CN114540368B - 一种烟草氨基酸转运蛋白相关基因NtLHT1的应用及方法 - Google Patents
一种烟草氨基酸转运蛋白相关基因NtLHT1的应用及方法 Download PDFInfo
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Abstract
本发明涉及一种烟草氨基酸转运蛋白相关基因NtLHT1的应用及方法,属于植物基因工程技术领域。一种烟草氨基酸转运蛋白相关的基因NtLHT1在调控烟草叶片中氨基酸含量中的应用。本发明通过CRISPR/Cas9介导的基因编辑技术,构建了用于敲除,NtLHT1基因的CRISPR/Cas9编辑载体,经编辑素材创制和分子检测鉴定后获得了,NtLHT1基因敲除的烟草植株。本发明获得的,NtLHT1基因敲除的编辑烟草植株,相比于对照烟草植株,叶片中氨基酸含量显著增加。
Description
技术领域
本发明属于植物基因工程技术领域,具体涉及一种烟草氨基酸转运蛋白基因NtLHT1的应用及方法。
背景技术
氨基酸转运蛋白基因(Amino acid transporters,AATs)是植物生长代谢过程中的关键调控因子,对植物的生长代谢和产量起着重要作用。近年来,AATs的研究对象逐渐由原来的模式植物拟南芥发展到以重要粮食作物为主,例如水稻、马铃薯等。烟草作为我国重要的经济作物和模式作物,研究烟草氨基酸转运的分子机制具有重要意义。烟草叶片中氨基酸含量及其组成与香气量、劲头等烟气指标关系密切,影响着烟叶的品质及风味。专利201210462382.0报道,人参氨基酸转运相关基因PgLHT在增强人参植株的抗性等方面有显著的效果;专利202010169968.2报道,敲除水稻氨基酸转运蛋白OsLHT1基因,提高了籽粒中蛋白质和氨基酸的含量,增强了对稻瘟病的抗性;专利201811196540.6报道,烟草氨基酸转运蛋白基因NtTAT通过氨基酸转运能力的改变而影响氨基酸的代谢基因,提高了烟叶中缬氨酸、脯氨酸、苏氨酸和苯丙氨酸等氨基酸的含量,进而影响烟叶的品质。本发明的基因在烟叶氨基酸含量调控中的功能目前没有报道。
发明内容
本发明要解决现有技术的不足,提供一种烟草氨基酸转运蛋白基因NtLHT1的应用,为烟草氨基酸转运蛋白研究及烟草品质调控提供材料和借鉴。
为实现上述目的,本发明采用的技术方案如下:
一种烟草氨基酸转运蛋白相关的基因NtLHT1在调控烟草叶片中氨基酸含量中的应用。
本发明上述技术方案中,所述的烟草氨基酸转运蛋白相关基因NtLHT1,核苷酸序列如SEQ ID NO.1所示,包括1368 bp个碱基,该基因来源于烟草(Nicotiana tabacum,命名为NtLHT1)。
SEQ ID No.1:
ATGCAACCTGCAACTATGGGAACGCAAACTCCAAATATTTCCAATTACTACTGCTCCAAGAGTGCTGATGAAAGATCTGCAGAAGAGAGGGCAATAGACGCATGGCTTCCTGTTACTTCAAACAGGAATGCGAAATGGTGGTATTCAGCTTTCCACAATGTTACTGCCATGGTCGGAGCTGGTGTCCTTGGTCTTCCTTATGCCATGGCACAACTTGGATGGGGACCAGGAGTAGCAGTGCTGGTGATTTCTTGGATTATAACATTTTACACATTATGGCAAATGGTTGAGATGCACGAAATGGTTCCTGGGAAACGTTTTGACAGATATCATGAACTTGGGCAGCATGCTTTTGGGAAAAAACTTGGGCTATGGATTATTGTGCCACAACAGTTGATTGTTGAAGTTGGAGTTGACATAGTTTATATGGTAACCGGAGGACAATCACTCCAGAAATTCTATGATCTAGTCTGTAAAAAAGATTGTAAAGACATAAAACTTACCTACTTCATTATGATCTTTGCCTCTGTCCATTTTGTTATCTCTCATCTTCCTGATTTCAATTCCATAGTAGGTGTGTCTTTGGCTGCAGCTGTCATGTCCTTAAGTTACTCAACAATTGCTTGGGGAGCTTCAGTTAAGAAAGGTGTAGTACCAGATGTGGAATATGGATACAAGGCAAAGTCAACAGCAGGAACAATTTTCAACTTTTTCAGTGCATTGGGAGATGTTGCTTTTGCTTATGCTGGCCATAATGTGGTGTTAGAAATTCAAGCTACAATCCCTTCAACACCTGAAAAGCCTTCAAAAGGACCTATGTGGAAAGGAGTTATTGTTGCTTATATAATTGTTGCTTTCTGTTATTTCCCTGTTGCTCTTATTGGCTACTGGATGTTTGGGAATCAAGTGAAAGACAACATTCTTAAGACTTTGGAGAAACCTACCTGGCTCATTGCTATGGCTAACTTGTTTGTTGTTATTCATGTTATTGGGAGTTATCAGATATATGCAATGCCAGTATTTGACATGATAGAAACAGTGCTTGTCAGAAAACTTAAGTTCAAGCCAAGCTGGATGTTGCGCTTTGTTACTAGGAACATTTATGTAGCTTTCACAATGTTTGTTGGCATTACCTTCCCTTTCTTCAATGGGCTGCTTGGATTCTTTGGAGGATTTGCTTTTGCCCCAACAACCTATTTTCTCCCTTGCATCATGTGGCTAGCAATCTGCAAACCAAAGAAATTCAGTCTCTCTTGGATTATTAATTGGATTTGCATTATTCTTGGAGTACTATTAATGGTTATAGCACCAATTGGTGGCCTAAGATCCATAATCATGCAAGCCAAGGGCTACAAATTTTACTCTTAA。
所述烟草氨基酸转运蛋白相关基因NtLHT1表达的编码蛋白,氨基酸序列如SEQ IDNO.2所示,包括455个氨基酸。
SEQ ID No.2:
MQPATMGTQTPNISNYYCSKSADERSAEERAIDAWLPVTSNRNAKWWYSAFHNVTAMVGAGVLGLPYAMAQLGWGPGVAVLVISWIITFYTLWQMVEMHEMVPGKRFDRYHELGQHAFGKKLGLWIIVPQQLIVEVGVDIVYMVTGGQSLQKFYDLVCKKDCKDIKLTYFIMIFASVHFVISHLPDFNSIVGVSLAAAVMSLSYSTIAWGASVKKGVVPDVEYGYKAKSTAGTIFNFFSALGDVAFAYAGHNVVLEIQATIPSTPEKPSKGPMWKGVIVAYIIVAFCYFPVALIGYWMFGNQVKDNILKTLEKPTWLIAMANLFVVIHVIGSYQIYAMPVFDMIETVLVRKLKFKPSWMLRFVTRNIYVAFTMFVGITFPFFNGLLGFFGGFAFAPTTYFLPCIMWLAICKPKKFSLSWIINWICIILGVLLMVIAPIGGLRSIIMQAKGYKFYS。
本发明还提供上述烟草氨基酸转运调控相关基因NtLHT1在烟草氨基酸含量调控中应用的方法:通过CRISPR/Cas9介导的基因编辑,构建用于敲除NtLHT1基因的CRISPR/Cas9编辑载体,经遗传转化后获得NtLHT1基因发生编辑的成长后叶片中氨基酸含量显著增加的烟草植株。该植株叶片中,天冬氨酸、丝氨酸、甘氨酸、精氨酸、脯氨酸、组氨酸、甲硫氨酸、亮氨酸、赖氨酸和总氨基酸等指标均显著增加。
本发明的第二个目的是提供上述烟草氨基酸转运调控相关基因NtLHT1在提高重金属胁迫抗性中的应用。
作为上述技术方案的优选,更具体是提供一种烟草氨基酸转运蛋白相关的基因NtLHT1在提高硫酸铜胁迫抗性中的应用。
作为上述技术方案的优选,更具体是提供一种烟草氨基酸转运蛋白相关的基因NtLHT1在提高0.5~2.0 mg/L硫酸铜胁迫抗性中的应用。
作为上述技术方案的优选,更具体是提供一种烟草氨基酸转运蛋白相关的基因NtLHT1在提高0.8~1.2 mg/L硫酸铜胁迫抗性中的应用。
本发明还提供上述烟草氨基酸转运调控相关基因NtLHT1在提高重金属胁迫抗性中应用的方法:通过CRISPR/Cas9介导的基因编辑,构建用于敲除NtLHT1基因的CRISPR/Cas9编辑载体,经遗传转化后获得NtLHT1基因发生编辑的成长后叶片中氨基酸含量显著增加的烟草植株。NtLHT1基因敲除的烟草植株,相比于对照烟草植株,在重金属胁迫下其种子萌发率较好,种子重金属胁迫抗性提高。
