CN114350685A - 烟草NtTAC1基因在叶片夹角调控中的应用 - Google Patents
烟草NtTAC1基因在叶片夹角调控中的应用 Download PDFInfo
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Abstract
本发明属于植物基因工程技术领域,具体涉及一个烟草NtTAC1基因在叶片夹角调控中应用的专利申请。烟草NtTAC1基因长度为969bp,其编码序列如SEQ ID No.1所示。该基因在翻译表达后,用于调控烟草叶片夹角。本申请中,发明人对NtTAC1基因在根、茎、叶、花、顶芽、叶枕(叶夹角)等组织中的表达模式进行了检测分析,结果表明,烟草NtTAC1基因在叶枕(叶夹角)处表达量最高。NtTAC1基因表达量下降的转基因株系中,叶片生长呈直立,叶夹角明显减小。基于对NtTAC1基因调控机理的深入研究,可为烟草株型调控、提高烟叶光合作用效率、改善烟叶品质等奠定良好的技术理论基础。
Description
技术领域
本发明属于植物基因工程技术领域,具体涉及一个烟草NtTAC1基因在叶片夹角调控中应用的专利申请。
背景技术
植物株型对于植物的健康生长具有重要决定作用。植物株型包括多个评价角度:株高、枝杈数量及角度、叶片高度及数量等等。叶片夹角是指叶片与茎之间形成的角度,叶片夹角作为植物株型评价中重要组成部分之一,直接决定着叶片直立程度,而这又与叶片对于光接收、光合效率直接相关,因此对于植物最终生物量积累、农作物产量、品质和抗逆性等具有重要影响。
已有研究表明,直立叶片群体的光合效率高于平展或弯垂叶,叶夹角小有利于叶片两面受光,提高适宜叶面积指数,从而提高冠层光合速率,增加生物量积累,同时对于增强根系活力、提高抗倒性也具有积极作用。同时,叶片夹角大小受植物生长发育进程、环境、激素以及遗传因素共同协调,因此,深入研究叶片夹角与植物生长代谢之间关系,对于优化采光面积及提高光合效率具有重要的技术意义。
随着基因工程技术发展,已有研究认为,相关基因合调控是决定叶片夹角最重要的因素之一。部分研究人员对于水稻、玉米、大豆等农作物中有关叶夹角基因进行了初步鉴定,并对相关分子调节机制进行了分析研究。但与水稻、玉米、大豆等农作物关注种子产量不同,烟草是以叶片作为直接生产利用部分,因此,对于烟草中相关叶片夹角基因进行深入研究,对于改良烟草叶片产量和提高烟叶品质具有更为重要的现实意义和技术价值。
发明内容
本申请目的在于提供一个与烟草叶片夹角调控相同的烟草NtTAC1基因,从而为烟草株型调控、提高烟叶品质等奠定一定技术基础。
本申请所采取的技术方案详述如下。
烟草NtTAC1基因,该基因与烟草叶片夹角调控相同;所述烟草NtTAC1基因,长度为969bp,其编码序列如SEQ ID No.1所示,具体如下:
ATGAAGATCTTCAATTGGGTGCACCGCAAATTGTATCAGAAAGATGGATTGGTCAGTCGGAATGTGAAGAAAGATGAGCTCAGGATTAGCAATGAGTTTATTGGTGACGCACAAGTTCTTCTTCAAGATGCATCCATTGCACATATGTTAGATAGTTGGAGAGGAGGAATCCTAACAATTGGCACATTTGGGTTTGATCCATTGAAAAATGTGCAAGATCAAAGTGTCATAGACATTGAAGAAGAAGAAGTAGAAGAATCACTTGAGGATGAATATTACTCAGTGGAAAGTATTGGACAATGTCAAATTACTGTCACTAAGGAAAATGAAGAAGTGTACCCATTGATATATGCAAGTGGAGGTGAAGTGATTGAATATCCAAAACAAAATGAGATACTGGCCATTGAGTTAAACAATTCTTCAGAGAGCAATAAGATGCTGAAAAAAGAAAGGATTACTCTAGCAGACCTTTTTTCAGCTGATTCTGATCATCACCAAAACCTTAAGCCAAATCCAAGCAAAAAGGAAGAAGAGTTTTACACAAAGAAACCTTGTTCACAAGTGAAGAATGGAACATTTTTCGCCAAAAAGCTAATTCCTCGAGTTAAGGAGGATTCTCGTCCGATCCAAAAACTACAGCAATTGATGACGAGGGTGATGAAAAGGAAGGTTCATCCAGATATTGAAAGCAAAATAGGCAAGAACAACAACATTACTACTCATCAAGTGAAAGCAGCAGCTAGCATGCTTGGGCTTTCCTGCGTTAAGCATGTAAGAGTTGACTCTGTTTCCCTTCTGCAACTTGATCAAGATTTTCGATTTCAGGTCGAAATGTCTAACTCAGTGAACGGAGGTGTAAACAACAATCTTGAGGACCACGGAGAAAACAGTGTGGCTATTCCAGGTGTTGGTGTGCCACCACGAAACCCTGAGAATGCATCGGACCCAATCCCCATGGATGCGATCTAA。
所述烟草NtTAC1基因,对应所编码的烟草NtTAC1蛋白,长度为322AA,氨基酸序列如SEQ ID No.2 所示,具体如下:
MKIFNWVHRKLYQKDGLVSRNVKKDELRISNEFIGDAQVLLQDASIAHMLDSWRGGILTIGTFGFDPLKNVQDQSVIDIEEEEVEESLEDEYYSVESIGQCQITVTKENEEVYPLIYASGGEVIEYPKQNEILAIELNNSSESNKMLKKERITLADLFSADSDHHQNLKPNPSKKEEEFYTKKPCSQVKNGTFFAKKLIPRVKEDSRPIQKLQQLMTRVMKRKVHPDIESKIGKNNNITTHQVKAAASMLGLSCVKHVRVDSVSLLQLDQDFRFQVEMSNSVNGGVNNNLEDHGENSVAIPGVGVPPRNPENASDPIPMDAI。
烟草NtTAC1基因在叶片夹角调控中应用,该基因在翻译表达后,用于调控烟草叶片夹角;具体应用时,在将NtTAC1基因沉默后,基因沉默植株中,烟草叶片直立生长,烟草叶片夹角明显减小,也即,通过降低NtTAC1基因表达量,可以减小烟草叶片夹角;换言之,通过调节叶片夹角,可以实现对烟草株型调控。
针对烟草NtTAC1基因的基因沉默用序列,序列如SEQ ID No.3所示,具体如下:
CTTCAAGATGCATCCATTGCACATATGTTAGATAGTTGGAGAGGAGGAATCCTAACAATTGGCACATTTGGGTTTGATCCATTGAAAAATGTGCAAGATCAAAGTGTCATAGACATTGAAGAAGAAGAAGTAGAAGAATCACTTGAGGATGAATATTACTCAGTGGAAAGTATTGGACAATGTCAAATTACTGTCACTAAGGAAAATGAAGAAGTGTACCCATTGATATATGCAAGTGGAGGTGAAGTGATTGAATATCCAAAACAAAATGAGATACTGGCCATTGAGTTAAACAATTCT。
用于敲低NtTAC1基因表达的重组表达载体,将其命名为:pBWA(V)KS-RNAi-TAC1,该重组表达载体以pBWA(V)KS-RNAi质粒为出发质粒,具体通过如下步骤构建获得:
(一)获得目的序列
设计正反向引物序列如下:
正向引物:
TAC F(+):5’-cagtGGTCTCacaacCTTCAAGATGCATCCATTGCACATA-3’,
TAC F(-):5’-cgatGGTCTCacaggAGAATTGTTTAACTCAATGGCCAGT-3’;
反向引物:
TAC R(+):5’-cagtGGTCTCagggcAGAATTGTTTAACTCAATGGCCAGT-3’,
TAC R(-):5’-cagtGGTCTCatacaCTTCAAGATGCATCCATTGCACATA-3’;
以烟草cDNA为模板,进行PCR扩增,获得构建干扰载体的目标序列片段;
具体具体目的序列为:
