CN114426973B - 一种烟草输导蛋白相关基因NtImpα4及其应用 - Google Patents
一种烟草输导蛋白相关基因NtImpα4及其应用 Download PDFInfo
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Abstract
本发明涉及一种烟草输导蛋白相关基因NtImpα4及其应用,属于植物基因工程技术领域。一种烟草输导蛋白相关的基因,核苷酸序列如SEQ ID NO.1所示。本发明还提供上述烟草输导蛋白相关基因在烟草叶片总氮和氨基酸含量调控中的应用。本发明通过CRISPR/Cas9介导的基因编辑技术,构建了用于敲除NtImpα4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NtImpα4基因敲除的烟草植株。本发明获得的NtImpα4基因敲除的编辑烟草植株,相比于对照烟草植株,对逆境的响应稍好,总氮和总氨基酸等指标均显著降低。
Description
技术领域
本发明属于植物基因工程技术领域,具体涉及一种烟草输导蛋白基因NtImpα4及其应用。
背景技术
核质转运是真核生物必需的过程,依赖于核质转运受体的介导。核质转运受体高度保守,其中一类蛋白质入核是由importin β受体及其结合蛋白importin α共同介导。Importin α是adaptor蛋白,负责识别核定位信号,将底物和importin β受体组装成转运复合体,介导转运复合体入核。张玉国报道拟南芥importin α1α2和α2α3双突变体对盐、葡萄糖、PAC等处理高度敏感和不同程度的发育缺陷,α1α2α3三重突变体表型比α1α2和α2α3双突变体的表型更加明显。(张玉国. 拟南芥importin alpha调控逆境应答和根发育的研究[D]. 2010. 中国科学院研究生院.)本发明的输导蛋白基因在烟草逆境调控和叶片氨基酸含量调控中的功能目前没有报道。
发明内容
本发明的目的是为了解决现有技术的不足,提供一种烟草输导蛋白基因NtImpα4,为烟草逆境调控和叶片含氮类成分含量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:
一种烟草输导蛋白相关的基因,核苷酸序列如SEQ ID NO.1所示,包括1602 bp个碱基,该基因来源于烟草(Nicotiana tabacum,命名为NtImpα4)。
SEQ ID NO.1:
ATGTCACTTCGACCCAGCAGTAGAACAGAGGTGAGGAAGAAATCTTATAAAATTGGGGTAGATGCGGATGAGGCTCGTCGTAGGAGGGAAGACAATTTGGTGGAGATCAGGAAGAACAAGCGAGAAGACAATCTCCTTAAGAAACGCCGTGAAGGCCTTCTTCACTCCCAACAGCTTCCTGATGCTTCTCAATCCCCTGCTCTTCTCGAGAAAAGATTGGAAAGTATTCCTGCTATGGTCCAAGGAGTGTGTTCGGAAGATCCTGCTACACAAATCGAAGCAACTACGCATTTTAGGAAGCTCTTATCAATTGAGCGCAGCCCTCCAATTGATGAGGTGATTAATGCTGGAGTTGTTCCTCGATTTGTGGAATTCCTCGGGAGGCATGACCTACCTCAACTGCAATTCGAAGCTGCATGGGCTTTGACCAATGTTGCATCTGGGTCTTCAGAACATACTCGAGCTGTGATCGAACATGGAGCTGTCCCTAAGTTTATTCAACTTCTAAGTTCAGCCAGTGATGATGTGCGTGAGCAGGCAGTCTGGGCTTTGGGCAACGTTGCTGGTGATTCCCCTAGTTGTAGGGATCTTGTGCTTGGTCAAGGCGCACTCATGCCATTGCTAGCTCAGTTGAATGAACACTCAAAGCTTTCAATGTTGAGAAATGCTACATGGACACTCTCCAACTTCTGTCGAGGCAAACCACCAACACCATTTGAGGCGGTCAAACCTGCATTGCCAATTCTTCAACAGCTTATCCACATGAATGATGAAGAGGTTTTGACAGATGCTTGTTGGGCCCTTTCTTATCTATCTGATGGCCCAAATGATAAGATTCAAGCTGTAATCGAGGCGGGTGTCTGTCCCCGACTTGTGGAGCTTCTTCTTCATCCATCACCTACAGTTCTTATACCTGCTCTTCGGACTGCGGGGAATATAGTCACGGGTGATGATGCTCAAACACAGTACATGATTGACAACCAAGTCTTGCCATGTCTCTATCAATTGCTATCTGAAAATCATAAGAAAAGCATCAAGAAGGAGGCTTGTTGGACAATATCTAATATCACCGCTGGAAATAGAGCCCAAATACAGGCTGTTATCGAGGCCAATATCATTCTTCCCTTGGTACACCTCTTGCAGCATGCAGAATTTGATATTAAGAAAGAGGCGGCATGGGCTATCTCAAATGCTGCTTCTGGAGGATCGCATGACCAGATCAGGTTCTTGGCAAGTCAAGGTTGCATTAAGCCACTTTGTGATCTCCTGATCTGTCCGGATCCCAGAATTGTTATTGTATGTCTGGAGGGGCTTGAAAATATATTAAAGGTGGGTGAAGCAGACAGAGAAGCAGGCATGAATGGTGGAATTAATTTATATGCACAAAGGATTGATGAATGTGAAGGTTTGGACAAGATCGAGAATCTCCAAACTCATGACAACAATGAGATATATGAGAAAGTTGTAAAGATCTTGGAGAAATATTGGGCTGAAGAAGATGAAGAACAAAATCTTTCTGATGGTTTTGATAGAAATCAGCAAGGTTTCCAATTCGGAAACAACCAACCTAATGTTCCTGCAGGTGGCTTCAAATTTGGATAA。
本发明的另一个目的是提供一种烟草输导蛋白相关的基因NtImpα4编码的蛋白,包括533个氨基酸;所述编码蛋白的氨基酸序列如SEQ ID NO.2所示:
MSLRPSSRTEVRKKSYKIGVDADEARRRREDNLVEIRKNKREDNLLKKRREGLLHSQQLPDASQSPALLEKRLESIPAMVQGVCSEDPATQIEATTHFRKLLSIERSPPIDEVINAGVVPRFVEFLGRHDLPQLQFEAAWALTNVASGSSEHTRAVIEHGAVPKFIQLLSSASDDVREQAVWALGNVAGDSPSCRDLVLGQGALMPLLAQLNEHSKLSMLRNATWTLSNFCRGKPPTPFEAVKPALPILQQLIHMNDEEVLTDACWALSYLSDGPNDKIQAVIEAGVCPRLVELLLHPSPTVLIPALRTAGNIVTGDDAQTQYMIDNQVLPCLYQLLSENHKKSIKKEACWTISNITAGNRAQIQAVIEANIILPLVHLLQHAEFDIKKEAAWAISNAASGGSHDQIRFLASQGCIKPLCDLLICPDPRIVIVCLEGLENILKVGEADREAGMNGGINLYAQRIDECEGLDKIENLQTHDNNEIYEKVVKILEKYWAEEDEEQNLSDGFDRNQQGFQFGNNQPNVPAGGFKFG。
本发明的再一个目的是提供一种烟草输导蛋白相关基因NtImpα4在调控烟草响应逆境中的应用。该基因编辑后,NtImpα4基因敲除编辑植株生长稍好于野生型红花大金元。
本发明还提供上述烟草输导蛋白相关基因在烟草叶片总氮和氨基酸含量调控中的应用。该基因编辑后,NtImpα4基因敲除编辑植株的叶片中,总氮、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降低。
本发明还提供烟草输导蛋白相关的基因NtImpα4调控烟草响应逆境的方法,通过CRISPR/Cas9介导的基因编辑,构建用于敲除NtImpα4基因的CRISPR/Cas9编辑载体,经遗传转化后获得了NtImpα4基因发生编辑的烟草植株。
本发明还提供一种烟草输导蛋白相关基因NtImpα4调控烟草叶片中总氮和氨基酸含量的方法,通过CRISPR/Cas9介导的基因编辑,构建用于敲除NtImpα4基因的CRISPR/Cas9编辑载体,经遗传转化后获得了NtImpα4基因发生编辑的烟草植株。
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NtImpα4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NtImpα4基因敲除的烟草植株。本发明获得的NtImpα4基因敲除的编辑烟草植株,相比于对照烟草植株,对逆境的响应稍好,为培育调控逆境的烟草品种提供了新的遗传材料;
2、本发明利用CRISPR/Cas9介导的基因编辑技术,能够敲除NtImpα4基因,使得叶片中总氮和氨基酸含量显著降低;这为进一步阐明烟草氨基酸调控机理提供了理论依据,为培育氨基酸含量显著改变的烟草品种提供了新的遗传材料。
附图说明
图1为NtIMPα4基因的编码蛋白的系统发育分析结果;
图2为NtIMPα4基因编辑材料在不同PEG6000处理下的结果;
图3为NtIMPα4基因编辑植株叶片总氮含量降低示意图;
图4为NtIMPα4基因编辑植株叶片氨基酸含量降低示意图。
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NtImpα4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'- ATGTCAAGTCGCCCTCAA -3',(SEQ ID No.3)
R:5'- AAGTAAACCAGCCATAACCA -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NtImpα4序列,其碱基序列如SEQ ID No.1所示,共包括1602 bp个碱基。
SEQ ID No.1:
