CN110423759A - 棉花GhMADS36-A11基因在促进植物开花中的应用 - Google Patents
棉花GhMADS36-A11基因在促进植物开花中的应用 Download PDFInfo
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- CN110423759A CN110423759A CN201910629345.6A CN201910629345A CN110423759A CN 110423759 A CN110423759 A CN 110423759A CN 201910629345 A CN201910629345 A CN 201910629345A CN 110423759 A CN110423759 A CN 110423759A
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Abstract
本发明公开了棉花GhMADS36‑A11基因在促进植物开花中的应用，属于植物基因工程技术领域。GhMADS36‑A11基因具有SEQ ID NO:1所示的核苷酸序列并可编码SEQ ID NO:2所示氨基酸序列。本发明为短季棉培育提供了有利的基因资源。
Description
技术领域
本发明属于植物基因工程技术领域，具体地，涉及GhMADS36-A11基因在促进植物开花中的应用。
背景技术
随着我国耕地面积日益减少，棉花作为我国最重要的经济作物，无法与粮食作物争用土地面积，因此，早熟性的研究趋势已成为必然。越来越多的早熟棉品种的出现，给分子育种提供了更多的材料，研究其早熟分子相关机理，分析早熟材料开花通路中花发育相关调控基因的关系，最终更好的服务于早熟品种的培育。
MADS-box基因是一类序列特异的转录因子家族，其编码的蛋白质序列包含一个非常保守的MADS盒、一个相对保守的K盒、一个低度保守的I区和多变的C区。根据不同MDAS-box基因的序列特点，研究者将MADS-box基因分为了13个亚族。作为转录因子，MADS-box基因调控着植物各种生长发育过程，花发育是其重要作用方面之一。
发明内容
发明人从陆地棉中克隆出棉花GhMADS36-A11基因。表达模式发现其在棉花幼苗顶芽中优势表达，并且在棉花三叶期(花芽分化时期)表达量升高，并在早熟品种的表达量显著高于晚熟品种，结果表明GhMADS36-A11可能在花芽的分化，生长阶段的转变和花器官的形成上起着重要作用；利用病毒诱导的基因沉默(VIGS)技术进一步验证了GhMADS36-A11在棉花中的功能，并观察到被沉默的植株与在相同生长状态下生长的CK相比出现明显晚花。因此认为 GhMADS36-A11在促进棉花开花方面可能具有关键作用，可以作为短季棉培育的有利基因资源。从而完成本发明。
本发明提供了GhMADS36-A11基因在提高促进植物开花中的应用，所述 GhMADS36-A11基因具有SEQ ID NO:1所示的核苷酸序列。该基因的开放阅读框为630bp。
在本发明的一些实施方案中，SEQ ID NO:1所示的核苷酸序列能够编码 SEQ IDNO:2所示氨基酸序列。该氨基酸序列包含209个氨基酸，蛋白的相对分子量为24.31kDa，等电点为9.95。
在本发明的一些实施方案中，在植物中提高GhMADS36-A11基因的表达量，以促进植物开花。
在本发明的一些具体实施方案中，所述的在植物中提高GhMADS36-A11基因的表达量是通过如下方法实现：提高植物内源GhMADS36-A11基因的表达，或在植物中过表达外源GhMADS36-A11基因。
在本发明的一个具体要求实施方案中所述过表达外源GhMADS36-A11基因是指将所述GhMADS36-A11基因利用植物表达载体，经农杆菌介导转化到植物中进行表达。
进一步地，所述GhMADS36-A11基因通过植物表达载体导入植物细胞、组织或器官。
更进一步地，所述植物表达载体通过一种组成型或诱导型启动子驱动所述GhMADS36-A11基因的表达。
再进一步地，所述组成型启动子是35S启动子。
在本发明中，所述促进开花是指促使植物开花期提前。
在本发明中，所述植物是棉花、玉米、水稻、小麦或拟南芥。
本发明的有益效果
本发明通过沉默棉花中GhMADS36-A11基因，结果表明GhMADS36-A11基因在促进棉花开花方面可能具有关键作用。本发明为短季棉培育提供了有利的基因资源。
附图说明
图1示出了电泳凝胶检测结果。第1泳道为GhMADS36-A11PCR目的片段，大小为630bp，M泳道为MARKER III。
图2示出了GhMADS36-A11在不同组织的表达量(A)和GhMADS36-A11在不同生育期材料的表达量(B)。
图3示出了病毒诱导的GhMADS36-A11基因沉默及表达分析。
具体实施方式
为了使本发明所解决的技术问题、技术方案及有益效果更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。
