CN113913440A - GhD1119基因调控陆地棉开花方面的应用 - Google Patents
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Abstract
本发明提供了GhD1119基因调控陆地棉开花方面的应用，将GhD1119基因构建到过表达载体和CRISPR基因编辑载体中，进行棉花遗传转化，验证该基因功能。发现陆地棉GhD1119基因能够促进过表达转基因棉花株系提早现蕾2～12d，提早开花8～24d，使得CRISPR棉花编辑株系现蕾期推迟3～9d。这将为挖掘棉花调控开花时间相关的重要基因，解析棉花开花调控途径提供有效支撑，为利用这些基因改良优质中晚熟品种的早熟性，创制出早熟优质棉花种质资源，实现早熟性和优良纤维品质的协同改良，进而培育出早熟性强的优质棉品种具有重要作用。
Description
技术领域
本发明属于棉花种质资源培育和分子生物学领域，具体涉及：GhD1119调控陆地棉开花方面的应用。
背景技术
棉花(Gossypium spp.)是可以提供天然纤维的重要经济作物，在我国国民经济中占有重要地位。当前我国棉花种植主要分布于西北内陆棉区以及黄河、长江流域棉区。以新疆为代表的西北内陆棉区秋季降温快、初霜出现早，春季升温慢、终霜结束晚。在这样的气候条件下，种植早熟棉品种可以有效利用该地区有限的无霜期进行生产。与此同时，西北内陆棉区开展机械化采收也需要更早熟的优质棉品种[1]。在黄河、长江流域棉区较突出的问题是粮棉争地矛盾，种植早熟棉品种既可以实现麦(油)棉一年两熟生产，又能增加农民种植收益[2]。因此，针对我国棉花种植区域特点，关注棉花早熟性状选择，已成为我国棉花育种的重要目标之一。棉花早熟性是一个受环境影响的综合性状，主要表现在果枝始节位、现蕾期、开花期、盛絮期、霜前花率等方面[3-4]，其中开花和吐絮(棉铃开裂)集中程度不仅影响棉花收获时间，还影响能否适时开展机械化采收、麦(油)后直播。然而，我国现有棉花品种纤维品质整体上呈现早熟棉低于中晚熟棉品种，如何实现棉花早熟与纤维优质的同步改良，是我们需要解决的重要问题。鉴于传统育种手段很难在解决此问题上有突破的考虑，我们希望通过挖掘、鉴定调控棉花开花及吐絮的关键基因，在阐明其调控机制的基础上，进一步利用这些基因改良优质中晚熟品种的早熟性，创制出早熟优质棉种质资源，实现早熟性和优良纤维品质的协同改良，进而培育出早熟性强的优质棉品种具有重要的意义。
植物开花是育种家选育品种关注的重要农艺性状。Yanofsky等人[5]首次从拟南芥中克隆出了控制花器官发育的AGAMOUS基因，之后对开花机制的研究有了不断发展。通过分子生物学和生理学等方法研究开花机制发现，开花受到各种环境及内源基因等调控[6-7]。迄今为止，科学家们已经在植物中，发现了8种调控开花的途径，包括光周期、春化、昼夜节律钟、赤霉素、自主、环境、年龄和蔗糖途径[8-9]。这些调控途径整合了环境信号和内源信号，形成一个复杂的激活与抑制、正负反馈共存的调控网络，并通过作用于一些关键节点基因，如SOC1(SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1)、FT(FLOWERING LOCUS T)、FLC(FLOWERING LOCUS C)等，激活花分生组织特性基因AP1(APETALA1)、LFY(LEAFY)的表达，启动植物开花进程[8,10]。
棉花的野生祖先属于多年生、无限生长植物，生长地域分布广泛，其具有复杂的生长模式[11]。近十年来，科学家们先后研究了调控陆地棉开花的GhCOLs[12]、GhPEBPs[13]、GhMIKCs[14]和GhAAIs[15]等基因家族，通过遗传转化拟南芥初步验证了部分基因控制棉花开花的功能[16-17]。另一些研究则通过结合陆地棉自然突变、早熟性状QTL定位或花发育转录组等方法，研究基因GhCEN-Dt[18]、GhEMF2B[19]和GhCAL[20]在调控棉花开花进程中的生物学功能。从现有文献报道来看，棉花调控开花的关键基因尚需进一步挖掘，其参与调控开花的分子机制有待继续探寻。
