CN117286149A - 过表达GhCIB1基因在促进棉花开花中的应用 - Google Patents
过表达GhCIB1基因在促进棉花开花中的应用 Download PDFInfo
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Abstract
本发明提供了过表达GhCIB1基因在促进棉花开花中的应用,属于基因工程技术领域。本发明从陆地棉中克隆出GhCIB1基因,该基因通过构建过表达载体,在拟南芥中异源表达得到的过表达转基因株系相比于野生型开花提前,说明GhCIB1基因在控制棉花开花期方面起到了重要的调控作用。
Description
技术领域
本发明涉及基因工程技术领域,特别是涉及过表达GhCIB1基因在促进棉花开花中的应用。
背景技术
棉花是我国重要的经济作物和关系国民经济的战略重要物资。我国是全球最大的棉花生产国和消费国,在纺织服装的出口量也位居世界前列。早熟陆地棉生育周期短,生长发育快,利用其适宜晚春播、初夏播和夏播的特性,可以和冬季作物如冬小麦、油麦菜等实现粮棉轮作,通过优化作物种植指数,有效提高土地复种指数(王雪颖等,2022)。此外,早熟棉的选育可以有效提高棉花的霜前花率,在光热条件差和气温变化大的高纬度地区如辽宁、甘肃等地区提高棉花的品质(喻树迅等,1991)。因此,鉴定棉花开花相关基因,阐明其调控机制,并创制早熟棉优异种质资源,对于我国棉花产业健康稳定发展具有重要的意义。
研究人员利用双杂交筛库的方法首次在植物中筛选出与蓝光受体CRY2蓝光特异性相互作用的bHLH蛋白,命名为CIB1。CIB1位于bHLH转录因子家族第18亚族,该亚族包括17个成员,其中6个成员与CIB1亲缘关系更近,分别命名为CIB2、CIB3、CIB4、CIB5、CIL1(CIB1Like)和CIL2。CIB1的N端结构域与CRY2仅在蓝光下发生特异性相互作用,并通过BiFC实验进一步验证,长日照下cibl功能缺失突变体没有晚花表型,推测植物中存在与CIB1功能冗余的bHLH蛋白。
通过对cib2、cib3、cib5和cill突变体研究发现,拟南芥的单突变体都没有明显的开花表型(LIU et al.,2008)。但在长日照野生型背景下过表达CIB1、CIB2、CIB4、CIB5和CIL1,拟南芥开花显著早于野生型,进一步研究发现,CIB1,CIB2、CIB4和CIB5都具有促进开花的功能,且CIB2和CIB5都与CRY2互作,CIB4不与CRY2发生体内互作,但CIB4促进开花的功能却依赖于CRY2(LIU et al.,2013)。进一步研究发现,CIB1能够与CIB2、CIB4和CIB5形成二聚体,结合FT启动子中的E-box基序;CIB1、CIB2、CIB4和CIB5过表达都引起转基因拟南芥中FT基因表达量上升,植株提早开花(LIU et al.,2013)。结果表明,上述CIB蛋白存在功能冗余,并形成异源二聚体,从而促进FT的mRNA的表达,促进植物开花。并且CIB1蛋白在蓝光下积累,蓝光受体ZTL和LKP2在蓝光下抑制了CIB1的泛素化降解(LIU et al.,2018)。此外,CIB1、CO及CRY2可形成复合体CRY-CIB1-CO,促进FT的转录(LIU et al.,2018)。
发明内容
为了解决上述问题,本发明提供了过表达GhCIB1基因在促进棉花开花中的应用,本发明从陆地棉中克隆出GhCIB1基因,该基因通过构建过表达载体,在拟南芥中异源表达得到的过表达转基因株系相比于野生型开花提前,说明GhCIB1基因在控制棉花开花期方面起到了重要的调控作用。
为了实现上述目的,本发明提供如下技术方案:
本发明提供了过表达GhCIB1基因在促进棉花开花中的应用,所述GhCIB1基因的核苷酸序列如SEQ ID No.1所示。
本发明还提供了过表达GhCIB1基因在促进棉花生殖生长中的应用,所述GhCIB1基因的核苷酸序列如SEQ ID No.1所示。
优选的,所述GhCIB1基因的氨基酸序列如SEQ ID No.2所示。
本发明的有益效果为:
本发明从陆地棉中克隆出GhCIB1基因,该基因通过构建过表达载体,在拟南芥中异源表达得到的过表达转基因株系相比于野生型开花提前,说明GhCIB1基因在控制棉花开花期方面起到了重要的调控作用。