作为上述技术方案的优选,所述重金属胁迫为铜离子胁迫。
综上所述,本发明具有以下有益效果:
1、本发明通过CRISPR/Cas9介导的基因编辑技术,构建了用于敲除,NtLHT1基因的CRISPR/Cas9编辑载体,经编辑素材创制和分子检测鉴定后获得了,NtLHT1基因敲除的烟草植株。本发明获得的,NtLHT1基因敲除的编辑烟草植株,相比于对照烟草植株,叶片中氨基酸含量显著增加。
2、本发明获得的,NtLHT1基因敲除的烟草植株,相比于对照烟草植株,在重金属胁迫下其种子萌发率较好,种子重金属胁迫抗性提高。
3、本发明利用CRISPR/Cas9介导的基因编辑技术敲除NtLHT1基因获得了叶片氨基酸含量显著增加、种子重金属胁迫抗性提高的编辑烟草素材,这为进一步阐明烟草氨基酸调控机理提供了理论依据,为培育氨基酸含量改变、抗性提高的烟草品种提供了新的遗传材料。
附图说明
图1为NtLHT1基因克隆电泳图;
图2为对照(未转化)植株叶片(H)和NtLHT1基因编辑植株(3#)
的表型;
图3为对照(未转化)植株叶片和NtLHT1基因编辑植株叶片氨基酸含量示意图;
图2中,其中a为蒸馏水处理,b1和b2为1mg/L CuSO4处理。
具体实施方式
以下通过实施例来详细说明本发明的技术方案,以下的实施例仅是示例性的,仅能用来解释和说明本发明的技术方案,而不能解释为是对本发明技术方案的限制。
在本申请的各实施例中,没有注明具体技术或条件者,按照本领域内现有技术或条件进行,所使用的材料或设备未注明生产厂商者,均为可以通过购买获得的常规产品。
本发明除非另有说明,否则百分号为体积百分数,比例为体积比。
本申请中所使用的烟草品种为红花大金元,一种商品化烟草品种。
实施例1
本实施例主要就烟草氨基酸转运相关的基因NtLHT1的获得过程,简要介绍如下。
以栽培种烟草红花大金元叶片为样品,利用RNA提取试剂盒提取烟草叶片总RNA,反转录为cDNA备用:
按照植物RNA提取试剂盒说明书提取烟草总RNA。
1μg从叶片中提取总RNA用于反转录,转录体系如下:
Total RNA 1μg
Oligo(dT) (10μM) 1.5μL
ddH2O up to 15μL
将上述体系混匀后置于PCR中,70℃保温5min,去除后立即置于冰上5min,之后向体系中加入以下试剂:
M-MLV Buffer(5X) 5μL
M-MLV逆转录酶 0.5μL
RNase 抑制剂 0.5μL
dNTP Mixture 4μL
ddH2O up to 25μL
上述体系放入PCR仪中,42℃ 65min,65℃ 10min,4℃保温,然后置于-20℃冰箱中保存使用。
通过同源比对的方法,参考拟南芥基因的序列及已知烟草部分基因序列,设计扩增引物序列如下:
F:5'-ATGCAACCTGCAACTATGGGAACGC-3',(SEQ ID No.3)
R:5'-TTAAGAGTAAAATTTGTAGCCCTTGGC-3';(SEQ ID No.4)
以上述所制备cDNA为模板,利用上述引物进行PCR扩增:
扩增体系(50μL):
cDNA 0.5μL
5×Reaction Buffer 10μL
上游引物(10mmol/L) 2.5μL
下游引物(10mmol/L) 2.5μL
dNTP (10 mM) 5μL
Phusion DNA Polymerase 0.5μL
ddH2O up to 50μL
混匀离心后进行PCR 扩增,PCR反应条件为:95℃ 10sec,52℃ 30sec,72℃2.5min,共30个循环;72℃ 10min;12℃ Hold。
对扩增产物进行提纯后测序,获得烟草氨基酸转运相关的基因NtLHT1序列,其碱基序列如SEQ ID No.1所示,共包括1368 bp个碱基。
SEQ ID No.1:
ATGCAACCTGCAACTATGGGAACGCAAACTCCAAATATTTCCAATTACTACTGCTCCAAGAGTGCTGATGAAAGATCTGCAGAAGAGAGGGCAATAGACGCATGGCTTCCTGTTACTTCAAACAGGAATGCGAAATGGTGGTATTCAGCTTTCCACAATGTTACTGCCATGGTCGGAGCTGGTGTCCTTGGTCTTCCTTATGCCATGGCACAACTTGGATGGGGACCAGGAGTAGCAGTGCTGGTGATTTCTTGGATTATAACATTTTACACATTATGGCAAATGGTTGAGATGCACGAAATGGTTCCTGGGAAACGTTTTGACAGATATCATGAACTTGGGCAGCATGCTTTTGGGAAAAAACTTGGGCTATGGATTATTGTGCCACAACAGTTGATTGTTGAAGTTGGAGTTGACATAGTTTATATGGTAACCGGAGGACAATCACTCCAGAAATTCTATGATCTAGTCTGTAAAAAAGATTGTAAAGACATAAAACTTACCTACTTCATTATGATCTTTGCCTCTGTCCATTTTGTTATCTCTCATCTTCCTGATTTCAATTCCATAGTAGGTGTGTCTTTGGCTGCAGCTGTCATGTCCTTAAGTTACTCAACAATTGCTTGGGGAGCTTCAGTTAAGAAAGGTGTAGTACCAGATGTGGAATATGGATACAAGGCAAAGTCAACAGCAGGAACAATTTTCAACTTTTTCAGTGCATTGGGAGATGTTGCTTTTGCTTATGCTGGCCATAATGTGGTGTTAGAAATTCAAGCTACAATCCCTTCAACACCTGAAAAGCCTTCAAAAGGACCTATGTGGAAAGGAGTTATTGTTGCTTATATAATTGTTGCTTTCTGTTATTTCCCTGTTGCTCTTATTGGCTACTGGATGTTTGGGAATCAAGTGAAAGACAACATTCTTAAGACTTTGGAGAAACCTACCTGGCTCATTGCTATGGCTAACTTGTTTGTTGTTATTCATGTTATTGGGAGTTATCAGATATATGCAATGCCAGTATTTGACATGATAGAAACAGTGCTTGTCAGAAAACTTAAGTTCAAGCCAAGCTGGATGTTGCGCTTTGTTACTAGGAACATTTATGTAGCTTTCACAATGTTTGTTGGCATTACCTTCCCTTTCTTCAATGGGCTGCTTGGATTCTTTGGAGGATTTGCTTTTGCCCCAACAACCTATTTTCTCCCTTGCATCATGTGGCTAGCAATCTGCAAACCAAAGAAATTCAGTCTCTCTTGGATTATTAATTGGATTTGCATTATTCTTGGAGTACTATTAATGGTTATAGCACCAATTGGTGGCCTAAGATCCATAATCATGCAAGCCAAGGGCTACAAATTTTACTCTTAA。
对该基因序列进行翻译后,其所编码蛋白序列如SEQ ID No.2所示,共包括455个氨基酸,进一步对比分析表明,该蛋白含有同源性很高的序列,高度保守。
SEQ ID No.2:
MQPATMGTQTPNISNYYCSKSADERSAEERAIDAWLPVTSNRNAKWWYSAFHNVTAMVGAGVLGLPYAMAQLGWGPGVAVLVISWIITFYTLWQMVEMHEMVPGKRFDRYHELGQHAFGKKLGLWIIVPQQLIVEVGVDIVYMVTGGQSLQKFYDLVCKKDCKDIKLTYFIMIFASVHFVISHLPDFNSIVGVSLAAAVMSLSYSTIAWGASVKKGVVPDVEYGYKAKSTAGTIFNFFSALGDVAFAYAGHNVVLEIQATIPSTPEKPSKGPMWKGVIVAYIIVAFCYFPVALIGYWMFGNQVKDNILKTLEKPTWLIAMANLFVVIHVIGSYQIYAMPVFDMIETVLVRKLKFKPSWMLRFVTRNIYVAFTMFVGITFPFFNGLLGFFGGFAFAPTTYFLPCIMWLAICKPKKFSLSWIINWICIILGVLLMVIAPIGGLRSIIMQAKGYKFYS。
实施例2
利用实施例1中所获得烟草氨基酸转运相关的基因NtLHT1,本发明进一步构建了CRISPR/Cas9载体,以及利用叶盘法转化获得基因编辑植株。