本发明以陆地棉GhD1119(GH_D03G1119)基因为切入点，通过载体构建、棉花转化和CRISPR基因编辑等方法研究陆地棉GhD1119基因的功能，旨在为基因资源挖掘利用，探究棉花的开花调控通路，促进早熟棉的培育提供理论和技术依据。
发明内容
本发明要解决的关键技术问题在于提供GhD1119调控陆地棉开花方面的应用。为解决上述技术问题，本发明采用如下技术方案：
1.陆地棉GhD1119的CDS序列如SEQ ID NO.1所示，全长转录本序列如序列表SEQID NO.2所示。
2.陆地棉GhD1119基因表达载体构建方法，包括：
(1)植株材料和试剂选择；(2)RNA提取与反转录；(3)引物设计和基因克隆；(4)构建过表达载体及CRISPR基因编辑载体。
3.陆地棉GhD1119基因功能验证方法，包括：
(1)棉花遗传转化、CRISPR基因编辑陆地棉；(2)过表达转基因棉花获得及表型分析；(3)CRISPR基因编辑陆地棉植株获得及表型分析；(4)GhD1119基因在转基因材料中影响其他开花调控基因表达情况。
4.陆地棉GhD1119基因的应用，所述基因的应用是促进陆地棉开花。
5.陆地棉GhD1119基因的应用，所述基因通过正调控SOC1和SVP基因促进陆地棉开花。
有益效果：本发明将GhD1119基因构建过表达载体和CRISPR基因编辑载体，在棉花中过表达基因GhD1119发现，转基因棉花比对照早现蕾2～12d，早开花8～24d。利用CRISPR基因编辑GhD1119基因，发现编辑植株比对照组植株迟现蕾3～9d。说明，GhD1119基因能够促进陆地棉早开花。
进一步利用荧光定量发现，在过表达棉花转基因株系中表达量明显增加的有SOC1基因和SVP基因，略微增加的有FT基因和LFY基因，而CAL基因表达量下降。在CRISPR基因编辑棉花材料中，SOC1基因表达量显著增加，FT基因表达量有所增加，CAL基因和LFY基因表达量下降，SVP基因表达量显著下降。推测FT基因和SOC1基因处于GhD1119基因调控通路的上游，GhD1119基因在开花期之前正调控SVP基因和LFY基因。
过表达基因GhD1119能够促进棉花早现蕾2～12d，早开花8～24d，CRISPR基因编辑GhD1119能够使得陆地棉迟现蕾3～9d。说明基因GhD1119能够促进植株开花。此外，基因GhD1119正调控SVP基因和LFY基因。
附图说明
图1为pBI121载体结构图；
图2为CRISPR基因编辑结构图；
图3为过表达转基因棉花株系鉴定及表型分析；其中，A：过表达转基因棉花阳性株系PCR检测；B：野生型及转基因棉花阳性株系开花期表型；C：目的基因在野生型及转基因棉花阳性株系中的相对表达量；D：野生型及转基因棉花阳性株系现蕾期统计；E：野生型及转基因棉花阳性株系开花期统计。
图4为CRISPR基因编辑陆地棉株系鉴定及表型分析；其中，A：野生型及CRISPR基因编辑棉花阳性株系现蕾期表型；B：CRISPR基因编辑棉花阳性株系PCR检测；C：野生型及CRISPR基因编辑棉花阳性株系现蕾期统计。
图5为过表达转基因棉花和中CRISPR基因编辑棉花阳性株系中SOC1、FT、CAL、LFY及SVP基因表达量分析；其中，A-E：过表达转基因棉花阳性株系中SOC1、FT、CAL、LFY及SVP基因表达量分析；F-J：CRISPR基因编辑棉花阳性株系中SOC1、FT、CAL、LFY及SVP基因表达量分析。
具体实施方法
本发明专利下述实施例中使用方法和装置，如无特殊说明，均为常规方法和装置；所用器材、试剂均为试剂公司购买的常规器材和试剂。为使本发明专利的目的、技术方案和优点更加清楚，下面结合具体实施例对本发明专利的具体实施方式进行详细说明。这些优选实施方式的示例在具体实施例中进行了例示。在此，还需要说明的是，为了避免因不必要的细节而模糊了本发明专利的技术方案，在实施例中仅仅示出了与根据本发明专利的方案密切相关的技术方案和/或处理步骤，而省略了关系不大的其他细节。
实施例1
本实施例提供陆地棉GhD1119基因克隆和载体构建方法，包括：
1.植株材料和试剂选择
