CN112481268B - 一种棉花启动子PGhPGF及其重组载体和应用 - Google Patents
一种棉花启动子PGhPGF及其重组载体和应用 Download PDFInfo
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Abstract
本发明属于基因工程技术领域,涉及棉花启动子的克隆与重组载体的构和应用,特别是指一种棉花启动子PGhPGF及其重组载体和应用。其核苷酸序列如SEQ ID NO.1所示。本发明所述的PGhPGF启动子被证明是调控基因在棉花色素腺体细胞中特异表达的一个启动子,该启动子在植物基因工程领域具有重要的应用价值,将会推进棉花育种产业的发展,提高棉花的综合利用价值。
Description
技术领域
本发明属于基因工程技术领域,涉及棉花启动子的克隆与重组载体的构和应用,特别是指一种棉花启动子PGhPGF及其重组载体和应用。
背景技术
启动子是RNA 聚合酶识别、结合并启动转录的一段DNA 序列,它含有RNA 聚合酶特异性结合和转录起始所需的保守序列,多数位于基因转录起始位点的上游。启动子的结构直接影响它与RNA聚合酶的结合能力,进而影响基因的表达水平(焦勇等,2019)。因此,研究启动子的结构、功能和表达模式对于基因在转录水平上的调控机制研究具有重要意义。
色素腺体,又称黑腺体或棉酚腺体,是棉属及其近缘植物所特有的腔体结构,分布在植株各个组织的表面,即肉眼可见的黑色或褐色点状结构。一般认为,成熟的色素腺体由1-3层分泌细胞包围一个含有分泌物的腔体构成。后来刘文哲团队用实验证明棉花色素腺体腔的形成属于典型的溶生发育,并且是由属于分生组织的某些原始细胞在经历了细胞溶生过程和细胞程序性死亡过程后形成的(Liuet al.,2010)。这些腺体将大量次级代谢产物限制在其空腔中,是棉花抵御害虫和病菌侵染的重要防御物质。研究表明,棉酚是棉花色素腺体中研究最多的次生代谢物,也是抵御病虫害的重要代谢物质,同时还具有药用价值。棉酚是一种倍半萜烯类化合物,在棉花各组织中均有分布,其中棉籽中含量最高。棉籽中含有大量蛋白质、脂肪、碳水化合物等营养物质,因而在食用、保健、饲用和工业应用方面具有很大的应用前景。然而,棉酚对某些单胃动物和人有毒害作用,需经过多重工艺才能去除棉酚,这严重限制了人们对棉籽的综合利用(钱玉源等,zhang et al., 2007)。人们通常认为,棉酚及其衍生物在植物根尖合成,然后运输到植物的地上部分并储存在色素腺体中,棉酚含量与腺体数量呈正相关关系(Smith, F.H., 1962)。后来随着研究的深入,人们发现在一些新棉种中,棉酚含量与腺体之间并非是绝对的正相关关系。这使得棉酚与腺体之间的关系更为复杂。因此,研究棉花色素腺体发育和棉酚代谢的调控机制对低酚棉分子育种具有重要参考依据和应用价值。到目前为止,关于棉花色素腺体发育和代谢相关的研究甚少,GoPGF是人们克隆到的第一个调控腺体发育的基因,而该基因的表达模式和调控机制尚不清楚(Ma et al., 2016)。
发明内容
本发明提出一种棉花启动子PGhPGF及其重组载体和应用,为棉花色素腺体发育调控机制和物质代谢等相关研究提供参考依据。
本发明的技术方案是这样实现的:
一种棉花启动子PGhPGF,其核苷酸序列如SEQ ID NO.1所示。
或其核苷酸序列与SEQ ID NO.1所示的序列存在大于70 %的同源性。
或其核苷酸序列为与SEQ ID NO.1所示的序列具有类似功能的亚片段。
含有上述任一种情况的棉花启动子PGhPGF的重组载体,包括植物表达载体和RNAi载体。
上述的重组载体在调控目的基因在棉花腺体细胞中表达的应用。
含有上述的重组载体的菌株。
上述的菌株在制备转基因植株中的应用。
本发明提供两个PGhPGF驱动的植物表达载体PGhPGF::GUS和PGhPGF-RNAi。所述的两个载体包含PGhPGF序列,其中PGhPGF扩增引物序列为:正向引物PGhPGF-F1:5’-GAAGCTGCCCTTCGCTGCAG-3’和反向引物PGhPGF-R1:5’-TCGTCTAGATATTGAATATG-3’。
以本申请发明人提供的上述重组载体的两个重组菌株为例,其中PGhPGF::GUS转化的是LBA4404农杆菌,PGhPGF-RNAi转化的是大肠杆菌。PGhPGF::GUS的应用如实施例4。可在PGhPGF-RNAi载体基础上采用Gateway技术连入任何感兴趣的基因,再利用遗传转化方法转入棉花植株,即可得到在棉花腺体细胞中特异沉默该基因的转基因株系。
本发明具有以下有益效果:
1、本发明所述的PGhPGF启动子被证明是调控基因在棉花色素腺体细胞中特异表达的一个启动子,该启动子在植物基因工程领域具有重要的应用价值,将会推进棉花育种产业的发展,提高棉花的综合利用价值。
2、本发明提供了一种组织特异性表达启动子,该启动子为利用分子育种提供了一种新方法。这种方法可通过以下步骤实现,将PGhPGF启动子与目标基因连接后的重组载体转入棉花即可驱动该基因在腺体细胞中表达,从而提高了该基因在色素腺体细胞中的表达。也可以利用该启动子的重组载体特异性沉默目标基因在腺体细胞中的表达。本发明可以提供或利用上述重组载体获得的转基因植株和相应的种子,然后利用杂交技术将本发明的启动子转入其它植株,避免了繁琐的遗传转化过程。