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,下面将对实施例中所需要使用的附图作简单地介绍。
图1为GhCIB1基因在早熟材料顶芽中的表达量;
图2为GhCIB1基因在晚熟材料中的表达量;
图3为GhCIB1超表达拟南芥表型;
图4为GhCIB1超表达拟南芥抽薹天数统计;
图5为GhCIB1超表达拟南芥抽薹时莲座叶数量统计。
具体实施方式
本发明提供了过表达GhCIB1基因在促进棉花开花中的应用,所述GhCIB1基因的核苷酸序列如SEQ ID No.1所示。
本发明还提供了过表达GhCIB1基因在促进棉花生殖生长中的应用,所述GhCIB1基因的核苷酸序列如SEQ ID No.1所示。
SEQ ID No.1:
ATGAATAGAGCATTAGCAGAGACATTGACAGTGCTGGATAGACAAAGAGCACGCGTGAAATGGCAACAAGAAAGCTATTTCAGCGAATTAAGTGGGGTGTTTTCGACCCAAACCAGCACCCATGTTCATGGCTTTCAGGGTGATTTAATAAGCGATGAGTCGGTGTTGGATGACTTGGTGATGACTCGGCAAGTGAAGCCTGACCCTAGCTTGGAGACATCGTGGCCGGAGTTGGGGAAGGTTGACATGGCTGGCATGGGGTTTGGGCCATGCGGCTACAGTAATGGACCGAGTTTTGATATGAATTACGCCATTTCTAGGACTTTTAGCTGCCCTCCAGCTGTGGCGGCGACTATAGCCAAAGAGGCGATGGAGGTCAAGGGCAAAGAGTCGATTGTCTCTGAGAACATGGGTTCAGCCGTTGCAAGAGAAAGCTCCAAGAAAAGGAAAGCTGACAAGTTACATAATTCAAAGGTTGCTGCGGAAGATGACTCTAAGAAGACCAAAGCCTGTGGAGAAGAAGAGGAAGAGTCAAAAATTACAGGACCACCCAACACCAACAAAAGCAGCACCAAGCAGGAACCTTCTGCTGATACTTCCAAGGAGAATTCAAAGCTCACTGAGGTTCAAAAGCCTGATTATATTCACGTTAGGGCGCGTCGTGGCCAAGCCACTGATAGCCATAGCTTAGCTGAGAGAGTTAGAAGGGAAAAGATCAGTGAAAGAATGAAATATCTGCAAGATTTAGTTCCAGGGTGTAATAAAATCACTGGGAAAGCTGGAATGCTTGATGAAATAATCAATTATGTTCAATCTCTTCAACGACAAGTTGAGTTCCTATCCATGAAACTAGCTGCTGTAAATCCAAGGCTTGATTTCGACATTGACAATCTTTTTGCCAAAGATGTATTTCCTCCTTGTATGACTAATTTCCCAACAGTTGGGATGTCATCAGAAATGGCAAATCCTTCTTATCTTCACTTCAATCCAGTTCAACAAGTGGTTGCTTGTTCTGGAGTTGAAATGGGATTGAACTCTCCGGACATTGCTCTTCGGAGAACCATTAGTGCTCCCAAATCGACAATCCCGGACGCATCATTTCTAGATACATCCTGTTTCACTCAAATTCAGCCCTCACCAACATGGGACGTTGAATTGCAAAACGTTTACAATGTGGCATTCGAACATGGAAGATCAACAACACCCTTCCCATCTCAACCATTTGCAGCTGCAGGTTCCATTGAAGCTAGCCATCTAAAGATGGAGATGTGA。
在本发明中,所述GhCIB1基因的氨基酸序列如SEQ ID No.2所示,具体如下:
MNRALAETLTVLDRQRARVKWQQESYFSELSGVFSTQTSTHVHGFQGDLISDESVLDDLVMTRQVKPDPSLETSWPELGKVDMAGMGFGPCGYSNGPSFDMNYAISRTFSCPPAVAATIAKEAMEVKGKESIVSENMGSAVARESSKKRKADKLHNSKVAAEDDSKKTKACGEEEEESKITGPPNTNKSSTKQEPSADTSKENSKLTEVQKPDYIHVRARRGQATDSHSLAERVRREKISERMKYLQDLVPGCNKITGKAGMLDEIINYVQSLQRQVEFLSMKLAAVNPRLDFDIDNLFAKDVFPPCMTNFPTVGMSSEMANPSYLHFNPVQQVVACSGVEMGLNSPDIALRRTISAPKSTIPDASFLDTSCFTQIQPSPTWDVELQNVYNVAFEHGRSTTPFPSQPFAAAGSIEASHLKMEM。
为了进一步说明本发明,下面结合实施例对本发明进行详细地描述,但不能将它们理解为对本发明保护范围的限定。
实施例1
一、试验材料
1.1棉花材料
本发明选取的棉花材料为陆地棉早熟品种中棉所36和晚熟品种国欣棉11,其中中棉所36和国欣棉11在开花时间和生育期存在着极显著差异,种植于中国农业科学院棉花研究所试验田(河南省安阳市白璧镇),管理措施为正常大田管理。取样方式为两个棉花品种一叶期到五叶期的幼芽,至于液氮中,在提取样品RNA之前放置-80℃留存。
1.2试剂和耗材
限制性内切酶,修饰酶、PCR反应体系相关酶、同源重组酶、胶回收试剂盒、克隆试剂盒、质粒小提试剂盒购自诺唯赞生物科技有限公司,荧光定量试剂盒购自康为世纪生物科技有限公司公司,RNA提取试剂盒购自北京天根生化科技公司
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、氯化钠等为国产分析纯,卡那霉素等索莱宝生物有限公司,大肠杆菌感受态细胞DH5α和农杆菌感受态购自擎科生物公司
培养基:LB液体培养基:胰蛋白胨(Tryptone)10g/L、酵母提取物(Yeast extract)5g/L、氯化钠(NaCl)10g/L;LB固体培养基:胰蛋白胨(Tryptone)10g/L、酵母提取物(Yeastextract)5g/L、氯化钠(NaCl)10g/L、琼脂粉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度时加入相应浓度抗生素,摇匀后铺平板;1/2MS固体培养基:1/2MS22g/L,琼脂粉(agarpowder)8g/L,蔗糖(sucrose)30g/L。
主要仪器:PCR扩增仪(BIO-RAD)、高速离心机(HettichMIKRO 200R)、电泳设备(BIO-RAD)、凝胶成像系统(BIO-RAD)、荧光定量PCR仪(ABI7500)、电热恒温培养箱(上海森信)、恒温培养振荡器(上海智城)、人工气候试验箱(赛福)、人工气候室。
二、试验方法和结果
2.1基因克隆和序列分析
从CottonFGD(http://www.cottonfgd.org/)上获得GhCIB1(Gohir.D12G028700)基因的CDS序列和编码的氨基酸序列,其开放阅读框为1272bp,编码423个氨基酸。我们克隆得到该基因,并将该基因命名为GhCIB1,并对其功能进行研究。
GhCIB1开放阅读框序列为(SEQ ID No.1):
ATGAATAGAGCATTAGCAGAGACATTGACAGTGCTGGATAGACAAAGAGCACGCGTGAAATGGCAACAAGAAAGCTATTTCAGCGAATTAAGTGGGGTGTTTTCGACCCAAACCAGCACCCATGTTCATGGCTTTCAGGGTGATTTAATAAGCGATGAGTCGGTGTTGGATGACTTGGTGATGACTCGGCAAGTGAAGCCTGACCCTAGCTTGGAGACATCGTGGCCGGAGTTGGGGAAGGTTGACATGGCTGGCATGGGGTTTGGGCCATGCGGCTACAGTAATGGACCGAGTTTTGATATGAATTACGCCATTTCTAGGACTTTTAGCTGCCCTCCAGCTGTGGCGGCGACTATAGCCAAAGAGGCGATGGAGGTCAAGGGCAAAGAGTCGATTGTCTCTGAGAACATGGGTTCAGCCGTTGCAAGAGAAAGCTCCAAGAAAAGGAAAGCTGACAAGTTACATAATTCAAAGGTTGCTGCGGAAGATGACTCTAAGAAGACCAAAGCCTGTGGAGAAGAAGAGGAAGAGTCAAAAATTACAGGACCACCCAACACCAACAAAAGCAGCACCAAGCAGGAACCTTCTGCTGATACTTCCAAGGAGAATTCAAAGCTCACTGAGGTTCAAAAGCCTGATTATATTCACGTTAGGGCGCGTCGTGGCCAAGCCACTGATAGCCATAGCTTAGCTGAGAGAGTTAGAAGGGAAAAGATCAGTGAAAGAATGAAATATCTGCAAGATTTAGTTCCAGGGTGTAATAAAATCACTGGGAAAGCTGGAATGCTTGATGAAATAATCAATTATGTTCAATCTCTTCAACGACAAGTTGAGTTCCTATCCATGAAACTAGCTGCTGTAAATCCAAGGCTTGATTTCGACATTGACAATCTTTTTGCCAAAGATGTATTTCCTCCTTGTATGACTAATTTCCCAACAGTTGGGATGTCATCAGAAATGGCAAATCCTTCTTATCTTCACTTCAATCCAGTTCAACAAGTGGTTGCTTGTTCTGGAGTTGAAATGGGATTGAACTCTCCGGACATTGCTCTTCGGAGAACCATTAGTGCTCCCAAATCGACAATCCCGGACGCATCATTTCTAGATACATCCTGTTTCACTCAAATTCAGCCCTCACCAACATGGGACGTTGAATTGCAAAACGTTTACAATGTGGCATTCGAACATGGAAGATCAACAACACCCTTCCCATCTCAACCATTTGCAGCTGCAGGTTCCATTGAAGCTAGCCATCTAAAGATGGAGATGTGA。
GhCIB1编码的氨基酸序列为(SEQ ID No.2):
MNRALAETLTVLDRQRARVKWQQESYFSELSGVFSTQTSTHVHGFQGDLISDESVLDDLVMTRQVKPDPSLETSWPELGKVDMAGMGFGPCGYSNGPSFDMNYAISRTFSCPPAVAATIAKEAMEVKGKESIVSENMGSAVARESSKKRKADKLHNSKVAAEDDSKKTKACGEEEEESKITGPPNTNKSSTKQEPSADTSKENSKLTEVQKPDYIHVRARRGQATDSHSLAERVRREKISERMKYLQDLVPGCNKITGKAGMLDEIINYVQSLQRQVEFLSMKLAAVNPRLDFDIDNLFAKDVFPPCMTNFPTVGMSSEMANPSYLHFNPVQQVVACSGVEMGLNSPDIALRRTISAPKSTIPDASFLDTSCFTQIQPSPTWDVELQNVYNVAFEHGRSTTPFPSQPFAAAGSIEASHLKMEM。
2.2模式分析
研究表明,棉花花芽分化与早熟性状密切相关,是棉花由营养生长过渡到生殖生长的标志。直接影响开花时间。我们选取早熟品种中棉所36和晚熟品种国欣棉11,提取了一叶期到五叶期的花芽RNA,采用qRT-PCR技术对GhCIB1表达量进行检测,发现该基因在早熟品种中36一叶期到五叶期的表达量是均显著高于晚熟品种国欣棉11。
2.2.1取样、磨样
选取中棉所36和国欣棉11的一叶期到五叶期的顶芽置于液氮中,使用研钵和研杵将其研磨至粉末,取大约1g样品于1.5ML离心管中。
2.2.2提取
RNA提取利用试剂盒FastPure Universal PlantTotal RNAIsolationKit(诺维赞,中国南京)进行,具体步骤如下:
(1)实验在常温下进行,在加有植物组织的离心管中立即加入600μl Buffer PSL(多酚多糖植物),剧烈涡旋振荡30sec,使样本与裂解液充分混合均匀,12,000rpm(134,00×g)离心5min,立即进行后续操作。
(2)取上清约500μl至FastPure gDNA-Filter Columns III(FastPure gDNA-Filter Columns III
