<220>
<223> 引物PR
<400> 3
TTTTGATGTG GTGTGGTGAA GAAGTT 26
<210> 4
<211>21
<212> DNA
<213>人工序列
<220>
<223> 引物RGUS502
<400> 4
GTCCGCATCT TCATGACGAC C 21
<210> 5
<211>21
<212> DNA
<213>人工序列
<220>
<223> 引物FBAR
<400> 5
CGACATCCGC CGTGCCACCG A 21
<210> 6
<211>21
<212> DNA
<213>人工序列
<220>
<223> 引物RBAR
<400> 6
TAGATCTCGG TGACGGGCAG G 21
Claims (10)
1.基于BnaCnng52950D基因启动子的核苷酸片段,其特征在于,所述核苷酸片段由BnaCnng52950D基因启动子克隆而来,核苷酸序列如SEQ ID NO:1所示。
2.一种权利要求1所述核苷酸片段的克隆方法,其特征在于,具体步骤如下:
1)设计如SEQ ID NO:2和SEQ ID NO:3所示序列引物一对,以甘蓝型油菜ZS11的基因组DNA为模板进行PCR扩增;
2)扩增引物中加入pENTR/D-TOPO载体,混匀后全部转化大肠杆菌TOP10感受态细胞;
3)在含有卡那霉素的LB平板上进行筛选,挑选出菌落培养,利用两个引物对组合,进行菌液PCR检测,挑选阳性克隆子测序,测序正确的菌液提取质粒,命名为pEntry-52950;
4)对启动子序列中的顺式作用元件进行分析。
3.根据权利要求2所述的方法,其特征在于,步骤3)中所述的引物对组合为:上游引物对:M13R23和SEQ ID NO:2;下游引物对:M13F26和SEQ ID NO:3。
4.重组表达载体,其特征在于,所述重组表达载体包含权利要求1所述的核苷酸片段,以及植物表达载体pEarly502。
5.一种权利要求4所述重组表达载体的构建方法,其特征在于,具体步骤如下:
1)酶切质粒pEntry-52950,将环形质粒变为线性;
2)将线性化的pEntry-52950质粒与植物表达载体pEarly502进行LR重组反应,将BnaCnng52950D启动子序列重组至pEarly502载体中;
3)将重组载体全部转入大肠杆菌TOP10感受态细胞,在含有卡那霉素的LB平板上进行筛选,挑选克隆子培养;
4)利用两个引物对组合,进行培养产物PCR检测,挑选阳性克隆子测序,测序正确的菌液提取质粒,命名为pEarly502-52950。
6.根据权利要求5所述的方法,其特征在于,步骤4)中所述引物对组合为:上游引物对:SEQ ID NO:2和SEQ ID NO:4;下游引物对:SEQ ID NO:5和SEQ ID NO:6。
7.权利要求1所述的启动子在培育短角果柄或长角果柄植物中的应用。
8.根据权利要求7所述的应用,其特征在于,所述启动子在培育短角果柄或长角果柄甘蓝型油菜中的应用。
9.基于BnaCnng52950D基因启动子PCR扩增的引物对,其特征在于,所述引物对序列如SEQ ID NO:2和SEQ ID NO:3所示。
10.基于权利要求4所述重组表达载体PCR扩增的引物对组合,其特征在于,所述引物对组合为SEQ ID NO:2和SEQ ID NO:4;SEQ ID NO:5和SEQ ID NO:6。
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