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些实施例,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,还可以根据这些附图获得其他的附图。
图1为本发明实施例中PGhPGF启动子PCR扩增的琼脂糖凝胶电泳检测图。
图2为PGhPGF启动子连接GUS(β-葡萄糖醛酸酶)报告基因的重组质粒的示意图。
图3为本发明所述的PGhPGF-RNAi重组质粒的示意图。
图4为棉花遗传转化植株中GUS的表达模式说明PGhPGF启动子启动基因在色素腺体细胞中特异表达。
具体实施方式
下面将结合本发明实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明所用的Taq DNA Polymerase、Phanta Max Super-Fidelity DNAPolymerase保真酶、ClonExpress II One Step Cloning Kit无缝克隆酶购于南京诺唯赞生物科技有限公司;Cycle-Pure Kit DNA纯化试剂盒购于北京普洛麦格生物技术有限公司;pEASY-Blunt Simple Cloning Kit购于北京全式金生物技术有限公司;BP、LR试剂盒,Gateway中间克隆载体PDNOR-221,PKGWFS7.0载体,pK7GWIWG2(II),0载体购自于美国Invitrogen公司;植物基因组DNA提取试剂盒、快速质粒小提试剂盒购于天根生化科技(北京)有限公司;植株转化、组织培养相关培养基及X-Gluc染色液购自北京酷来搏科技有限公司;所用引物和测序工作在上海生工生物工程有限公司完成。
实施例1:克隆PGhPGF启动子
1. 棉花DNA提取
按照植物基因组DNA提取试剂盒说明书提取陆地棉真叶组织的DNA。然后用NanoDrop 2000c紫外-可见光分光光度计(Tbermo Fisber,美国)测定提取的DNA浓度,最后用1.0 %的琼脂糖凝胶电泳检测DNA纯度。
2. 聚合酶链式反应扩增PGhPGF启动子
以DNA为模板扩增PGhPGF启动子的全长序列,PCR反应体系如下:
其中模板为实施例一中提取的棉花真叶DNA。PCR产物用琼脂糖凝胶电泳检测(图1),之后用Cycle-Pure Kit试剂盒纯化回收扩增产物,然后将PCR产物连到pEASY-BluntSimple Cloning Kit试剂盒的载体上,随后将酶连产物转化至大肠杆菌DH5α,利用Taq DNAPolymerase做PCR检测,并将阳性克隆送上海生工生物有限公司进行测序。测序正确的克隆利用快速质粒小提试剂盒提取质粒,记为PGhPGF,序列信息见SEQ ID NO.1。
实施例2:PGhPGF::GUS重组表达载体的构建
通过PCR扩增得到带接头的启动子片段,PCR反应体系如下所示:
扩增模板为实施例一中含PGhPGF序列的pEASY-Blunt Simple载体,扩增引物如下:PGhPGF-F2:
5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTGAAGCTGCCCTTCGCTGCAG-3’,
PGhPGF-R2:
5’-GGGGACCACTTTGTACAAGAAAGCTGGGTTCGTCTAGATATTGAATATG-3’, 其中下划线核苷酸序列是BP反应的接头序列。
PCR的程序同实施例一所用程序相同。纯化回收扩增的PCR产物,然后利用Gateway方法将PGhPGF连入入门克隆载体pDNOR221上,具体操作为:PCR产物0.5 μL;pDNOR221 vector0.5 μL;BP酶0.5 μL;TE(8.0)Buffer 1 μL,25℃反应2h。随后将酶连产物通过热激方法转化至大肠杆菌DH5α,利用Taq DNA Polymerase做PCR检测,并将阳性克隆送上海生工生物有限公司进行测序。测序正确的克隆用快速质粒小提试剂盒提取质粒,记为pDNOR221-PGhPGF。随后进行LR反应,LR反应体系为:pDNOR221-PGhPGF质粒0.5 μL;PKGWFS7.0载体0.5 μL;LR酶0.5 μL;10× TE(8.0)Buffer 1 μL,25℃反应2 h。连接产物转化大肠杆菌DH5α,经PCR检测,挑选阳性克隆提质粒,记为PGhPGF::GUS,载体具体信息如图2所示。最后,通过电击转化将LR质粒转入农杆菌LBA4404中,检测正确的阳性克隆加甘油保存于-80℃冰箱备用。
实施例3:PGhPGF驱动的干涉载体的构建