ATGTCACTTCGACCCAGCAGTAGAACAGAGGTGAGGAAGAAATCTTATAAAATTGGGGTAGATGCGGATGAGGCTCGTCGTAGGAGGGAAGACAATTTGGTGGAGATCAGGAAGAACAAGCGAGAAGACAATCTCCTTAAGAAACGCCGTGAAGGCCTTCTTCACTCCCAACAGCTTCCTGATGCTTCTCAATCCCCTGCTCTTCTCGAGAAAAGATTGGAAAGTATTCCTGCTATGGTCCAAGGAGTGTGTTCGGAAGATCCTGCTACACAAATCGAAGCAACTACGCATTTTAGGAAGCTCTTATCAATTGAGCGCAGCCCTCCAATTGATGAGGTGATTAATGCTGGAGTTGTTCCTCGATTTGTGGAATTCCTCGGGAGGCATGACCTACCTCAACTGCAATTCGAAGCTGCATGGGCTTTGACCAATGTTGCATCTGGGTCTTCAGAACATACTCGAGCTGTGATCGAACATGGAGCTGTCCCTAAGTTTATTCAACTTCTAAGTTCAGCCAGTGATGATGTGCGTGAGCAGGCAGTCTGGGCTTTGGGCAACGTTGCTGGTGATTCCCCTAGTTGTAGGGATCTTGTGCTTGGTCAAGGCGCACTCATGCCATTGCTAGCTCAGTTGAATGAACACTCAAAGCTTTCAATGTTGAGAAATGCTACATGGACACTCTCCAACTTCTGTCGAGGCAAACCACCAACACCATTTGAGGCGGTCAAACCTGCATTGCCAATTCTTCAACAGCTTATCCACATGAATGATGAAGAGGTTTTGACAGATGCTTGTTGGGCCCTTTCTTATCTATCTGATGGCCCAAATGATAAGATTCAAGCTGTAATCGAGGCGGGTGTCTGTCCCCGACTTGTGGAGCTTCTTCTTCATCCATCACCTACAGTTCTTATACCTGCTCTTCGGACTGCGGGGAATATAGTCACGGGTGATGATGCTCAAACACAGTACATGATTGACAACCAAGTCTTGCCATGTCTCTATCAATTGCTATCTGAAAATCATAAGAAAAGCATCAAGAAGGAGGCTTGTTGGACAATATCTAATATCACCGCTGGAAATAGAGCCCAAATACAGGCTGTTATCGAGGCCAATATCATTCTTCCCTTGGTACACCTCTTGCAGCATGCAGAATTTGATATTAAGAAAGAGGCGGCATGGGCTATCTCAAATGCTGCTTCTGGAGGATCGCATGACCAGATCAGGTTCTTGGCAAGTCAAGGTTGCATTAAGCCACTTTGTGATCTCCTGATCTGTCCGGATCCCAGAATTGTTATTGTATGTCTGGAGGGGCTTGAAAATATATTAAAGGTGGGTGAAGCAGACAGAGAAGCAGGCATGAATGGTGGAATTAATTTATATGCACAAAGGATTGATGAATGTGAAGGTTTGGACAAGATCGAGAATCTCCAAACTCATGACAACAATGAGATATATGAGAAAGTTGTAAAGATCTTGGAGAAATATTGGGCTGAAGAAGATGAAGAACAAAATCTTTCTGATGGTTTTGATAGAAATCAGCAAGGTTTCCAATTCGGAAACAACCAACCTAATGTTCCTGCAGGTGGCTTCAAATTTGGATAA。
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533个氨基酸,图1的系统发育分析结果表明,该蛋白含有同源性很高的序列,高度保守。
SEQ ID NO.2所示:
MSLRPSSRTEVRKKSYKIGVDADEARRRREDNLVEIRKNKREDNLLKKRREGLLHSQQLPDASQSPALLEKRLESIPAMVQGVCSEDPATQIEATTHFRKLLSIERSPPIDEVINAGVVPRFVEFLGRHDLPQLQFEAAWALTNVASGSSEHTRAVIEHGAVPKFIQLLSSASDDVREQAVWALGNVAGDSPSCRDLVLGQGALMPLLAQLNEHSKLSMLRNATWTLSNFCRGKPPTPFEAVKPALPILQQLIHMNDEEVLTDACWALSYLSDGPNDKIQAVIEAGVCPRLVELLLHPSPTVLIPALRTAGNIVTGDDAQTQYMIDNQVLPCLYQLLSENHKKSIKKEACWTISNITAGNRAQIQAVIEANIILPLVHLLQHAEFDIKKEAAWAISNAASGGSHDQIRFLASQGCIKPLCDLLICPDPRIVIVCLEGLENILKVGEADREAGMNGGINLYAQRIDECEGLDKIENLQTHDNNEIYEKVVKILEKYWAEEDEEQNLSDGFDRNQQGFQFGNNQPNVPAGGFKFG。
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NtImpα4,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NtImpα4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-NtImpα4编辑载体。