本发明所使用的陆地棉中棉所113，jin668由本实验室保存，种植于实验室植物培养间。本发明采用多糖多酚植物总RNA提取试剂盒、快速质粒小提试剂盒、通用型DNA纯化回收试剂盒均购自天根生化科技有限公司。反转录试剂盒Transcriptor FirstStrand cDNASynthesis Kit和荧光定量试剂盒FastStart Essential DNA Green Master购买自罗氏公司。抗生素购自索莱宝公司。限制酶购自宝日医生物技术有限公司。Taq预混液购自艾科瑞公司。
pGM-T克隆试剂盒，TOP10感受态细胞购自天根生化科技有限公司，过表达载体pBI121载体由本实验保存(图1)，CRISPR基因编辑载体(图2)购自武汉天问生物科技有限公司。GV3101农杆菌感受态细胞购买自上海唯地生物技术有限公司。
2.RNA提取与反转录
于陆地棉中棉所113四叶期时采集叶片，使用天根多糖多酚植物总RNA提取试剂盒提取RNA后，使用罗氏反转录试剂盒Transcriptor FirstStrand cDNA Synthesis Kit将RNA反转录为cDNA，步骤详见其说明书。
结果显示：提取中棉所113四叶期幼叶RNA，通过超微量紫外分光光度计检测显示，各RNA 260/280均在1.9～2.1之间，纯度较好。使用1.2％琼脂糖凝胶，150V电泳15min，检测结果同样显示RNA质量较好，可用于后续实验。
3.引物设计和基因克隆
使用NCBI Primer-BLAST(https://www.ncbi.nlm.nih.gov/tools/primer-blast/)设计克隆引物、过表达引物和CRISPR编辑引物，结果见表1。
表1 GhD1119克隆引物、过表达引物(含酶切位点及保护碱基)和CRISPR编辑引物
使用艾科瑞Taq预混液PCR扩增目的基因，利用天根通用型DNA纯化回收试剂盒回收目标片段，通过天根pGM-T克隆试剂盒连接目的基因片段与克隆载体，并转化TOP10大肠杆菌感受态进行蓝白斑筛选，最终使用天根快速质粒小提试剂盒提取阳性克隆质粒后送生工生物工程有限公司测序。具体步骤见各试剂盒说明书。
结果显示：以陆地棉中棉所113的cDNA为模板，使用克隆引物，PCR扩增GhD1119基因，将扩增产物于1.5％的琼脂糖凝胶，100V电泳30min。利用天根通用型琼脂糖凝胶回收试剂盒回收扩增产物后，16℃过夜连接回收的目的基因扩增产物和pGM-T载体。第二天将过夜连接的产物转化TOP10大肠杆菌，12～16h后进行蓝白斑筛选，将白斑挑至5ml含有氨苄青霉素(Amp)的LB液体培养基中，37℃，150rpm，12h后提取质粒，送测序。
4.过表达载体和CRISPR基因编辑载体的构建
以上述3中阳性质粒为模板，带酶切位点过表达引物扩增，对扩增产物与过表达载体pBI121用Xba I和Sac I分别进行双酶切。使用根通用型DNA纯化回收试剂盒回收酶切产物，将目的基因与过表达载体pBI121连接，并送测序。成功获得GhD1119基因的过表达载体。
根据GhD1119基因的mRNA序列及对应的基因组序列信息，设计2个CRISPR靶位点，以提高基因打靶效率。根据靶位点设计靶位点PCR扩增引物。引物由天一辉远生物科技有限公司合成。引物合成后，采用重叠延伸PCR扩增出含有靶位点的片段，PCR片段利用南京诺维赞生物科技有限公司的重组酶克隆至最终的CRISPR表达载体。
实施例2
本实施例提供陆地棉GhD1119基因在调节开花方面的应用，包括：
1.过表达和CRISPR基因编辑GhD1119基因的陆地棉转基因株系获得
将实施例1中构建完成的GhD1119基因过表达和CRISPR基因编辑载体，进行棉花下胚轴遗传转化：将剥好的棉花种子用升汞杀菌，无菌水清洗后放入无菌苗培养基中，28℃暗培养1d，挑去种皮，将苗扶正，再28℃暗培养4～5d；农杆菌菌液划线，2d后挑取单克隆，摇菌过夜，离心弃上清，加入10mL的MGL和25μL的AS，震荡使菌液悬浮，28℃摇床，200rpm/min活化至少30min；将下胚轴切成小茎段，用活化后的农杆菌侵染，弃菌液，并吹干；将下胚轴平铺在放有滤纸的共培养培养基中，20℃暗培养1～2d；下胚轴转入到2,4-D培养基中，放入光照培养室，20～30d左右继代一次；愈伤组织长成米粒状颗粒，转入分化培养基中(15d继代一次)，进一步分化成胚状体；将分化出的小苗继代到生根培养基中，直至长成生根良好健康的小苗；将苗子转到营养液中，进行炼苗，一周左右后，种到温室。
