CN112851781B - 柑橘bZIP转录因子在缩短植物童期中的应用 - Google Patents
柑橘bZIP转录因子在缩短植物童期中的应用 Download PDFInfo
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Abstract
本发明涉及植物基因工程技术领域,具体涉及一个柑橘bZIP转录因子的应用。本发明首次克隆了柑橘中的一个bZIP转录因子,并构建该基因的超表达载体,通过农杆菌介导的遗传转化方式将其转入到烟草和枳中,获得了转基因植株,经过表型观察和统计发现,转基因烟草和枳都有早花表型,表明本发明所克隆的bZIP转录因子具有早花功能,可以明显缩短童期。该基因的发现,为柑橘成花育种、缩短柑橘等多年生木本植物童期提供了新的遗传资源,该遗传资源的开发利用对于降低工业生产成本具有重要意义。
Description
技术领域
本发明涉及植物基因工程技术领域,具体涉及一个柑橘bZIP转录因子在缩短植物童期中的应用。
背景技术
柑橘是世界第一大水果,在我国园艺经济中具有十分重要的地位,我国柑橘的栽培面积和产量居世界首位。成花转变是果实形成的基础,柑橘童期较长,是柑橘育种的重要障碍之一,而创制短童期的新种质一直是柑橘育种的重要目标,柑橘成花转变机制的解析将为缩短童期提供重要的理论基础。
发明内容
本发明的目的之一在于提供一个编码柑橘bZIP转录因子的基因在缩短植物童期中的应用,该基因编码的蛋白的氨基酸序列如SEQ ID NO:1所示。
其中,所述基因是如下(1)至(2)中任一项所述的DNA分子:
(1)其核苷酸序列为SEQ ID NO:2所示的DNA分子;
(2)在严格条件下与(1)限定的DNA序列杂交且编码权利要求1中所述蛋白的DNA分子。
本发明的目的之二在于提供一种缩短植物童期的方法,所述方法包括上调编码柑橘bZIP转录因子基因的表达,所述编码柑橘bZIP转录因子的基因为权利要求2所述的基因。
具体地,所述方法包括将bZIP转录因子转入植物细胞、组织或器官,获得转入bZIP转录因子的植物细胞、组织或器官。
具体地,所述bZIP转录因子以包含在表达载体及农杆菌中的方式转入植物细胞、组织或器官。
本发明的目的之三在于提供一种含有bZIP转录因子的表达载体或农杆菌在缩短植物童期中的应用,其特征在于,所述bZIP转录因子为上述的基因。
其中,所述表达载体为PBI121载体。
其中,所述植物为烟草或枳。
本发明首次克隆了柑橘中的一个bZIP转录因子,并构建该基因的超表达载体,通过农杆菌介导的遗传转化方式将其转入到烟草和枳中,获得了转基因植株,经过表型观察和统计发现,转基因烟草和枳都有早花表型,表明本发明所克隆的bZIP转录因子具有早花功能,可以明显缩短童期。该基因的发现,为柑橘成花育种、缩短柑橘等多年生木本植物童期提供了新的遗传资源,该遗传资源的开发利用对于降低工业生产成本具有重要意义。
附图说明
图1为实施例2中转入了编码柑橘bZIP转录因子的烟草开花时间及叶片数统计结果,其中A为成花天数,B为叶片数;
图2为实施例2中转入了编码柑橘bZIP转录因子的烟草开花情况;
图3为实施例3中转入了编码柑橘bZIP转录因子的枳的开花情况。
具体实施方式
以下结合具体实施方式对本发明的原理和特征进行描述,所举实例只用于解释本发明,并非用于限定本发明的范围。
烟草遗传转化中所用培养基类型及配方:
MS共培养培养基:MS培养基+AS(乙酰丁香酮20mg/L);
MS筛选培养培养基:MS培养基+6-BA(2mg/L)+NAA(0.3mg/L)+Kan(50mg/L)+Cef(400mg/L);
MS生根培养基:MT培养基+Kan(50mg/L)+Cef(400mg/L)。
枳遗传转化中所用培养基类型及配方:
MT基本培养基:微量、铁盐、甘氨酸、肌醇、VB、VC各10mL,大量元素100mL,蔗糖40g/L,琼脂8g/L;
柑橘试管播种培养基:MT+蔗糖25g/L+Agar 8.0g/L;
悬浮培养基(LM):MT+麦芽浸粉0.5g/L+谷氨酰胺1.5g/L;
生芽培养基(SY):MT+BA 0.5mg/L+NAA 0.1mg/L+KT 0.5mg/L+Agar 8g/L;
共培养培养基(CM):SY+AS 50mg/L+Agar 8g/L;
筛选培养基(SM):SY+Cef400 mg/L+Km 50mg/L+Agar 8g/L;