实施例
以下例子在此用于示范本发明的优选实施方案。本领域内的技术人员会明白，下述例子中披露的技术代表发明人发现的可以用于实施本发明的技术，因此可以视为实施本发明的优选方案。但是本领域内的技术人员根据本说明书应该明白，这里所公开的特定实施例可以做很多修改，仍然能得到相同的或者类似的结果，而非背离本发明的精神或范围。
除非另有定义，所有在此使用的技术和科学的术语，和本发明所属领域内的技术人员所通常理解的意思相同，在此公开引用及他们引用的材料都将以引用的方式被并入。
那些本领域内的技术人员将意识到或者通过常规试验就能了解许多这里所描述的发明的特定实施方案的许多等同技术。这些等同将被包含在权利要求书中。
实施例1
1.棉花材料
本实施例选取的棉花材料为陆地棉中50、中棉所74、国欣棉11号、渤棉1 号，种植于中国农业科学院棉花研究所棉花生物学国家重点实验室试验田(安阳白璧)，管理措施为正常大田管理。
2.试剂与耗材
2.1酶及试剂盒： GXL DNA Polymerase高保真酶、胶回收试剂盒、PCR产物纯化试剂盒均购自Takara公司；RNA反转录试剂盒、KOD FX Neo 酶(Code.No.KFX-201)购自Toyobo公司； Ultra One Step Cloning Kit试剂盒购自Vazyme公司；质粒少量提取试剂盒购自Magen公司；限制性内切酶(BamH I、Sac I)购自NEB公司；DNAMarker III、植物总RNA提取试剂盒购自TIANGEN公司；荧光定量TransStart Top GreenqPCR SuperMix购自 TransGen公司。
2.2其他药品：琼脂糖为西班牙原装产品，蛋白胨、酵母提取物、氯仿、异戊醇、乙醇、异丙醇、氯化钠、蔗糖、silwet L-77、间苯三酚等为国产分析纯，卡纳霉素、硫酸链霉素、氨苄青霉素等购自宝生物工程(大连)有限公司，大肠杆菌感受态细胞Trans5α购自北京全式金生物技术有限公司，农杆菌感受态细胞LBA4404购自上海唯地生物技术有限公司。
2.3培养基：LB液体培养基：胰蛋白胨(Tryptone)10g/L、酵母提取物(Yeastextract)5g/L、氯化钠(NaCl)10g/L；LB固体培养基：胰蛋白胨(Tryptone) 10g/L、酵母提取物(Yeast extract)5g/L、氯化钠(NaCl)10g/L、琼脂粉(Agar) 15g/L，定容至1L；LB选择培养基：在LB铺平板前，待培养基高压灭菌冷却至 55℃时加入相应浓度抗生素，摇匀后铺平板。本文中提到的但未列出的各种试剂溶液均按《分子克隆实验指南》第三版上的方法配制，生化试剂为分析纯或以上级。
2.4主要仪器：PCR扩增仪(Eppendorf)、高速离心机(Eppendorf 5427R)、电泳设备(北京六一)、凝胶成像系统(BIO-RAD)、荧光定量PCR仪(ABI7500)、荧光显微镜(OlympusBX43)、恒温培养振荡器(上海智城)、人工气候试验箱(赛福)等。
实验方法与结果
1棉花GhMADS36-A11基因的生物信息学分析与克隆
1.1从NCBI上获得GhMADS36-A11的基因序列，采用Primer Premier 5.0软件设计引物，采用PCR(Polymerase Chain Reaction)的方法从陆地棉中50中扩增，其开放阅读框为630bp，编码209个氨基酸，蛋白的相对分子量为24.31kDa，等电点为9.95。基因cds序列为(SEQ ID NO:1)：
ATGGGGAGAGGCAAGGTTCAGTTGAAAAGAATTGAGAACCCAACAA ACAGGCAAGTTACCTTCTCAAAGAGAAGAAATGGGCTTCTCAAGAAAGC TTTTGAGTTGTCCATTCTTTGTGATGCTGAAGTTGCCTTGCTTATCTTCTCT TCTTCTGGAAAGGTTTACCAGTTTGCAAGTCATGACATGGATAGGACTGT TGCAAAGTATAGAAGGGAAGTAGGGCTGCCTGATTCAAGTAACCCCCAA TTTAGAACCATGGAGTTTTGGAGAAGTGAGATTGATGAGTTAAATAGATC CATAAACACCTTGGAAGCTAGACTTAAGCACTTATCTGGAGAAGACATCT TAGCATTAGGCATGAGAGACTTGAAACAGCTTGAGCGCCAGTTGAAAAT AGGCGTTGAACGTGTCCGCTCCAGAAAGAGACGCATTGTTTCGGATCACG CCACCTTGCTGAAGAGAAGGCACAAACAATTGCAAGAGGAGAACAGTCG CCTCCATAAAAGATTGAAAGAGTTACAAGATGGCAATATCAGCTCAGGA CTTGTGGGTGAAAATGCTTGCACCATGTTTCATCAAAGGATCGTTCATGA AGAAGATTTCCATAACGAAACAGGCCTCCCACTATGA