CTTCAAGATGCATCCATTGCACATATGTTAGATAGTTGGAGAGGAGGAATCCTAACAATTGGCACATTTGGGTTTGATCCATTGAAAAATGTGCAAGATCAAAGTGTCATAGACATTGAAGAAGAAGAAGTAGAAGAATCACTTGAGGATGAATATTACTCAGTGGAAAGTATTGGACAATGTCAAATTACTGTCACTAAGGAAAATGAAGAAGTGTACCCATTGATATATGCAAGTGGAGGTGAAGTGATTGAATATCCAAAACAAAATGAGATACTGGCCATTGAGTTAAACAATTCT;
(二)酶切、连接
将步骤(一)所或目的序列与pBWA(V)KS质粒分包进行BsaI、Eco31I双酶切,并回收酶切产物,随后T4 DNA连接酶将所回收酶切产物进行连接;
(三)转化合筛选
将步骤(二)中连接产物转化大肠杆菌感受态细胞,并进行筛选、鉴定,最终获得重组正确的用于敲低烟草叶片夹角基因TAC1的重组pBWA(V)KS-RNAi-TAC1表达载体。
所述用于敲低NtTAC1基因表达的重组表达载体在烟草中应用,将该重组载体转化烟草后,能够降低烟草TAC1基因的表达水平,进而通过降低烟草NtTAC1蛋白表达量来调控烟草叶片夹角。
利用所述用于敲低NtTAC1基因表达的重组表达载体的转基因烟草新品种培育方法,利用农杆菌介导的基因转化方法,首先将重组表达载体pBWA(V)KS-RNAi-TAC1转化农杆菌并制备浸染液;随后将烟草组培用组培体(一般采用消毒灭菌后叶片)置于浸染液中进行转染;最后,将侵染后组培体在无菌条件下进行组培、筛选、鉴定,获得NtTAC1基因表达降低的转化正确转基因植物新株系(新品种)。
本申请中,发明人克隆获得了烟草基因NtTAC1,并结合实时定量PCR技术,对NtTAC1基因在根、茎、叶、花、顶芽、叶枕(叶夹角)等组织中的表达模式进行了检测分析,结果表明,烟草NtTAC1基因在叶枕(叶夹角)处表达量最高,另外在茎、花、顶芽中有较高的表达量,在根和叶片中表达量最低。这一表达模式也直接反应了该基因与叶片夹角的生理调控可能直接相关。
为进一步研究和明确烟草NtTAC1基因(NtTAC1蛋白)在烟草叶夹角大小调控中的作用,发明人通过构建重组表达载体pBWA(V)KS-RNAi-TAC1 并转化烟草植株,而NtTAC1基因表达量下降的转基因株系中,叶片生长呈直立,叶夹角明显减小。这一结果充分证明NtTAC1基因(NtTAC1蛋白)具有调控烟草叶片夹角的作用,通过降低或敲除NtTAC1基因表达量,可达到减小烟草叶片夹角的作用。进一步地,通过外源施加NtTAC1蛋白或者超表达NtTAC1基因,有望增加烟草叶片夹角。总体上,基于对NtTAC1基因(NtTAC1蛋白)调控机理的深入研究,可为烟草株型调控、提高烟叶光合作用效率、改善烟叶品质等奠定良好的技术理论基础。
附图说明
图1为NtTAC1基因在不同组织中的表达特征;
图2为NtTAC1干扰植株中NtTAC1基因表达量分析结果;
图3为NtTAC1干扰植株的表型,其中:A为基因表达水平检测结果,B为实际表型观察结果。
具体实施方式
下面结合实施例对本申请做进一步的解释说明。在介绍具体实施例前,就下述实施例中部分实验背景情况简要介绍说明如下。
生物材料:
烟草品种:K326,一种普通栽培烟草品种,可由公开渠道获得;
pEASY-T1 Simple 载体、Trans1-T1化学感受态细胞(大肠杆菌),购自北京全式金生物技术有限公司;
pBWA(V)KS-RNAi载体,由武汉伯远生物科技有限公司提供;
LBA4404农杆菌菌株,生物实验中常用菌株,可由公开渠道获得;
相关引物的合成和DNA测序工作,由北京六合华大基因科技股份有限公司提供完成;
实验试剂:
荧光定量PCR酶(SYBR qPCR kit),购自郑州安赛生物科技有限公司;
反转录试剂盒、T4连接酶、限制性内切酶等,购自宝生物工程(大连)有限公司;
DNA扩增酶,购自北京全式金生物技术有限公司;
植物基因组提取试剂盒、DNA纯化试剂盒购自QIAGEN公司;
实验设备:
PCR合成仪Tprofessional Thermocycler,Biometra公司产品;
定量PCR仪CFX96,Bio-Rad公司产品;
紫外凝胶成像系统BioSpectrum,UVP公司产品。
实施例1
鉴于叶片夹角在烟草栽培中的重要作用,结合现有其他作物中叶片夹角相关基因研究,以及基于发明人前期相关研究合实验总结,发明人初步猜测烟草NtTAC1基因与烟草叶片夹角调控相关,为此,发明人首先克隆获得了烟草NtTAC1基因。下面就该基因的克隆获得过程简介如下。
(1)制备cDNA
取旺长期烟草(K326)叶片100mg作为样品,在液氮中充分研磨,参照RNA提取试剂盒说明书提取总RNA,然后反转录为cDNA备用。
(2)设计引物,进行PCR扩增
设计用于扩增NtTAC1基因的引物序列如下:
NtTAC1-F: 5'-ATGAAGATCTTCAATTGGGTGCACC-3',
NtTAC1-R: 5'-TTAGATCGCATCCATGGGGATTGGG-3';
以步骤(1)中所制备cDNA为模板,利用上述引物进行PCR扩增,PCR扩增条件为:94℃预变性4min;94℃变性30s,56℃退火30s,72℃延伸40s,共30个循环;再72℃终延伸10min;PCR扩增4℃保存备用,或者直接进行电泳检测分析,并参照胶回收试剂盒说明书回收纯化PCR扩增后产物。
(3)连接、转化,并进行测序分析
将步骤(2)中所得PCR扩展产物连接至pEASY-T1载体上,连接体系参考如下:
DNA扩增产物,6μL;
pEASY-T1 载体,1μL;
混匀后,25℃连接25 min。
随后,将上述连接产物转化至大肠杆菌感受态细胞中,具体转化操作参考如下:
从-80℃冰箱中取出感受态细胞,置于冰上使其溶解,加连接产物50μL于溶解后Trans1-T1感受态细胞中,轻弹混匀,冰浴30 min;42℃水浴中热激30s,立即置于冰上2min;加入250μL平衡至室温的LB(不含抗生素),37℃摇荡培养1h;混合后均匀地涂布到LB固体平板(含60μg/μL 氨苄青霉素)平板上,倒置培养皿,37℃培养过夜。
最后,挑取白斑扩增培养后,提取质粒DNA,并进行PCR鉴定和测序鉴定和分析,最终获得如SEQ ID No.1所示的含有969 bp个核苷酸的NtTAC1基因编码序列,以及获得如SEQID No.2所示的NtTAC1蛋白的氨基酸序列。
实施例2
在实施例1基础上,发明人通过采集烟草不同生长期的组织、器官,利用荧光定量PCR技术,对NtTAC1基因的表达模式进行了分析,相关实验简要介绍如下。
采集现蕾期烟草的根、茎、叶、花、顶芽、叶枕(叶夹角)作为样品,提取RNA后,利用反转录试剂盒合成cDNA作为模板(参考试剂盒说明书操作即可),以烟草NtL25基因为内参,进行荧光定量PCR检测,检测时,引物序列设计如下:
检测NtTAC1基因的荧光定量引物,引物序列为:
NtTAC1-q-F: 5’- AGCAATTGATGACGAGGGTG-3’,
NtTAC1-q-R: 5’- ACACCTCCGTTCACTGAGTT-3’;
检测烟草NtL25基因时,具体引物为:
NtL25-F: 5’-CAAAAGTTACATTCCACCG-3’,
NtL25-F: 5’-TTTCTTCGTCCCATCAGGC-3’。
荧光定量PCR的条件为:第一步预变性,95℃ 10 s;第二步PCR反应,95℃ 5 s, 60℃ 30 s,39个循环;第三步溶解曲线。
每个样品进行3次生物学重复,通过2-△△CT方法分析相对基因表达差异。
具体结果如图1所示。可以看出,烟草NtTAC1基因在叶枕(叶夹角)处表达量最高,另外在茎、花、顶芽中有相对较高的表达量,在根和叶片中表达量最低。
实施例3
为进一步准确明确NtTAC1基因在植物株型形成忠的作用,发明人构建了用于敲低NtTAC1基因的重组表达载体pBWA(V)KS-RNAi-TAC1,并进一步转化烟草和获得了相关转基因株系,具体实验过程简介如下。
(一)构建重组表达载体pBWA(V)KS-RNAi-TAC1
(1)获得目的序列
首先,根据实施例1中测序所得NtTAC1基因编码序列,以及基于RNAi干扰技术特点,选择NtTAC1基因编码区域300bp长度序列作为靶位点(即SEQ ID No.3序列),具体设计PCR扩增引物序列如下:
正向引物:
TAC F(+):cagtGGTCTCacaacCTTCAAGATGCATCCATTGCACATA