已放入收集管中)中,12,000rpm(13,400×g)离心30sec,弃掉FastPure gDNA-Filter Columns III,收集滤液。
(3)向收集管中加入0.5倍滤液体积的无水乙醇(约250μl,根据上清实际情况调整),振荡混匀15sec。将上述混合液转移至FastPure RNA Columns V(FastPureRNAColumns V已放入收集管中,12,000rpm(13,400×g)离心30sec,弃滤液。
(4)向FastPure RNA Columns V中加入700μl Buffer RWA,12,000rpm(13,400×g)离心30sec,弃滤液。
(5)向FastPure RNAColumns V中加入500μl BufferRWB(使用前请检查是否已加入48ml无水乙醇,12,000rpm(13,400×g)离心30sec,弃滤液。
(6)重复步骤6。
(7)将FastPure RNAColumns V放回收集管中,12,000rpm(13,400×g)离心2min。
(8)将FastPure RNAColumns V转移至新的RNase-free CollectionTubes 1.5ml离心管中,向吸附柱膜中央悬空滴加30-100μl的RNase-free ddH2O,12,000rpm(13,400×g)离心1min。
▲洗脱体积建议不少于30μl,体积过小会影响核酸回收效率。
▲以下步骤都可以帮助提高RNA产物浓度:滴加RNase-free ddH2O后室温静置5min;将第一次洗脱液重新加入吸附柱进行洗脱。
(9)提取的RNA可直接用于下游实验或-85~-65℃保存。
2.2.3反转录cDNA的合成
反转录cDNA的合成利用试剂盒IIQ RT SuperMix for qPCR(+gDNAwiper)(诺维赞,中国南京)进行,可以分为基因组gDNA的去除和RNA的反转录两部分,反应在冰上进行,反应步骤如下:
(1)基因组gDNA的去除
表1反应体系配置
试剂 | 用量 |
RNase-free ddH2O | to16μl |
4×gDNA wiper Mix | 4μl |
模板RNA | 1pg-1μg |
用移液器轻轻吹打混匀。42℃2min。
(2)配制逆转录反应体系
表2反应体系
试剂 | 用量 |
第一步的反应液 | 16μl |
5×HiScript II qRT SuperMix II | 4.0μl |
用移液器轻轻吹打混匀,将上述混合溶液20μl在PCR仪中50℃15min,85℃5sec。产物可立即用于qPCR反应,或在-20℃保存,并在半年内使用。
2.2.4荧光定量PCR
(1)利用Oligo 7软件设计GhCIB1基因的特异性引物,用棉花His3基因为内参基因。
表3引物序列
(2)荧光定量PCR
利用Cwbio(China)的UltraSYBR Mixture(Low ROX)试剂盒和AppliedBiosystems 7500仪器完成。具体过程如下:
1)将上述的cDNA原液稀释5倍;
2)反应体系的配置(冰上操作):
表4反应体系
将配置好的体系混匀,离心至无气泡,然后利用Applied Biosystems 7500进行荧光定量PCR:按照两步法设置PCR程序:预变性:95℃2min;95℃,5s;60℃,34s(这一步收集荧光信号),这两步设置40个循环;最后溶解曲线分析:95℃,15s;60℃,20s;95℃,15s。利用Microsoft Excel 2019软件处理数据,计算基因的表达量,Origin 2022软件绘图。
2.2.5GhCIB1定量结果分析
荧光定量的结果数据按照2-ΔCt计算,得到GhCIB1的相对表达量。由图1和2可以看到GhCIB1在早熟品种一叶期到五叶期的表达量均随着时期变化而升高,而在晚熟材料品种一叶期到五叶期的表达量均随着时期变化而降低。
2.3拟南芥异源表达
将GhCIB1 CDS序列全长连接pCambia2300-HA载体,构建35S启动子载体。将35S::GhCIB1重组载体通过拟南芥花序侵染法侵染拟南芥,通过对后代种子进行阳性筛选和纯化加代,得到T3代纯系植株。对后代表型和表达量分析,发现过表达GhCIB1促进拟南芥早花。
2.3.1基因引物设计