选择NtLHT1基因中较特异的23nt核苷酸序列(SEQ ID No.5)为CRISPR/Cas9的引导序列,并将该序列片段与CRISPR/Cas9载体(由西南大学提供)进行连接、转化和PCR扩增检测,PCR阳性克隆送测序公司进行测序确认,最后得到CRISPR/Cas9-NtLHT1编辑载体。
利用上一步所构建的CRISPR/Cas9-NtLHT1编辑载体质粒,以红花大金元为例,进行遗传转化和组培,以获得烟草氨基酸转运相关的基因NtLHT1发生敲除编辑的植株,相关实验过程简要介绍如下。
将烟草种子表面消毒后点种至MS培养基上,待长到4片子叶(15~20 d),移入含MS固体培养基的培养瓶中,于25±1℃、光照强度30~50 μmol/(m2·s),光照时间为16h/d条件继续培养35~40 d,备用。
取出-80℃保存的LBA4404电转化感受态农杆菌细胞,置于冰上冻融。待感受态刚刚解冻时,加入CRISPR/Cas9-NtLHT1编辑载体质粒的2μL,混匀,置于冰上。后将混匀的感受态转移至预冷的电转杯中,将电转杯置于电转仪中进行转化,转化完成后加入1mL的YEB液体培养基与转化液进行混合,后置于摇床28℃,200rpm培养1.5~2h。8000rpm离心菌体弃掉上清培养基,然后用200μL的YEB液体培养基悬浮菌体,涂于含50mg/L利福平、50mg/L链霉素和50mg/L卡那霉素的YEB固体培养基上28℃倒置黑暗培养2~3d。
在超净工作台中制作烟草叶盘成边长为1cm的方形叶盘,用MS液体制备含有CRISPR/Cas9-NtLHT1编辑载体的农杆菌菌落成悬浮菌液(OD600=0.6~0.8)。利用悬浮农杆菌菌液浸泡侵染烟草叶盘10min。之后将叶盘置于含2.0mg/L NAA+0.5mg/L 6-BA的MS固体培养基上,28℃,黑暗,共培养3d。之后进行继代培养,放置于含2.0mg/L NAA+0.5mg/L 6-BA+250mg/L Cb+50mg/L Kan的MS固体培养基上,培养条件为:28℃光照培养16h/d,光照强度30~50μmol/(m2·s),25℃黑暗培养8h/d,培养45~60d,直至分化芽形成,每7~10d更换一次分化培养培养基,更换3~4次;培养至分化芽形成;将已有分化芽形成的愈伤组织切下,置于含有500mg/L羧苄青霉素与50mg/L卡那霉素的MS培养基上进行培养,待愈伤组织上分化芽培养长至2~4cm高,培养条件与分化培养条件一致,培养8-14d;再生植株生根培养,将分化芽切下,插入含有500mg/L羧苄青霉素与50mg/L卡那霉素的MS培养基上进行生根培养,培养条件与分化培养条件一致,培养20-30d,再生移栽至花盆后进行培养,后进行转化植株叶片取样,送华大基因进行分子检测,确定获得NtLHT1基因编辑植株,之后进行收种获得T0代编辑植株种子。T0代种子按23倍进行自交纯合扩繁,待植株长到5~6片叶时,单株的叶片取样,送华大基因进行分子检测,确定获得NtLHT1基因发生纯合编辑的植株,之后进行收种获得NtLHT1基因纯合编辑的T1代种子。
本发明所述的烟草氨基酸转运基因NtLHT1的应用为在烟草植株体内降低所述NtLHT1基因的表达,可调控烟草叶片氨基酸含量。现有技术领域内常用的降低基因表达或者基因沉默的方法均适用于本发明。
实施例3
例2中获得NtLHT1基因纯合编辑的T1代种子,在水和1mg/L CuSO4条件下进行种子萌发实验,生长7d后观察表型情况。在蒸馏水条件处理下NtLHT1基因编辑材料和对照红花大金元种子的萌发率情况没有显著差异;在1mg/L CuSO4条件下处理后,NtLHT1基因编辑材料的种子萌发率更好,重金属胁迫抗性提高。
实施例4
利用实施例2中分子检测确定为NtLHT1基因纯合敲除的植株,进行收种获得基因纯合编辑素材。然后以GC-MS进行NtLHT1基因纯合敲除素材叶片的氨基酸含量的检测试验。
选择成熟期的烟株,采集5株对照(未编辑)烟草植株样本,采集同一叶位的叶片;选择现蕾期的烟株,采集5株NtLHT1基因纯合编辑的烟草植株样本;叶片去主筋,锡箔纸包裹液氮保存运输,实验室超低温(-70℃)保存,冻干磨粉过筛。
标准品的衍生过程:准确吸取1 mL混合标准品样品,加入1mL 1mol/L三乙胺乙腈溶液,并加入1mL 0.1mol/L苯基异硫氰酸酯乙腈溶液,涡旋混匀1min,室温反应半个小时,待反应完全后加入2mL正己烷溶液,涡旋1min,静置10min,取下层清液200μL置于样品瓶加入800μL超纯水,混匀15s,过滤,供液相分析用。(标曲范围:2.5ug/mL~50ug/mL)