2.过表达转基因棉花阳性株系获得及表型分析
在陆地棉jin668中过表达GhD1119基因，共获得17个独立过表达株系(图3A)，在上述转基因株系中检测目标基因表达情况，发现9个株系中GhD1119基因呈现上调表达(图3C)。其中六个株系表现出明显的早现蕾、早开花(图3B)，与比对照组植株相比提早现蕾2～12d(图3D)，提早开花8～24d(图3E)。
3.CRISPR基因编辑棉花阳性株系获得及表型分析
对陆地棉jin668中的GhD1119基因进行编辑，共获得6株独立的基因编辑株系(图4B)。其中三个株系表现出明显的晚现蕾(图4A)，与比对照组植株相比迟现蕾3～9d(图4C)。
4.GhD1119基因与其他开花调控基因的调控关系
为了进一步探究GhD1119基因与SVP、CAL、FT、SOC1、LFY基因间的调控关系，使用NCBI Primer-BLAST设计引物(表2)，利用荧光定量分析转基因过表达和基因编辑陆地棉中各基因的表达情况。在过表达棉花转基因株系中表达量明显增加的有SOC1基因和SVP基因(图5A和5E)，略微增加的有FT基因和LFY基因(图5B和5D)，而CAL基因表达量下降(图5C)。在CRISPR基因编辑棉花材料中，SOC1基因表达量显著增加(图5F)，FT基因表达量有所增加(图5G)，CAL基因和LFY基因表达量下降(图5H和5I)，SVP基因表达量显著下降(图5J)。推测FT基因和SOC1基因处于GhD1119基因调控通路的上游，GhD1119基因在开花期之前正调控SVP基因和LFY基因。
表2陆地棉中SVP、CAL、FT、SOC1、LFY基因荧光定量引物
以上所述仅是本申请的具体实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本申请原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本申请的保护范围。
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<110> 甘肃农业大学
<120> GhD1119基因调控陆地棉开花方面的应用
<160> 2
<210> 1
<211> 696
<212> DNA
<213> 陆地棉（Gossypium hirsutum）
<400> 1
1 ATGGGAAGGG GTAGGGTTCA ACTGAAGAGA ATAGAGAATA AGATCAACAG GCAAGTGACG
61 TTTTCGAAAC GAAGGTCGGG CTTGTTGAAG AAAGCCCATG AAATCTCTGT GCTTTGTGAT
121 GCTCAAGTCG CTTTGATGGT CTTCTCTTCG AAAGGCAAAC TCTTTGAATA CGCGACTGAG
181 TCTTGCATGG AAAGGATCCT TGAACGATAT GAAAGAAACT CGTATACTGA GATCCAATGT
241 GCTACAGATG AAATTCAACA AAATGGAAAC TGGACCTGGG AACATGCAAA ACTTAAAGCT
301 AGAATGGAGA CTTTACAAAG AAACCTGAGG CATTACGAAG GAGAAGATAT CCAGAATTTG
361 AGTCTTAGAG AGCTTCAAAA TTTGGAGCAA CAACTTGATT CTGCCCTTAA ACGCATAAGA
421 TCCAGAAAGA ATCAACTTAT GCTTGAATCA ATTTCTGAGC TTCAGAAAAA GGACAAAGCA
481 CTGCAAGAAC AGAATAACAT ACTTGCAAAG AAGCTGAAGG AAAAGGAGAA AACTAATGTG
541 GAGCAGGCAC ATTGGCAGCT GAACAACAAT TGCCAAGATT CATCCTCCAT GCTTCTGCCC