氨基酸序列为(SEQ ID NO:2)：
MGRGKVQLKRIENPTNRQVTFSKRRNGLLKKAFELSILCDAEVALLIFSS SGKVYQFASHDMDRTVAKYRREVGLPDSSNPQFRTMEFWRSEIDELNRSINT LEARLKHLSGEDILALGMRDLKQLERQLKIGVERVRSRKRRIVSDHATLLKR RHKQLQEENSRLHKRLKELQDGNISSGLVGENACTMFHQRIVHEEDFHNETG LPL
1.2具体克隆基因的过程为：
(1)试验材料陆地棉早熟品种中50、中棉所74以及晚熟品种国欣棉11号、渤棉1号种植于中国农业科学院棉花研究所安阳试验基地，按一般大田管理。取样方式为从子叶展平时开始取棉一苗顶尖，每展平一片真叶取一次样，每次三个生物学重复，所取材料迅速放入液氮中冷冻，保存于-80℃冰箱备用。子叶展平时标记为0TLS(0true-leaf stage)，一片真叶展平时标记为1TLS(1true-leaf stage)，往后类推,一直取到第五片真叶完全展开。植物总RNA提取采用 TIANGEN公司试剂盒。
(2)RNA的提取步骤为：
1)匀浆处理：取适量纤维样品在液氮中迅速研磨成粉末，加入700μL SL (使用前加入β-巯基乙醇)，立即剧烈震荡使样品混匀；
2)12,000rpm离心2min；
3)将上清液转移至过滤柱CS上，12,000rpm离心2min，小心吸取收集管中的上清至新的RNase-Free的离心管中，吸头避免接触收集管中的细胞碎片；
4)加入0.4倍上清体积的无水乙醇，混匀，将混合物转入吸附柱CR3中， 12,000rpm离心15sec，倒掉收集管中的废液，将吸附柱CR3放回收集管中；
5)向吸附柱CR3中加入350μL去蛋白液RW1，12,000rpm离心15sec，倒掉收集管中的废液，将吸附柱CR3放回收集管中；
6)DNase I工作液：取10μL DNase I储存液和70μL RDD溶液轻柔混匀；
7)向CR3中加入80μL的DNase I工作液，室温静止15min；
8)静置完后，向CR3中加入350μL去蛋白液RW1，12,000rpm离心15sec，倒掉收集管中的废液，将吸附柱CR3放回收集管中；
9)向吸附柱CR3中加入500μL漂洗液RW(使用前加入乙醇)，12,000rpm 离心15sec，倒掉收集管中的废液，将吸附柱CR3放回收集管中；
10)重复步骤9；
11)12,000rpm(～13,400×g)离心2min，将吸附柱CR3放入一个新的 RNase-Free离心管中，向吸附膜的中间部位悬空滴加30-50μL RNase-Free ddH2O，室温放置2min，12,000rpm(～13，400×g)离心1min，得到RNA溶液。注意：洗脱缓冲液体积不应少于30μL，体积过小影响回收效率。RNA样品请在 -70℃中保存。如果预期RNA得率大于30μg，可将步骤11中离心得到的RNA溶液再加入吸附柱CR3中，室温放置2min，12,000rpm(～13，400×g)离心1min，得到RNA溶液。
(3)cDNA的合成。将500ng RNA反转录为cDNA，采用Toyobo的反转录试剂盒FSQ-201，反转录体系为：
按下列组份配制RT反应液(反应液配制在冰上进行)：
反转录反应条件如下：
37℃15min(反转录反应)，
98℃5s(反转录酶的失活反应)；
将反转录产物cDNA溶液稀释6倍作为PCR反应模板。
(4)PCR扩增目的基因
在冰上配制以下体系，用TM-1的cDNA为模板扩增目的基因 GhMADS36-A11。根据TaKaRa GXL DNA Polymerase高保真酶说明书，PCR反应体系如下：
PCR扩增程序为：
引物序列：
GhMADS36-A11-F：5′-ATGGGGAGAGGCAAGGTTCAG-3′(SEQ ID NO:3)
GhMADS36-A11-R：5′-ATTATGCAATAGTAACGCCGGTG-3′(SEQ ID NO: 4)
反应结束后4℃保存，用1％的琼脂糖电泳进行检测，条带大小符合预期设计则视为有效结果(如图1所示)。
(5)对目的片段使用胶回收试剂盒进行切胶回收。
(6)将上述胶回收的产物根据 Ultra One Step Cloning Kit试剂盒进行连接T载体构建和转化大肠杆菌。
(7)37℃过夜培养从抗性LB培养基上挑取单克隆后37℃摇菌培养。
(8)菌液PCR验证，挑取阳性克隆样品，送样至金唯智生物科技有限公司测序，测序正确的菌液中加入一定量的甘油，使甘油终浓度在20％左右，-70℃保存。
2 GhMADS36-A11基因棉花VIGS载体构建及侵染