PGhPGF-RNAi重组载体是在pK7GWIWG2(II),0干涉载体基础上改造得到的特异性干涉载体。具体方法如下:首先通过SacI和MluI两个酶切位点将pK7GWIWG2(II),0载体上的p35S启动子和临近的attR1,CCDB,attR2切除,形成一个线性载体备用。然后,利用一步克隆技术设计带有接头的引物,引物序列为:PGhPGF-F3正向引物:5’GCTCAAGCTAAGCTTGAGCTCGAAGCTGCCCTTCGCTGCAGTAGAAG 3’,PGhPGF-R3反向引物:5’TAGTGCGGCCGCCTGCAGGGAGCTCTCGTCTAGATATTGAATATGATAGTG 3’。CCDB-F正向引物:5’TATCATATTCAATATCTAGACGAGAGCTCCCTGCAGGCGGCCGCACTAGTG 3’,CCDB-R反向引物:5’TGGCAGGGCGGGGCGTAAACGCGTGGATCAGCTTAATATGACTCTC3’。通过PCR扩增得到PGhPGF片段和包含attR1,CCDB和attR2的片段,纯化回收后利用一步克隆酶与上述SacI和MluI酶切后的线性载体进行酶连,转化,最终获得测序正确的阳性克隆,提质粒后保存备用,重组载体结构示意图如图3所示。此重组载体将原来的p35S启动子替换为PGhPGF启动子,保留Gateway方法需要的attR1,CCDB和attR2相关序列。因此,我们仍然可以利用Gateway方法将感兴趣的基因连入此载体中,便可以实现在棉花腺体细胞中特异的沉默目的基因。
实施例4:PGhPGF启动子的功能验证
1. 转基因材料创制
利用农杆菌介导的棉遗传转化方法将LBA4404农杆菌携带的PGhPGF::GUS导入棉花中(Jin等,2006)。具体方法如下,首先将需要转化的棉花脱壳,然后用0.1 %的升汞溶液消毒,消毒后的种子在无菌苗萌发培养基(1/2 MS)中于室温黑暗条件下培养8天。然后将活化好的携带PGhPGF::GUS质粒的LBA4404农杆菌用MGL培养基稀释至OD值为0.6,生长8天左右的无菌苗下胚轴切成0.6 cm段放置到备好的农杆菌中至下胚轴组织完全淹没并轻摇晃7min,去除菌液,最后再用无菌滤纸吸干组织表面的多余菌液并平铺于MSB培养基上。避光共培养72 h后将下胚轴组织置于含卡那霉素的诱导培养基上诱导生出胚性愈伤组织。以后约30天继代1次直至愈伤组织发生胚分化,形成转基因棉花幼苗。本发明最终获得6个独立转基因株系,且全部得到后代。
2. PGhPGF启动子驱动GUS在腺体细胞中特异表达
对获得的T0代阳性棉花株系的不同组织进行GUS染色实验。将剪取的棉花组织迅速加入预冷的80 %(v/v)丙酮固定15 min -30 min,然后用预冷的100 mM 的磷酸缓冲液冲洗三次,转移到含有X-Gluc反应底物的染色液中,真空处理5 min,在37℃培养箱中放置过夜,看染色效果,终止反应。随后在37℃条件下加入75 %酒精进行脱色,脱色结束后用体式显微镜(Leica MZFLⅢ)观察并照相。拍照后的样品再按照石蜡切片的步骤进行脱色、浸蜡、包埋,最后将样品切成10 μm的片子。脱蜡封片后用光学显微镜(OLYMPUS IX73,Japan)进行观察拍照。如图4所示,在这些组织中,GUS染色集中在色素腺体细胞中,说明PGhPGF启动子能够启动基因再腺体细胞中特异表达,是一个植物组织特异性启动子。
实施效果例
本发明提供两个PGhPGF驱动的植物表达载体PGhPGF::GUS和PGhPGF-RNAi。上述重组载体的两个重组菌株,其中PGhPGF::GUS转化的是LBA4404农杆菌,PGhPGF-RNAi转化的是大肠杆菌。PGhPGF::GUS的应用如实施例4。可在PGhPGF-RNAi载体基础上采用Gateway技术连入任何感兴趣的基因,再利用遗传转化方法转入棉花植株,即可得到在棉花腺体细胞中特异沉默该基因的转基因株系。本发明的PGhPGF启动子被证明是调控基因在棉花色素腺体细胞中特异表达的一个启动子,该启动子在植物基因工程领域具有重要的应用价值,将会推进棉花育种产业的发展,提高棉花的综合利用价值。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
<110> 河南大学
<120> 一种棉花启动子PGhPGF及其重组载体和应用
<141> 2021-01-25
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1847
<212> DNA
<213> 棉属(Gossypium spp)
<400> 1
gaagctgccc ttcgctgcag tagaagatgt gctgtatgag tatccaaaat ttaagatgct 60
ttaatcttgt tatgcttggc aaatagagtt ggtggattat aaacaatccc aatcctcttc 120
agagtcaaat attcaaagct aaatagttcc cgaattgtgg gtttcttgaa tctagtgaag 180
gattgattgt tgaaaactta gaccatgttt taatgatgta tatcaaagct caatttgtat 240