样品前处理及衍生:称取烟叶粉末0.3000g置于15mL离心管,加入5mL0.1mol/L盐酸水溶液,超声提取40min,8000r/min离心10min,准确移取1mL上清液,加入1mL 1mol/L三乙胺乙腈溶液,并加入1mL 0.1mol/L苯基异硫氰酸酯乙腈溶液,涡旋混匀1min,反应半个小时,待反应完全后加入2mL正己烷溶液,涡旋1min,静置10min,取下层清液200μL加入800μL超纯水,混匀15s,过滤后置于样品瓶,供液相分析用。
仪器方法:
A:50mmol/L乙酸钠(ph=6.5)(93:7乙腈)
B:甲醇:ACN:水=2:6:2
色谱柱:Dikma Endeavosil C18,100*2.1mm,1.8um
波长:254 nm
柱温:40℃
对照(未编辑)及NtLHT1基因纯合编辑烟草植株叶片中氨基酸含量比较(结果如图2所示)。与对照相比,NtLHT1基因纯合编辑烟草植株叶片中,天冬氨酸、丝氨酸、甘氨酸、精氨酸、脯氨酸、组氨酸、甲硫氨酸、亮氨酸、赖氨酸和总氨基酸等指标均显著增加。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
序列表
<110> 云南中烟工业有限责任公司
<120> 一种烟草氨基酸转运蛋白相关基因NtLHT1的应用及方法
<141> 2022-02-22
<160> 5
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1368
<212> DNA
<213> Artificial Sequence
<400> 2
atgcaacctg caactatggg aacgcaaact ccaaatattt ccaattacta ctgctccaag 60
agtgctgatg aaagatctgc agaagagagg gcaatagacg catggcttcc tgttacttca 120
aacaggaatg cgaaatggtg gtattcagct ttccacaatg ttactgccat ggtcggagct 180
ggtgtccttg gtcttcctta tgccatggca caacttggat ggggaccagg agtagcagtg 240
ctggtgattt cttggattat aacattttac acattatggc aaatggttga gatgcacgaa 300
atggttcctg ggaaacgttt tgacagatat catgaacttg ggcagcatgc ttttgggaaa 360
aaacttgggc tatggattat tgtgccacaa cagttgattg ttgaagttgg agttgacata 420
gtttatatgg taaccggagg acaatcactc cagaaattct atgatctagt ctgtaaaaaa 480
gattgtaaag acataaaact tacctacttc attatgatct ttgcctctgt ccattttgtt 540
atctctcatc ttcctgattt caattccata gtaggtgtgt ctttggctgc agctgtcatg 600
tccttaagtt actcaacaat tgcttgggga gcttcagtta agaaaggtgt agtaccagat 660
gtggaatatg gatacaaggc aaagtcaaca gcaggaacaa ttttcaactt tttcagtgca 720
ttgggagatg ttgcttttgc ttatgctggc cataatgtgg tgttagaaat tcaagctaca 780
atcccttcaa cacctgaaaa gccttcaaaa ggacctatgt ggaaaggagt tattgttgct 840
tatataattg ttgctttctg ttatttccct gttgctctta ttggctactg gatgtttggg 900