病毒诱导的基因沉默(Virus induced gene silence，VIGS)是一种依赖于植物对病毒的防御机制而产生的，由RNA介导的转录后基因沉默。将目标基因 GhMADS36-A11的一段长度为322bp的片段连接到穿梭质粒pCLCrV中，构建载体(pCLCrV-GhMADS36-A11)转化大肠杆菌(Escherichia Coli)，挑单克隆，送样测序(金唯智，苏州)。测序成功的单克隆，扩摇，提取质粒。将阳性对照载体(pCLCrV-VA)、阴性对照(pCLCrV)、辅助质粒(pCLCrV-VB)以及构建的含有GhMADS36-A11基因目标片段的载体(pCLCrV-GhMADS36-A11) 分别转化农杆菌(Agrobacterium tumefaciens)菌株LBA4404。在子叶展平而真叶还没有长出的时期，采用农杆菌注射、侵染棉花子叶的方法，详细操作步骤参加Gao等人(2013)关于棉花病毒诱导的基因沉默的报道(Gao et al.，Functional genomic analysis of cotton genes withagrobacterium-mediated virus-induced gene silencing.Methods Mol Biol(2013),975157-65；Gu et al.，A versatile system for functional analysis of genes andmicroRNAs in cotton，Plant Biotechnology Journal (2014)12,pp.638–649，通过引用全文并入此处)。侵染后的棉花幼苗避光处理24h，40天后提取棉花叶片的总RNA，利用qRT-PCR技术检测基因的沉默情况。
扩增引物为：
pCLCrV-GhMADS36-A11F(SEQ ID NO:5)：
pCLCrV-GhMADS36-A11R(SEQ ID NO:6)：
3 GhMADS36-A11在不同生育期材料中的表达模式分析
3.1试验材料陆地棉早熟品种中50、中棉所74以及晚熟品种国欣棉11号、渤棉1号种植于中国农业科学院棉花研究所安阳试验基地，按一般大田管理。取样方式为从子叶展平时开始取棉一苗顶尖，每展平一片真叶取一次样，每次三个生物学重复，所取材料迅速放入液氮中冷冻，保存于-80℃冰箱备用。子叶展平时标记为0TLS(0true-leaf stage)，一片真叶展平时标记为1TLS(1true-leaf stage)，往后类推，一直取到第五片真叶完全展开。
3.2取上述不同样品，提取RNA，反转录成cDNA，对不同材料中的 GhMADS36-A11基因的表达量进行分析，GhActin作为内参基因，其荧光定量 PCR的引物如下：
冰上配制qRT-PCR反应体系，进行荧光定量PCR反应。
qRT-PCR反应体系为：
qRT-PCR反应程序：
植物的顶端分生组织类似于动物的干细胞，不断分化出植物的各种地上组织器官，当内外条件合适时，顶端分生组织就会分化出花芽。从qRT-PCR的结果可以看出，GhMADS36-A11基因在茎、顶芽优势表达，在茎中表达量最高(图 2，A)。随着顶芽的发育，GhMADS36-A11基因在不同生育期材料中的表达量都呈上升趋势，从第三片真叶展开时期，在两个早熟品种中50和盐早2号的表达量显著高于晚熟材料中的表达(图2，B)。表明GhMADS36-A11基因可能与陆地棉花芽分化甚至花器官的发育有关。
4病毒诱导的GhMADS36-A11基因沉默
分别用阳性对照(pCLCrVA)，阴性对照(pCLCrV)以及GhMADS36-A11 的病毒载体(pCLCrV-GhMADS36-A11)的农杆菌菌液分别注射棉花幼苗的子叶。注射四周后检测被注射植株的GhMADS36-A11基因表达情况。如图3，被含有pCLCrV-GhMADS36-A11病毒载体侵染后，GhMADS36-A11基因的表达量与阴性对照相比较极显著下调。与空载体pCLCrVA植株相比，GhMADS36-A11基因表达量极显著下调的pCLCrV-GhMADS36-A11植株出现显著晚花表型，并且植株株高较对照显著降低，进一步证明GhMADS36-A11基因与陆地棉生长发育相关(图3，A)。研究表明，GhMADS36-A11在促进棉花开花方面可能具有关键作用，可以作为短季棉培育的有利基因资源。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
<110> 中国农业科学院棉花研究所
<120> 棉花GhMADS36-A11基因在促进植物开花中的应用
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gctgaagttg ccttgcttat cttctcttct tctggaaagg tttaccagtt tgcaagtcat 180