生根培养基(RIM):L/2MT+NAA 0.5mg/L+IBA 0.1mg/L+活性炭0.5g/L+Agar 8g/L+Km 25mg/L+Cef400 mg/L。
实施例1基因的分离与克隆
1.1编码柑橘bZIP转录因子基因的DNA全长扩增
以柑橘DNA为模板,采用高保真酶进行扩增,扩增引物序列为:5’-ATGTGGCCATCTCCAGCTAGAAACA-3’和5’-TTATTCTTACTTCTCAAAATGGAGC-3’。
扩增反应体系为:ddH2O 18μL、2×PhantaMax Buffer 25μL、Sense primer2μL、Antisense primer 2μL、dNTP mix 1μL、MaxSuper-FideLity DNA PoLymerase 1μL、TempLe(DNA)1μL、TotaL 50μL。
扩增反应程序为:95℃预变性3min,95℃变性30s、55℃退火30s、72℃延伸45s、35个循环;72℃延伸10min,16℃降温10min。
1.2连接克隆载体
采用上海捷瑞生物有限公司纯化回收试剂盒对扩增得到的产物进行纯化回收,将回收的目的片段连接到pEASY-BLunt载体(购自北京全式金生物技术有限公司)上,其连接体系为:回收产物0.5-4μL(按照1kb 20ng的量计算),但最多不超过4μL,载体加入1μL,用水补齐到5μL。在25℃条件下连接,连接时间为0.1-1Kb(含1Kb)为5-10min;1-2Kb(含2Kb)为10-15min;2-3Kb(含3Kb)为15-20min。
1.3转化大肠杆菌
将连接好的连接产物转入大肠杆菌DH5α感受态中,具体操作步骤如下:
a、连接结束前5分钟从-80℃冰箱中取出大肠感受态DH5a,在冰上解冻;
b、将连接产物全部加入感受态细胞中,并用枪头轻轻吸打混匀,置于冰上30min;
c、在混匀仪中42℃热激1min30s,热击完后立即置于冰上2min;
d、在感受态细胞中加入400μL的空白LB液体培养基,在37℃摇床上220r/min摇菌1h;
e、摇完后6000r/min,离心2min,吸出上清液留100μL菌液,吸打混匀,涂布于km的LB培养基上,晾干封口,37℃恒温箱中培养过夜;
f、挑斑进行PCR检测,将检测的阳性克隆送公司测序,然后对序列进行比对分析。
编码柑橘bZIP转录因子的基因序列分析结果如下:该基因DNA长度为1022bp,碱基序列如SEQ ID NO:2所示,包含3个外显子,2个内含子,开放阅读框为774bp,编码257个氨基酸,氨基酸序列如SEQ ID NO:1所示。编码的蛋白质等电点为9.514,预测的分子量为28.12kDa。
1.4超表达载体的构建
以测序正确的质粒为模板,设计重组引物将编码柑橘bZIP转录因子的基因全长扩增并插入至pBI121载体上的BamHⅠ和SacⅠ两个酶切位点中间,引物设计如下:
与童期相关的-pBI121-F:5’-ACGGGGGACTCTAGAGGATCCATGTGGCCATCTCCAGCTAGA-3’;
与童期相关的-pBI121-R:5’-CGATCGGGGAAATTCGAGCTCTTATTCTTACTTCTCAAAAT-3’。
根据重组试剂盒(购自南京诺唯赞生物科技有限公司)使用说明书在37℃条件下进行重组反应30min,再将重组反应产物转入大肠杆菌DH5α感受态中,后挑斑检测测序,重组反应体系为:线性化载体1.0μL、目的片段10ng~200ng、Exnase Entry 1.0μL、5×CEEntry Buffer2.0μL、ddH2O To 10μL。
1.5质粒转化农杆菌
将测序正确连有编码柑橘bZIP转录因子的大肠杆菌菌液PBI121质粒进行抽提,质粒的提取方法参照艾德莱公司质粒提取说明书。将抽提的质粒转入农杆菌感受态GV3101或EHA105中,具体操作步骤如下:
a、将农杆菌感受态在冰上冻融,取3-4μL质粒加入到感受态细胞中,冰上静置5min,于液氮中速冻5min,迅速取出于37℃恒温仪中温浴5min,再冰上放置5min;
b、向感受态细胞中加入800μL液体空白LB培养基,28℃,220r/min摇菌复苏3h;
c、将摇好的菌液以5000r/min的速度离心2min;
d、吸出上清液留100μL菌液,吸打混匀,涂布在相应抗性的固体LB培养基上于28℃培养箱中培养2~3d;
e、长斑后挑斑检测,将阳性菌液1:1加50%甘油于-80℃保存备用。
实施例2烟草遗传转化及表型分析
采用农杆菌介导的遗传转化法对栽培型烟草进行侵染,将连接PBI121超表达载体的农杆菌采用叶盘法转化野生型烟草叶片,具体步骤如下:
(1)准备侵染液:将实施例1中保存的农杆菌在卡那和利福平的LB固体培养基上划线,28℃条件下培养2d,2d后挑取单克隆进行摇菌活化后再次划线,2d后将农杆菌刮至MS液体中,调OD600值为0.6~0.8,加入20mg/mLAS(乙酰丁香酮);
(2)共培养:从含MS培养基罐中取出野生型烟草叶片,用灭菌的刀将烟草叶片两端及主叶脉去除后切成1cm x 1cm大小的叶片,而后放入MS侵染液中,28℃摇15min后倒去侵染液,再将叶片铺于无菌滤纸上吹干,叶背朝上均匀铺在加有50mg/mLAS的MS固体培养基上,于20℃暗培养3天;
(3)筛选培养:将共培养完的叶片用头孢的水清洗两次,再用无菌水清洗3次,吹干后铺在加相应抗生素的MS筛选培养基上,轻轻的用镊子压一压,于28℃光照培养;
(4)切芽:叶片筛选培养一个月后,将从愈伤中生长出的带有生长点的芽切至加抗生素的MS培养基罐子上壮苗生根,生根后即可提取DNA进行阳性鉴定。其中,抗生素为载体抗性和头孢。
将鉴定为阳性的三个系T1代转基因烟草与野生型烟草同时播种,放在同样条件下生长,待第一朵花露白时进行开花时间及叶片数统计,统计结果如图1。从图中我们可以看出,三个转基因系(2#、4#、5#)都较野生型开花时间早,开花时叶片数少,其中5#的表型最明显,开花时间在1-2月间,而野生型需要4-5个月;5#开花时的叶片数平均约为11片,而野生型叶片数平均多达38片。待三个系都开花后进行拍照,照片如图2,从图中也可以明显的看出,转基因系都较野生型开花时间早。
实施例3枳遗传转化及表型分析
3.1枳实生苗的准备
(1)将种子从新鲜的枳果实中取出,冲洗掉附着其上的果肉;
(2)将种子用1moL/L的NaOH(4g NaOH,100mL蒸馏水)中浸泡10-15分钟;
(3)倒掉NaOH溶液,清水清洗种子4-5次;
(4)下面操作在超净工作台上进行,将种子转入2.5%的NaClO(50mL5%NaClO,50mL无菌水),浸泡杀菌20分钟,中途摇晃几次;
(5)倒掉NaClO溶液,用无菌水冲洗种子4-5次,用无菌水浸泡保存备用;
(6)待播种子置于已高温灭菌的滤纸上,剥去内外种皮后插入播种培养基,长出胚根的尖头朝下,每管1-2颗,封口暗室培养4-6周,待下胚轴长到一定高度时取出光照培养7-10天,返绿后即可用于转化。
3.2农杆菌侵染液体
(1)菌株活化:将连接PBI121超表达载体的与童期相关的农杆菌菌体接种于固体LB培养基(Kan50 mg/L,Rif50mg/L),28℃暗培养36h以上;
(2)菌落的繁殖:挑取农杆菌单克隆进行摇菌,摇混后重新接种于固体LB培养基(Kan50 mg/L,Rif50mg/L),28℃暗培养36h以上,即进行第二次活化;
(3)制备侵染液:刮取所有菌体于LM悬浮培养基内,置于220r/min,28℃恒温摇床内40min-1h,使菌体分散;
(4)用紫外分光光度计测定悬浮菌液的浓度,调整至OD600在0.6-0.8之间;侵染液中添加AS(50mg/L),备用。
3.3侵染与共培养
(1)在无菌超净台上,将返绿一周的枳实生苗上胚轴在无菌滤纸上用刀片切成1cm左右的梯形茎段,并放入LM悬浮培养基中,防止茎段切口风干失水,影响再生率及转化率;
(2)将全部切好的的茎段浸泡于已准备好的农杆菌菌液中,封口,摇床上150rpm,20min;
(3)20min后,倒掉菌液,用无菌滤纸吸干枳茎段表面残留的农杆菌菌液,切口向上摆放在CM培养基中,21℃恒温箱暗处共培3天。
3.4筛选培养
(1)将共培养3天后的枳茎段移入装有无菌水的锥形瓶中,用无菌水清洗茎段3-5次,每次在摇床上摇5min,后倒掉无菌水,再用无菌滤纸吸干茎段表面水分;
(2)将枳茎段切口向上平铺在SY培养基上,26℃+2℃暗培6-7天;
转入光培条件下,16/8h光照,20天左右继代一次;
(3)切芽:茎段筛选培养1~2个月后,将带有生长点的芽切至筛选培养基SM的罐子上壮苗筛选;
(4)生根:待芽生长1个月后,将未黄化的芽移至生根培养基中。
将切下的芽移至生根培养基中约两个月后,部分株系在罐子中就出现了极早花现象,如图3所示。从图中我们可以看出,转基因枳(1#、2#、3#)的顶端形成了花苞,这进一步说明了柑橘bZIP转录因子具有早花功能。但我们发现转基因枳的花为畸形花,花较小,且花器官结构不完整,花凋谢后植株即死亡,推测可能是由于转基因系开花时间太早,营养生长不足导致。