tgcatattgt tgggttaagg ctattgcaag attcaacctt ggagtttctc tattaccacc 300
ttaatatgca agatgataat gagctgagtt ggtggttagg tgtagcatgg agtatatgga 360
cacataggaa ccagtttatt tagaataaaa aaagatcata atgtagtcca aattgttcaa 420
tttgaatatt cttacatgga ggcttggtag ggagctcaca cgacgatgac tcgtattcaa 480
ccttccaggt taagttgttc tacttctaag tagagttggt gtaaatcgct aggtaatggg 540
ttcaaattta acatcgatat ggttattttt ataagaatgt tgccacgagc tgatcgacta 600
tcattcataa tgaaaatggt gattttgaca aaggtcgcac aggttttatc caagcaacta 660
tggctccaaa agttacacat gattgttatt tgaaaggcat tgctttagtt gaagtcgttg 720
caaattgaga gagtgatcat tgagacggat tgtttgattg taccgcaaga tttaacgagt 780
aaaaaaaggt tatctctatg atgggtttga ttattgaagg ctgtctcatg ttgaaagctt 840
tttttttttt caatttatgt ccttatgttg aatgagtaga aacattgata agatcgctca 900
tgctcttgca agggtagctt tatgtcatgc aaatccttat gtttagaatt cttctcctag 960
ttgcatgtct tccattctta tttaagatgt tcgtactctt ttatctaatg aggagtcgag 1020
tgtataggat aagcccatta gcttaagaaa ataattttcc aatccttatt ttgggtaatt 1080
ttgtttgttt cattccgatt tgataataat ctattttctt ctttccttaa aaaaaaaggc 1140
taggaagagt tatttgaata aattaacaaa gttgaagagc taatttaata gattaacatt 1200
tgtatcaaat attaaaacaa caaggtagtt aattgggagt tgagttgggg ttaaaggtga 1260
cacgataagc tgaataatta acaaaatatg gatgggaatg gtcctaatat ctaaaaatag 1320
ttaaaaatca tgggaaatag aaaaaagaca gataaaagat gatcacgaaa tcgccccata 1380
ataactggtg tgaattaagt taagatcatg agtggagggg ttaagacgcc aacagaagca 1440
ccgcaactcc acatcaaata gagtaaacca tccatgccat aaagaacaaa aaagaagaag 1500
aaaattcaga ccggtcatca tccttatttg gagagaatct ggaacaggag atttccacgt 1560
gataatgatg tcaagatttg actgacaata ggaccatgag gttgatggtc ctttatttcc 1620
ccggaaaatt atactttgtt ttctccaaag cacttcactt aataattata gttttcctct 1680
tttttctcat ctgctccatg gggtttatag ccacctagtg acgactttga tcatcactct 1740
ttttcctctt tatttattct tcttgctgct gagtccgctt tggttagttt ttcttttttc 1800
tctttgaaaa acggtagtac acactatcat attcaatatc tagacga 1847
Claims (6)
1.一种棉花启动子PGhPGF,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所述的棉花启动子PGhPGF的重组载体,其特征在于:所述重组载体为植物表达载体。
3.含有权利要求1所述的棉花启动子PGhPGF的重组载体,其特征在于:所述重组载体为RNAi载体。
4.权利要求2或3所述的重组载体在调控目的基因在棉花腺体细胞中表达的应用。
5.含有权利要求2或3所述的重组载体的菌株。
6.权利要求5所述的菌株在制备转基因植株中的应用。
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