TAC F(-):cgatGGTCTCacaggAGAATTGTTTAACTCAATGGCCAGT
反向引物:
TAC R(+):cagtGGTCTCagggcAGAATTGTTTAACTCAATGGCCAGT
TAC R(-):cagtGGTCTCatacaCTTCAAGATGCATCCATTGCACATA
以烟草基因组cDNA模板为模板,进行PCR扩增,获得构建干扰载体的目标序列片段;
具体目的序列为:
CTTCAAGATGCATCCATTGCACATATGTTAGATAGTTGGAGAGGAGGAATCCTAACAATTGGCACATTTGGGTTTGATCCATTGAAAAATGTGCAAGATCAAAGTGTCATAGACATTGAAGAAGAAGAAGTAGAAGAATCACTTGAGGATGAATATTACTCAGTGGAAAGTATTGGACAATGTCAAATTACTGTCACTAAGGAAAATGAAGAAGTGTACCCATTGATATATGCAAGTGGAGGTGAAGTGATTGAATATCCAAAACAAAATGAGATACTGGCCATTGAGTTAAACAATTCT。
(2)酶切、连接
将步骤(一)所或目的序列与pBWA(V)KS质粒分包进行BsaI、Eco31I双酶切,并回收酶切产物,随后T4 DNA连接酶将所回收酶切产物进行连接。
(3)转化合筛选
将步骤(二)中连接产物转化大肠杆菌感受态细胞,并进行筛选、鉴定,最终获得重组正确的用于敲低烟草叶片夹角基因TAC1的重组pBWA(V)KS-RNAi-TAC1表达载体。
相关操作未详述部分,参考现有技术常规操作既可,不再赘述。
(二)制备转染用浸染液
将上述步骤(一)所制备pBWA(V)KS-RNAi-TAC1表达载体转化农杆菌,并制备转染用浸染液备用。具体操作可参考如下:
在冰上溶解100 μl农杆菌感受态细胞,加入6 μL的pBWA(V)KS-RNAi-TAC1表达载体,轻弹混匀;随后,将混合物置于预冷的电转杯中,冰上放置5 min;
将电转仪参数调至:电压2.5 kV,电容25 μF,电阻200 Ω,把电转杯放入电击槽中,电击5 ms;
再后,迅速加入800 μL的预热至28℃的YEB液体培养基,220 rpm、28℃振荡复苏3h;
再后,将菌液4500 rpm离心1 min,弃一半体积的上清,重新悬浮后均匀涂于含有Rif(100 μg/mL)、Str(50 μg/mL)和Kan(50 μg/mL)的YEB固体培养基上,28℃倒置培养约2~3 d,直至单菌落形成;
挑取单菌落,扩培后对菌液进行PCR鉴定,鉴定正确的阳性克隆菌株,即为转化正确的工程菌,并可进一步扩增用于制备浸染液。
制备侵染液时,将转化正确的工程菌首先扩增培养至OD600=0.6左右,随后,4000rpm离心5 min收集菌体,再用20 mL的MS液体培养基悬浮菌体,此即为浸染液。
(三)转化烟草和筛选
预先取生长一个月左右的烟草无菌苗叶片,用打孔器将叶片处理成直径0.5 cm大小的叶盘,将处理后叶盘在MS固体培养基上事先预培养3 d;
具体转化烟草时,将上述预培养后叶盘置于步骤(二)所制备浸染菌液中,侵染10min;
随后,用无菌的滤纸吸干浸染后叶盘周围多余的菌液,在MS+6-BA(2 mg/L)+NAA(0.5 mg/L)的固体培养基上暗培养3 d;
再后,用含有Cef(400 mg/L)的无菌水清洗叶盘,并用无菌滤纸吸去多余的液体,清洗完成后,将叶盘转接到含有6-BA(2 mg/L)、NAA(0.5 mg/L)、Cef(200 mg/L)和Kan(50mg/L)的MS固体筛选培养基上,28℃光照培养;
待不定芽长到0.5cm时,转移到含Cef(200 mg/L)和Kan(50 mg/L)的MS固体培养基上生根。
待生长一个月左右,取少量叶片作为样品,提取DNA,通过PCR扩增、克隆、测序的方法,检测阳性转基因株系。具体鉴定方法为:
设计一对PCR检测引物,用于通过PCR方式来鉴定阳性转化植株:具体为:
NtTAC1-J-F: 5’-TTCATTTGGAGAGAACACGGGGGAC-3’,
NtTAC1-J-R: 5’-ATGGCCAGTATCTCATTTTGTTTTG-3’;