aatcaagtga aagacaacat tcttaagact ttggagaaac ctacctggct cattgctatg 960
gctaacttgt ttgttgttat tcatgttatt gggagttatc agatatatgc aatgccagta 1020
tttgacatga tagaaacagt gcttgtcaga aaacttaagt tcaagccaag ctggatgttg 1080
cgctttgtta ctaggaacat ttatgtagct ttcacaatgt ttgttggcat taccttccct 1140
ttcttcaatg ggctgcttgg attctttgga ggatttgctt ttgccccaac aacctatttt 1200
ctcccttgca tcatgtggct agcaatctgc aaaccaaaga aattcagtct ctcttggatt 1260
attaattgga tttgcattat tcttggagta ctattaatgg ttatagcacc aattggtggc 1320
ctaagatcca taatcatgca agccaagggc tacaaatttt actcttaa 1368
<210> 2
<211> 455
<212> PRT
<213> Artificial Sequence
<400> 2
Met Gln Pro Ala Thr Met Gly Thr Gln Thr Pro Asn Ile Ser Asn Tyr
1 5 10 15
Tyr Cys Ser Lys Ser Ala Asp Glu Arg Ser Ala Glu Glu Arg Ala Ile
20 25 30
Asp Ala Trp Leu Pro Val Thr Ser Asn Arg Asn Ala Lys Trp Trp Tyr
35 40 45
Ser Ala Phe His Asn Val Thr Ala Met Val Gly Ala Gly Val Leu Gly
50 55 60
Leu Pro Tyr Ala Met Ala Gln Leu Gly Trp Gly Pro Gly Val Ala Val
65 70 75 80
Leu Val Ile Ser Trp Ile Ile Thr Phe Tyr Thr Leu Trp Gln Met Val
85 90 95
Glu Met His Glu Met Val Pro Gly Lys Arg Phe Asp Arg Tyr His Glu
100 105 110
Leu Gly Gln His Ala Phe Gly Lys Lys Leu Gly Leu Trp Ile Ile Val
115 120 125
Pro Gln Gln Leu Ile Val Glu Val Gly Val Asp Ile Val Tyr Met Val
130 135 140
Thr Gly Gly Gln Ser Leu Gln Lys Phe Tyr Asp Leu Val Cys Lys Lys
145 150 155 160
Asp Cys Lys Asp Ile Lys Leu Thr Tyr Phe Ile Met Ile Phe Ala Ser
165 170 175
Val His Phe Val Ile Ser His Leu Pro Asp Phe Asn Ser Ile Val Gly
180 185 190
Val Ser Leu Ala Ala Ala Val Met Ser Leu Ser Tyr Ser Thr Ile Ala
195 200 205
Trp Gly Ala Ser Val Lys Lys Gly Val Val Pro Asp Val Glu Tyr Gly
210 215 220
Tyr Lys Ala Lys Ser Thr Ala Gly Thr Ile Phe Asn Phe Phe Ser Ala
225 230 235 240
Leu Gly Asp Val Ala Phe Ala Tyr Ala Gly His Asn Val Val Leu Glu
245 250 255
Ile Gln Ala Thr Ile Pro Ser Thr Pro Glu Lys Pro Ser Lys Gly Pro