利用上一步所构建的CRISPR/Cas9-NtImpα4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NtImpα4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-NtImpα4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-NtImpα4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NtImpα4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NtImpα4基因发生纯合编辑的植株,之后进行收种获得NtImpα4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NtImpα4的应用为在烟草植株体内降低所述NtImpα4基因的表达,可调控烟草对逆境应答和叶片中总氮和氨基酸含量。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
将NtImpα4基因编辑材料和红花大金元的种子进行表面消毒(首先用无菌水清洗种子2次,再利用75%酒精对其进行消毒45 s,然后用无菌水清洗2次,再用1% AgNO3消毒10min,最后无菌水清洗3~4次)后,布于MS培养基中,生长约5~6 d后,待种子露白时选取同一时期种子(6~10颗)小心移于干旱胁迫(3%、5%、7% PEG6000)处理培养基,置于培养室中培养7~15 d后观察表型。从图2可以看出,3%、5%、7% PEG6000条件处理后,基因编辑材料的种子萌发及植株生长稍好于野生型红花大金元。
实施例4
利用实施例2中分子检测确定为NtImpα4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NtImpα4基因纯合敲除素材的叶片的氨基酸含量的检测试验。
选择的烟株,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NtImpα4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*2.1 mm,1.8 μm
波长:254 nm
柱温:40℃
NtImpα4基因纯合编辑烟草及对照(未编辑)植株叶片中总氮和氨基酸含量比较。从图3和4可以看出:与对照相比,NtImpα4基因纯合编辑烟草植株叶片中,总氮、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降低。
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> 一种烟草输导蛋白相关基因NtImpα4及其应用
<141> 2022-02-22
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1602
<212> DNA
<213> Artificial Sequence
<400> 1
atgtcacttc gacccagcag tagaacagag gtgaggaaga aatcttataa aattggggta 60
gatgcggatg aggctcgtcg taggagggaa gacaatttgg tggagatcag gaagaacaag 120
cgagaagaca atctccttaa gaaacgccgt gaaggccttc ttcactccca acagcttcct 180
gatgcttctc aatcccctgc tcttctcgag aaaagattgg aaagtattcc tgctatggtc 240
caaggagtgt gttcggaaga tcctgctaca caaatcgaag caactacgca ttttaggaag 300
ctcttatcaa ttgagcgcag ccctccaatt gatgaggtga ttaatgctgg agttgttcct 360
cgatttgtgg aattcctcgg gaggcatgac ctacctcaac tgcaattcga agctgcatgg 420
gctttgacca atgttgcatc tgggtcttca gaacatactc gagctgtgat cgaacatgga 480
gctgtcccta agtttattca acttctaagt tcagccagtg atgatgtgcg tgagcaggca 540
gtctgggctt tgggcaacgt tgctggtgat tcccctagtt gtagggatct tgtgcttggt 600
caaggcgcac tcatgccatt gctagctcag ttgaatgaac actcaaagct ttcaatgttg 660
agaaatgcta catggacact ctccaacttc tgtcgaggca aaccaccaac accatttgag 720
gcggtcaaac ctgcattgcc aattcttcaa cagcttatcc acatgaatga tgaagaggtt 780
ttgacagatg cttgttgggc cctttcttat ctatctgatg gcccaaatga taagattcaa 840