以T0代转基因株系DNA模板,进行PCR扩增;PCR条件为:94℃预变性4 min;94℃变性30 s,56℃退火30 s,72℃延伸40 s,共25个循环;再72 ℃终延伸10 min;
取PCR扩增进行琼脂糖凝胶电泳检测,鉴定阳性转化植株。
鉴定结果如图2所示。可以看出,在T0代植株中,检测到重组pBWA(V)KS-RNAi-TAC1载体的目的检测片段,而在野生型植株K326中,未检测到目的片段。这说明在所检测T0代植株中已经成功转入了pBWA(V)KS-RNAi-TAC1重组载体,确定为阳性遗传转化植株。
转基因株系中基因表达量及表型变化情况:
基于实时定量PCR技术(具体操作参考前述或者现有技术常规操作即可,不再赘述),就NtTAC1基因表达情况而言,与野生型K326相比,在RNAi植株中,烟草NtTAC1基因表达水平明显降低(图3A)。
分别将野生型和转基因株系的T0代植株生根幼苗移栽至盆中,温室培养6周左右,表型观察结果显示:与野生型烟草相比较,基因沉默的阳性植株的叶片夹角明显变小。当生长至12周时(图3B),阳性株的叶片直立程度更加明显,叶片夹角更小。
从上述结果可以看出,NtTAC1基因的表达水平可以控制烟草叶片夹角大小,基于这一结果,可为烟草株型调控及最终烟草产量和品质提升奠定一定理论基础和技术基础。
SEQUENCE LISTING
<110> 中国烟草总公司郑州烟草研究院
<120> 烟草NtTAC1基因在叶片夹角调控中的应用
<130> none
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 969
<212> DNA
<213> Nicotiana tabacum
<400> 1
atgaagatct tcaattgggt gcaccgcaaa ttgtatcaga aagatggatt ggtcagtcgg 60
aatgtgaaga aagatgagct caggattagc aatgagttta ttggtgacgc acaagttctt 120
cttcaagatg catccattgc acatatgtta gatagttgga gaggaggaat cctaacaatt 180
ggcacatttg ggtttgatcc attgaaaaat gtgcaagatc aaagtgtcat agacattgaa 240
gaagaagaag tagaagaatc acttgaggat gaatattact cagtggaaag tattggacaa 300
tgtcaaatta ctgtcactaa ggaaaatgaa gaagtgtacc cattgatata tgcaagtgga 360
ggtgaagtga ttgaatatcc aaaacaaaat gagatactgg ccattgagtt aaacaattct 420
tcagagagca ataagatgct gaaaaaagaa aggattactc tagcagacct tttttcagct 480
gattctgatc atcaccaaaa ccttaagcca aatccaagca aaaaggaaga agagttttac 540
acaaagaaac cttgttcaca agtgaagaat ggaacatttt tcgccaaaaa gctaattcct 600
cgagttaagg aggattctcg tccgatccaa aaactacagc aattgatgac gagggtgatg 660
aaaaggaagg ttcatccaga tattgaaagc aaaataggca agaacaacaa cattactact 720
catcaagtga aagcagcagc tagcatgctt gggctttcct gcgttaagca tgtaagagtt 780
gactctgttt cccttctgca acttgatcaa gattttcgat ttcaggtcga aatgtctaac 840
tcagtgaacg gaggtgtaaa caacaatctt gaggaccacg gagaaaacag tgtggctatt 900