601 CTTAACATCA GCTCCAATGG AAGGGAGAAG GAAGATAATG AAACCACCAA CAGTGGCGTC
661 TTGCTGCCAT GGATGATTCG CCACCACCTT GAATAA
<210> 2
<211> 896
<212> DNA
<213> 陆地棉（Gossypium hirsutum）
<400> 2
1 CCTATATATA CACACGCAGA TTTACACCAT TTCTTTCGCA ACCTGCCCAA ACATACCCTT
61 ATAACTCTCC TTCCGCTGTC AGTCGTATAA AGAGAAAGAA ATGGGAAGGG GTAGGGTTCA
121 ACTGAAGAGA ATAGAGAATA AGATCAACAG GCAAGTGACG TTTTCGAAAC GAAGGTCGGG
181 CTTGTTGAAG AAAGCCCATG AAATCTCTGT GCTTTGTGAT GCTCAAGTCG CTTTGATGGT
241 CTTCTCTTCG AAAGGCAAAC TCTTTGAATA CGCGACTGAG TCTTGCATGG AAAGGATCCT
301 TGAACGATAT GAAAGAAACT CGTATACTGA GATCCAATGT GCTACAGATG AAATTCAACA
361 AAATGGAAAC TGGACCTGGG AACATGCAAA ACTTAAAGCT AGAATGGAGA CTTTACAAAG
421 AAACCTGAGG CATTACGAAG GAGAAGATAT CCAGAATTTG AGTCTTAGAG AGCTTCAAAA
481 TTTGGAGCAA CAACTTGATT CTGCCCTTAA ACGCATAAGA TCCAGAAAGA ATCAACTTAT
541 GCTTGAATCA ATTTCTGAGC TTCAGAAAAA GGACAAAGCA CTGCAAGAAC AGAATAACAT
601 ACTTGCAAAG AAGCTGAAGG AAAAGGAGAA AACTAATGTG GAGCAGGCAC ATTGGCAGCT
661 GAACAACAAT TGCCAAGATT CATCCTCCAT GCTTCTGCCC CTTAACATCA GCTCCAATGG
721 AAGGGAGAAG GAAGATAATG AAACCACCAA CAGTGGCGTC TTGCTGCCAT GGATGATTCG
781 CCACCACCTT GAATAACCTA AACAAATAAT GGAGACTCCT TTTATTTTTA GTATATCTTT
841 AATATGTATT ATATGTTTGT TTTCATGAAT AGATACAAAC ATACTCATCA CTTCGA
Claims (6)
1.陆地棉GhD1119基因，其特征在于所述GhD1119基因的CDS序列如SEQ ID NO.1所示，全长转录本序列如序列表SEQ ID NO.2所示。
2.权利要求1所述的陆地棉GhD1119基因的应用，其特征在于所述GhD1119基因促进陆地棉早开花的应用。
3.根据权利要求2所述的应用，其特征在于所述促进陆地棉早开花的应用采用过表达GhD1119基因实现。
4.陆地棉GhD1119基因表达载体构建方法，包括：
(1)植株材料和试剂选择；(2)RNA提取与反转录；(3)引物设计和基因克隆；(4)构建过表达载体及CRISPR基因编辑载体。
5.陆地棉GhD1119基因功能验证方法，包括：
(1)棉花遗传转化、CRISPR基因编辑陆地棉及qPCR检测；(2)过表达转基因棉花获得及表型分析；(3)CRISPR基因编辑陆地棉植株获得及表型分析；(4)GhD1119基因影响其他开花调控基因的关系。
6.权利要求1所述的陆地棉GhD1119基因的应用，其特征在于所述GhD1119基因正调控SOC1和SVP基因。
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