以上所述仅为本发明的较佳实施例,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。
序列表
<110> 华中农业大学
<120> 柑橘bZIP转录因子在缩短植物童期中的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 257
<212> PRT
<213> 柑橘(Citrus)
<400> 1
Met Trp Pro Ser Pro Ala Arg Asn Asn Lys Asn Gly Ile Ser Ser Ser
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Ile Ser Lys Ser Ser Ser Ser Cys Ser Ser Pro Ser Ser Pro Gly Thr
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Pro Asn Lys Lys Ser Met Glu Glu Val Trp Gln Asp Ile Ser Leu Thr
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Ser Leu Gln Asp His Ala Asn Thr Ala Ile Pro Asn Thr Thr Gly Ile
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Pro Asn Thr Ser Ala Ala Leu Ile Phe Gln Asp Phe Phe Ala Arg Pro
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Phe Asn Lys Asp Pro Pro Ile Thr Lys Ala Ser Pro Ala Ala His Pro
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Ser Thr Ala Glu Pro Ser Asn Ser Ser Cys Phe Gly Asn Leu Ala Pro
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His His Gly Gly Ala Leu Leu Ser Leu Asn Ser Gly Ser Gly Phe Asn
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Tyr Leu Glu Asn Val Pro Ala Pro Leu Ala His His His Gln Arg Pro
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Ser His Gln Leu Leu Gln Gly Phe Pro Leu Asn Asn Cys Asn Ser Pro
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Phe Asp Asp Ala Leu Ala Pro Ala His Val Val Ser Ser Ile Cys Phe
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Lys Arg Pro Gln Glu His Glu Gly His Leu Thr Asp Arg Arg His Lys
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Arg Met Met Lys Asn Arg Glu Ser Ala Ala Arg Ser Arg Ala Arg Lys
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Gln Ala Tyr Thr Thr Glu Leu Glu Gln Glu Val Ala His Leu Glu Gln
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Phe
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<212> DNA
<213> 柑橘(Citrus)