260 265 270
Met Trp Lys Gly Val Ile Val Ala Tyr Ile Ile Val Ala Phe Cys Tyr
275 280 285
Phe Pro Val Ala Leu Ile Gly Tyr Trp Met Phe Gly Asn Gln Val Lys
290 295 300
Asp Asn Ile Leu Lys Thr Leu Glu Lys Pro Thr Trp Leu Ile Ala Met
305 310 315 320
Ala Asn Leu Phe Val Val Ile His Val Ile Gly Ser Tyr Gln Ile Tyr
325 330 335
Ala Met Pro Val Phe Asp Met Ile Glu Thr Val Leu Val Arg Lys Leu
340 345 350
Lys Phe Lys Pro Ser Trp Met Leu Arg Phe Val Thr Arg Asn Ile Tyr
355 360 365
Val Ala Phe Thr Met Phe Val Gly Ile Thr Phe Pro Phe Phe Asn Gly
370 375 380
Leu Leu Gly Phe Phe Gly Gly Phe Ala Phe Ala Pro Thr Thr Tyr Phe
385 390 395 400
Leu Pro Cys Ile Met Trp Leu Ala Ile Cys Lys Pro Lys Lys Phe Ser
405 410 415
Leu Ser Trp Ile Ile Asn Trp Ile Cys Ile Ile Leu Gly Val Leu Leu
420 425 430
Met Val Ile Ala Pro Ile Gly Gly Leu Arg Ser Ile Ile Met Gln Ala
435 440 445
Lys Gly Tyr Lys Phe Tyr Ser
450 455
<210> 3
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 3
atgcaacctg caactatggg aacgc 25
<210> 4
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 4
ttaagagtaa aatttgtagc ccttggc 27
<210> 5
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 5
agactagatc atagaatttc tgg 23
Claims (4)
1.敲除烟草氨基酸转运蛋白相关基因NtLHT1在提高烟草重金属胁迫抗性中的应用,其特征在于:所述的重金属胁迫为铜离子胁迫;所述氨基酸转运蛋白相关基因NtLHT1的核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的应用,其特征在于:所述的铜离子胁迫为硫酸铜胁迫。
3.根据权利要求2所述的应用,其特征在于:所述的硫酸铜胁迫为1.0 mg/L的硫酸铜胁迫。
4.敲除烟草氨基酸转运蛋白相关基因NtLHT1在提高烟草重金属胁迫抗性中应用的方法,其特征在于:通过CRISPR/Cas9介导的基因编辑,构建用于敲除NtLHT1基因的CRISPR/Cas9编辑载体,经遗传转化后获得NtLHT1基因发生编辑的成长后叶片中氨基酸含量显著增加的烟草植株;所述的重金属胁迫为铜离子胁迫;所述NtLHT1基因的核苷酸序列如SEQ IDNO.1所示。
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PREDICTED: Nicotiana tabacum lysine histidine transporter 1-like (LOC107761281), transcript variant X1;GenBank;《GenBank》;第1-2页 * |
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