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ccccaattta gaaccatgga gttttggaga agtgagattg atgagttaaa tagatccata 300
aacaccttgg aagctagact taagcactta tctggagaag acatcttagc attaggcatg 360
agagacttga aacagcttga gcgccagttg aaaataggcg ttgaacgtgt ccgctccaga 420
aagagacgca ttgtttcgga tcacgccacc ttgctgaaga gaaggcacaa acaattgcaa 480
gaggagaaca gtcgcctcca taaaagattg aaagagttac aagatggcaa tatcagctca 540
ggacttgtgg gtgaaaatgc ttgcaccatg tttcatcaaa ggatcgttca tgaagaagat 600
ttccataacg aaacaggcct cccactatga 630
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Claims (9)
1.GhMADS36-A11基因在提高促进植物开花中的应用，其特征在于，所述GhMADS36-A11基因具有SEQ ID NO: 1所示的核苷酸序列。
2.根据权利要求1所述的应用，其特征在于，SEQ ID NO: 1所示的核苷酸序列能够编码SEQ ID NO: 2所示氨基酸序列。
3.根据权利要求1或2任一所述的应用，其特征在于：在植物中提高GhMADS36-A11基因的表达量，以促进植物开花。
4.根据权利要求3所述的应用，其特征在于，所述的在植物中提高GhMADS36-A11基因的表达量是通过如下方法实现：提高植物内源GhMADS36-A11基因的表达，或在植物中过表达外源GhMADS36-A11基因。
5.根据权利要求4所述的应用，其特征在于，所述过表达外源GhMADS36-A11基因是指将所述GhMADS36-A11基因利用植物表达载体，经农杆菌介导转化到植物中进行表达。
6.根据权利要求5所述的应用，其特征在于，所述GhMADS36-A11基因通过植物表达载体导入植物细胞、组织或器官。
7.根据权利要求6所述的应用，其特征在于，所述植物表达载体通过一种组成型或诱导型启动子驱动所述GhMADS36-A11基因的表达。
8.根据权利要求7所述的应用，其特征在于，所述组成型启动子是35S启动子。
9.根据权利要求5-8任一所述的应用，其特征在于，所述植物是棉花、玉米、水稻、小麦或拟南芥。
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| CN103074350A (zh) * | 2013-01-04 | 2013-05-01 | 华中师范大学 | 棉纤维特异表达的GhMADS14基因鉴定及应用 |
| CN103360484A (zh) * | 2013-06-24 | 2013-10-23 | 中国农业科学院棉花研究所 | 一种陆地棉蛋白GhMADS22及其编码基因与应用 |
| CN109748958A (zh) * | 2017-11-06 | 2019-05-14 | 中国农业科学院棉花研究所 | 植物发育相关蛋白GhWRKY42及其编码基因和应用 |
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| CN103074350A (zh) * | 2013-01-04 | 2013-05-01 | 华中师范大学 | 棉纤维特异表达的GhMADS14基因鉴定及应用 |
| CN103360484A (zh) * | 2013-06-24 | 2013-10-23 | 中国农业科学院棉花研究所 | 一种陆地棉蛋白GhMADS22及其编码基因与应用 |
| CN109748958A (zh) * | 2017-11-06 | 2019-05-14 | 中国农业科学院棉花研究所 | 植物发育相关蛋白GhWRKY42及其编码基因和应用 |
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| NONE: "NCBI Reference Sequence: XM_016853524.1", 《GENBANK》 * |
| WENXIANG ZHANG等: "Molecular Cloning and Function Analysis of Two SQUAMOSA‐Like MADS‐Box Genes From Gossypium hirsutum L.", 《JOURNAL OF INTEGRATIVE PLANT BIOLOGY》 * |
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