根据同源重组引物设计原则,使用Oligo 7软件设计特异性引物,扩增GhCIB1基因编码区全长。根据该基因CDS序列,在起始密码子ATG和终止密码子处添加相应酶切位点序列,使目的基因片段与酶切后的线性化载体具有相同末端序列。pCambia2300-HA载体酶切位点选择EcoRⅠ和KpnⅠ,所用cDNA模板为陆地棉TM-1,DNA模板为陆地棉TM-1。
35S::GhCIB1特异性引物序列如下:
表5引物序列
2.3.2基因克隆PCR体系、程序与产物检测
(1)反应在冰上进行,根据试剂盒Phanta Max Super-Fidelity DNA Polymerase(诺维赞,南京)设计反应体系如下:
表6反应体系
试剂名称 | 试剂用量 |
ddH2O | up to50μl |
2×Phanta Max Buffera | 25μl |
dNTP Mix(10mM each) | 1μl |
上游引物(10μM) | 2μl |
下游引物(10μM) | 2μl |
Phanta Max Super-Fidelity DNA Polymerase | 1μl |
模板DNA | 1μl |
(2)PCR反应程序:
(3)PCR产物的检测
取2μl PCR产物,加入2μl 5×Loading Buffer,混匀,点样于1%琼脂糖凝胶,电泳检测条带大小是否符合要求。
(4)PCR产物的胶回收
采用Vazyme产物纯化试剂盒,步骤如下:
1)DNA电泳结束后,在紫外灯快速切下含有目的DNA片段的凝胶,建议用纸巾吸尽凝胶表面液体并切碎,并尽量去除多余的凝胶。秤取凝胶中粮(去除空管的重量),100mg凝胶等同于100μl体积,作为一个凝胶体积;
2)加入等体积的Buffer GDP。50~55℃水浴7-10分钟,根据凝胶大小适当调整时间,确保凝胶块完全溶解。水浴期间颠倒混匀2次加速溶胶;
3)短暂离心收集管壁上的液滴。将FastPure DNA Mini Columns-G吸附柱置于Collection Tubes 2ml收集管中,把≤700μl溶胶液转移至吸附柱中,12,000Xg离心30-60sec。若溶胶体积大于700μl,把吸附柱置于收集管中,剩余的溶胶液转移至吸附柱中,12,000×g离心30-60sec。
4)弃滤液,把吸附柱置于收集管中。加入300μl Buffer GDP至吸附柱中。静置1min。12,000×g离心30-60sec。
5)弃滤液,把吸附柱置于收集管中。加入700μl Buffer GW(已加入无水乙醇)至吸附柱中。12,000×g离心30-60sec。
6)重复步骤5.
7)弃滤液,把吸附柱置于收集管中。12,000×g离心2min。
8)将吸附柱置于1.5ml灭菌的离心管中,加入20-30μl的灭菌水至吸附柱中央,放置2min。12,000Xg离心1min。弃去吸附柱,把DNA保存于-20℃。
2.3.3pCambia2300-HA植物表达载体的构建
(1)质粒的双酶切及胶回收
pCambia2300-HA将质粒双酶切,电泳回收载体的产物。酶切反应体系如下:
表7反应体系
试剂名称 | 试剂用量 |
酶1 | 1μl |
酶2 | 1μl |
Cut Smart | 5μl |
质粒 | 1μg |
ddH2O | Up to 50μl |
(2)PCR胶回收产物和线性化质粒的连接
把带有接头的PCR产物和线性化的质粒用诺唯赞同源重组酶试剂One Step Cloning Kit进行连接,连接反应如下:
体系配置于冰上进行:
表8反应体系
试剂名称 | 试剂用量 |
5XCE Ⅱ Buffer | 2μl |
Exnase Ⅱ | 1μl |
线性化载体 | 25~100ng |
PCR片段 | 10~100ng |
ddH2O | Up to 10μl |
体系完成后,吹打混匀各组分,37℃反应30min,立即冰水浴5min,转化或者-20℃保存。
(3)连接产物转化大肠杆菌
1)向连接反应体系中加入100ul大肠杆菌DH5a感受态,冰浴30min;
2)42℃水浴热激45~90s;
3)冰浴2min;加入900ul无抗性的LB液体培养基,37℃,190rpm,孵育1h;