ccaggtgttg gtgtgccacc acgaaaccct gagaatgcat cggacccaat ccccatggat 960
gcgatctaa 969
<210> 2
<211> 322
<212> PRT
<213> Nicotiana tabacum
<400> 2
Met Lys Ile Phe Asn Trp Val His Arg Lys Leu Tyr Gln Lys Asp Gly
1 5 10 15
Leu Val Ser Arg Asn Val Lys Lys Asp Glu Leu Arg Ile Ser Asn Glu
20 25 30
Phe Ile Gly Asp Ala Gln Val Leu Leu Gln Asp Ala Ser Ile Ala His
35 40 45
Met Leu Asp Ser Trp Arg Gly Gly Ile Leu Thr Ile Gly Thr Phe Gly
50 55 60
Phe Asp Pro Leu Lys Asn Val Gln Asp Gln Ser Val Ile Asp Ile Glu
65 70 75 80
Glu Glu Glu Val Glu Glu Ser Leu Glu Asp Glu Tyr Tyr Ser Val Glu
85 90 95
Ser Ile Gly Gln Cys Gln Ile Thr Val Thr Lys Glu Asn Glu Glu Val
100 105 110
Tyr Pro Leu Ile Tyr Ala Ser Gly Gly Glu Val Ile Glu Tyr Pro Lys
115 120 125
Gln Asn Glu Ile Leu Ala Ile Glu Leu Asn Asn Ser Ser Glu Ser Asn
130 135 140
Lys Met Leu Lys Lys Glu Arg Ile Thr Leu Ala Asp Leu Phe Ser Ala
145 150 155 160
Asp Ser Asp His His Gln Asn Leu Lys Pro Asn Pro Ser Lys Lys Glu
165 170 175
Glu Glu Phe Tyr Thr Lys Lys Pro Cys Ser Gln Val Lys Asn Gly Thr
180 185 190
Phe Phe Ala Lys Lys Leu Ile Pro Arg Val Lys Glu Asp Ser Arg Pro
195 200 205
Ile Gln Lys Leu Gln Gln Leu Met Thr Arg Val Met Lys Arg Lys Val
210 215 220
His Pro Asp Ile Glu Ser Lys Ile Gly Lys Asn Asn Asn Ile Thr Thr
225 230 235 240
His Gln Val Lys Ala Ala Ala Ser Met Leu Gly Leu Ser Cys Val Lys
245 250 255
His Val Arg Val Asp Ser Val Ser Leu Leu Gln Leu Asp Gln Asp Phe
260 265 270
Arg Phe Gln Val Glu Met Ser Asn Ser Val Asn Gly Gly Val Asn Asn
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Asn Leu Glu Asp His Gly Glu Asn Ser Val Ala Ile Pro Gly Val Gly
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Val Pro Pro Arg Asn Pro Glu Asn Ala Ser Asp Pro Ile Pro Met Asp
305 310 315 320
Ala Ile
<210> 3