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atgtggccat ctccagctag aaacaacaag aatggaatct caagctcaat ttcaaaatca 60
tcatcttcat gttcatctcc ttcatcgcca ggcaccccta acaaaaaatc catggaagaa 120
gtttggcagg acataagcct aacttctttg caagatcacg ctaacactgc catcccaaac 180
actaccggca tcccaaacac tagtgcagct ttaatatttc aagacttttt tgcaaggcca 240
tttaacaaag acccaccaat aacaaaagct tctcctgctg cacaccccag tactgctgag 300
ccctcaaaca gctcttgctt tggcaatttg gctccacatc acggcggcgc tttgttgagc 360
ctgaattctg ggtctggctt taattatctt gaaaatgtcc ctgcccctct ggctcatcat 420
catcaaaggc caagccatca attgctacag ggtttccctc tcaacaactg caactcccct 480
tttgatgatg ctttggctcc tgctcatgtt gtgtcctcta tttgcttcaa aaggcctcaa 540
gaacatgagg ggcatttgac tgatcgccga cataagcgca tgatgaagaa tcgagagtca 600
gccgctcgct ccagagccag aaagcaggaa cctcactttc ttttttttcc ccctcttttc 660
tattaaatca gaatgttcct ttctttcttt gtttttcatt ttactgaggg ttgttttttt 720
ttaattaaat ttttttttgg caaattatgt ttatgcaggc ttatacaact gagttggaac 780
aagaagttgc ccatttggag caagagaatg ccaagctgag aaggcagtta gagcaggtca 840
atgaacgagt gataattctt tatacagatt ttttttttta tgttaaaatt aattagcaat 900
aagccggcga tctaatccaa taataagccg cttgctgtcg tcatgtgtta cagttgttgg 960
cggcttctgg gcagcaaacg aaaaagccct cgctctatag aacctcaact gctccatttt 1020
ga 1022
Claims (8)
1.超表达编码柑橘bZIP转录因子的基因在缩短植物童期中的应用,其特征在于,该基因编码的蛋白的氨基酸序列如SEQ ID NO:1所示。
2.根据权利要求1所述的超表达编码柑橘bZIP转录因子的基因在缩短植物童期中的应用,其特征在于,所述基因是如下所述的DNA分子:其核苷酸序列为SEQ ID NO:2所示的DNA分子。
3.一种缩短植物童期的方法,其特征在于,所述方法包括上调编码柑橘bZIP转录因子的基因表达,编码柑橘bZIP转录因子的基因为权利要求2中 所述的基因。
4.根据权利要求3所述的缩短植物童期的方法,其特征在于,所述方法包括将编码bZIP转录因子的基因转入植物细胞、组织或器官,获得转入编码bZIP转录因子基因的植物细胞、组织或器官。
5.根据权利要求4所述的缩短植物童期的方法,其特征在于,所述编码bZIP转录因子的基因以包含在表达载体及农杆菌中的方式转入植物细胞、组织或器官。
6.含有编码bZIP转录因子基因的表达载体或农杆菌在缩短植物童期中的应用,其特征在于,所述编码bZIP转录因子的基因为权利要求2中 所述的基因。
7.根据权利要求6所述的含有编码bZIP转录因子基因的表达载体或农杆菌在缩短植物童期中的应用,其特征在于,所述表达载体为PBI121载体。
8.根据权利要求6所述的含有编码bZIP转录因子基因的表达载体或农杆菌在缩短植物童期中的应用,其特征在于,所述植物为烟草或枳。
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