4)离心收菌,4000rpm,3min,弃上层上清,留约100ul混匀后涂布含卡那抗性的LB平板;
5)37℃,恒温培养过夜;
(4)阳性克隆的检测及测序
1)从转化平板上挑取白色菌落,放入含有Kan的液体LB培养基中,37℃恒温摇床培养8小时;
2)菌落PCR验证阳性克隆,将验证正确的单克隆送到尚亚生物科技有限公司测序,每个序列测3个重复。
(5)阳性菌液的保存
菌液PCR验证且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,-80℃保存。返还测序正确的质粒用于转农杆菌。
(6)转化农杆菌
利用冻融法转化根癌农杆菌GV3101感受态细胞,具体转化过程如下:
1)-80℃农杆菌融化,冰水混合状态插入冰中。
2)100μl感受态中加入0.01~1μg质粒DNA,用手拨打管底混匀,依次于冰上静置5分钟,液氮5分钟,37℃五分钟,冰浴5分钟。
3)加入700ul无抗性的LB液体培养基,于28℃振荡培养2-3小时
4)取100-150ul菌液于含有卡那、利福平的LB平板上,倒置放于28℃培养箱2-3天。
5)挑选阳性克隆,在加有抗性的LB液体培养基上28度培养48h,菌液PCR验证条带正确的菌液甘油保存终浓度在20%左右,-80℃保存备用。
2.3.4农杆菌介导的拟南芥花序侵染
(1)拟南芥培养
长日照条件(光照16h,黑暗8h)下培养的哥伦比亚野生型拟南芥,选取5周龄左右,生长健壮的株系,剪去角果,侵染的前一天浇水保证拟南芥的状态和湿度。
(2)拟南芥花序侵染
1)菌液活化:取-80℃保存的对应重组载体的农杆菌菌液20μl,接种到1ml LB液体培养基(加入对应的抗生素:卡纳霉素、利福平和链霉素)中,28℃,180rpm,培养14-18h;
2)扩摇:取活化后的对应菌液200μl加入到50ml LB液体培养基(加入对应的抗生素);28℃,180rpm,培养至菌液OD600值约在1.2-1.6之间(约18-20h),5000g,离心8min,弃上清,收集菌体;
3)侵染转化的介质配制:1/2MS减半、6%蔗糖、0.02%的SilwetL-77,用NaOH将pH调至5.6-5.7;
4)用转化介质悬浮上述菌体,将OD600调至0.6-0.8;
5)浸染:将拟南芥花序(主要是未开放的花苞)置于转化介质中30-50s,浸染后,将拟南芥在弱光或者避光条件下平放24h;
6)将处理后的拟南芥放置正常条件下培养,并在侵染后的一周内每天给拟南芥叶片喷水;为了提高转化效率,可在约一周后进行重复侵染;
7)待成熟后,收获拟南芥种子,即为转基因的T0代种子。
2.3.5转基因拟南芥植株的表型鉴定
(1)将收获的种子消毒后种植在含卡那霉素的1/2MS上,后进行4℃春化2天,转移到人工气候试验箱中,10天左右会阳性植株生长正常,而阴性植株叶片变黄,不再生长。
(2)将阳性拟南芥植株移栽至小花盆中种植,待生长一个月后提取DNA用PCR进行检测。
检测时所用引物为:
表9引物序列
35S | SEQ ID No.9GACGCACAATCCCACTATCC |
GhCIB1-R | SEQ ID No.10CATCTCCATCTTTAGATGGCTA |
(3)将转基因T3代植株与非转基因植株进行消毒培养于1/2MS培养基上,4℃春化两天后,10天左右拟南芥幼苗长出真叶即移到小花盆里生长,同等条件下种植栽培,表型观察发现非转基因拟南芥开花明显晚于过表达转基因拟南芥(图3-5);说明过表达GhCIB1基因明显促进拟南芥开花和生殖生长发育。
尽管上述实施例对本发明做出了详尽的描述,但它仅仅是本发明一部分实施例,而不是全部实施例,人们还可以根据本实施例在不经创造性前提下获得其他实施例,这些实施例都属于本发明保护范围。
Claims (3)
1.过表达GhCIB1基因在促进棉花开花中的应用,所述GhCIB1基因的核苷酸序列如SEQID No.1所示。
2.过表达GhCIB1基因在促进棉花生殖生长中的应用,所述GhCIB1基因的核苷酸序列如SEQ ID No.1所示。
3.根据权利要求1或2所述的应用,其特征在于,所述GhCIB1基因的氨基酸序列如SEQID No.2所示。
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