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<212> DNA
<213> Nicotiana tabacum
<400> 3
cttcaagatg catccattgc acatatgtta gatagttgga gaggaggaat cctaacaatt 60
ggcacatttg ggtttgatcc attgaaaaat gtgcaagatc aaagtgtcat agacattgaa 120
gaagaagaag tagaagaatc acttgaggat gaatattact cagtggaaag tattggacaa 180
tgtcaaatta ctgtcactaa ggaaaatgaa gaagtgtacc cattgatata tgcaagtgga 240
ggtgaagtga ttgaatatcc aaaacaaaat gagatactgg ccattgagtt aaacaattct 300
Claims (8)
1.烟草NtTAC1基因,其特征在于,长度为969bp,其编码序列如SEQ ID No.1所示。
2.权利要求1所述烟草NtTAC1基因所编码的烟草NtTAC1蛋白,其特征在于,该蛋白长度为322AA,氨基酸序列如SEQ ID No.2 所示。
3.用于PCR扩增权利要求1所述烟草NtTAC1基因的引物对,其特征在于,引物序列如下:
NtTAC1-F: 5'-ATGAAGATCTTCAATTGGGTGCACC-3',
NtTAC1-R: 5'-TTAGATCGCATCCATGGGGATTGGG-3'。
4.权利要求1所述烟草NtTAC1基因在叶片夹角调控中应用,其特征在于,该基因在翻译表达后,用于调控烟草叶片夹角。
5.如权利要求4所述烟草NtTAC1基因在叶片夹角调控中应用,其特征在于,应用时,在将NtTAC1基因沉默后,通过降低NtTAC1基因表达量,用于减小烟草叶片夹角。
6.用于敲低权利要求1所述NtTAC1基因表达的重组表达载体,其特征在于,将其命名为:pBWA(V)KS-RNAi-TAC1,该重组表达载体以pBWA(V)KS-RNAi质粒为出发质粒,具体通过如下步骤构建获得:
(一)获得目的序列
设计正反向引物序列如下:
正向引物:
TAC F(+):5’-cagtGGTCTCacaacCTTCAAGATGCATCCATTGCACATA-3’,
TAC F(-):5’-cgatGGTCTCacaggAGAATTGTTTAACTCAATGGCCAGT-3’;
反向引物:
TAC R(+):5’-cagtGGTCTCagggcAGAATTGTTTAACTCAATGGCCAGT-3’,
TAC R(-):5’-cagtGGTCTCatacaCTTCAAGATGCATCCATTGCACATA-3’;
以烟草cDNA为模板进行PCR扩增,获得构建干扰载体的目标序列片段;
具体目的序列如SEQ ID No.3所示;
(二)酶切、连接
将步骤(一)所或目的序列与pBWA(V)KS质粒分包进行BsaI、Eco31I双酶切,并回收酶切产物,随后T4 DNA连接酶将所回收酶切产物进行连接;
(三)转化合筛选
将步骤(二)中连接产物转化大肠杆菌感受态细胞,并进行筛选、鉴定,最终获得重组正确的用于敲低烟草叶片夹角基因TAC1的重组pBWA(V)KS-RNAi-TAC1表达载体。
7.权利要求6所述用于敲低NtTAC1基因表达的重组表达载体在烟草中应用,其特征在于,将该重组载体转化烟草后,能够降低烟草TAC1基因的表达水平,进而通过降低烟草NtTAC1蛋白表达量来调控烟草叶片夹角。
8.利用权利要求6所述用于敲低NtTAC1基因表达的重组表达载体的转基因烟草新品种培育方法,其特征在于,利用农杆菌介导的基因转化方法,首先将重组表达载体pBWA(V)KS-RNAi-TAC1转化农杆菌并制备浸染液;随后将烟草组培用组培体置于浸染液中进行转染;最后,将侵染后组培体在无菌条件下进行组培、筛选、鉴定,获得NtTAC1基因表达降低的转化正确转基因植物新株系。
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