gctgtaatcg aggcgggtgt ctgtccccga cttgtggagc ttcttcttca tccatcacct 900
acagttctta tacctgctct tcggactgcg gggaatatag tcacgggtga tgatgctcaa 960
acacagtaca tgattgacaa ccaagtcttg ccatgtctct atcaattgct atctgaaaat 1020
cataagaaaa gcatcaagaa ggaggcttgt tggacaatat ctaatatcac cgctggaaat 1080
agagcccaaa tacaggctgt tatcgaggcc aatatcattc ttcccttggt acacctcttg 1140
cagcatgcag aatttgatat taagaaagag gcggcatggg ctatctcaaa tgctgcttct 1200
ggaggatcgc atgaccagat caggttcttg gcaagtcaag gttgcattaa gccactttgt 1260
gatctcctga tctgtccgga tcccagaatt gttattgtat gtctggaggg gcttgaaaat 1320
atattaaagg tgggtgaagc agacagagaa gcaggcatga atggtggaat taatttatat 1380
gcacaaagga ttgatgaatg tgaaggtttg gacaagatcg agaatctcca aactcatgac 1440
aacaatgaga tatatgagaa agttgtaaag atcttggaga aatattgggc tgaagaagat 1500
gaagaacaaa atctttctga tggttttgat agaaatcagc aaggtttcca attcggaaac 1560
aaccaaccta atgttcctgc aggtggcttc aaatttggat aa 1602
<210> 2
<211> 533
<212> PRT
<213> Artificial Sequence
<400> 2
Met Ser Leu Arg Pro Ser Ser Arg Thr Glu Val Arg Lys Lys Ser Tyr
1 5 10 15
Lys Ile Gly Val Asp Ala Asp Glu Ala Arg Arg Arg Arg Glu Asp Asn
20 25 30
Leu Val Glu Ile Arg Lys Asn Lys Arg Glu Asp Asn Leu Leu Lys Lys
35 40 45
Arg Arg Glu Gly Leu Leu His Ser Gln Gln Leu Pro Asp Ala Ser Gln
50 55 60
Ser Pro Ala Leu Leu Glu Lys Arg Leu Glu Ser Ile Pro Ala Met Val
65 70 75 80
Gln Gly Val Cys Ser Glu Asp Pro Ala Thr Gln Ile Glu Ala Thr Thr
85 90 95
His Phe Arg Lys Leu Leu Ser Ile Glu Arg Ser Pro Pro Ile Asp Glu
100 105 110
Val Ile Asn Ala Gly Val Val Pro Arg Phe Val Glu Phe Leu Gly Arg
115 120 125
His Asp Leu Pro Gln Leu Gln Phe Glu Ala Ala Trp Ala Leu Thr Asn
130 135 140
Val Ala Ser Gly Ser Ser Glu His Thr Arg Ala Val Ile Glu His Gly
145 150 155 160
Ala Val Pro Lys Phe Ile Gln Leu Leu Ser Ser Ala Ser Asp Asp Val
165 170 175
Arg Glu Gln Ala Val Trp Ala Leu Gly Asn Val Ala Gly Asp Ser Pro
180 185 190
Ser Cys Arg Asp Leu Val Leu Gly Gln Gly Ala Leu Met Pro Leu Leu
195 200 205
Ala Gln Leu Asn Glu His Ser Lys Leu Ser Met Leu Arg Asn Ala Thr
210 215 220
Trp Thr Leu Ser Asn Phe Cys Arg Gly Lys Pro Pro Thr Pro Phe Glu
225 230 235 240
Ala Val Lys Pro Ala Leu Pro Ile Leu Gln Gln Leu Ile His Met Asn
245 250 255
Asp Glu Glu Val Leu Thr Asp Ala Cys Trp Ala Leu Ser Tyr Leu Ser
260 265 270
Asp Gly Pro Asn Asp Lys Ile Gln Ala Val Ile Glu Ala Gly Val Cys
275 280 285
Pro Arg Leu Val Glu Leu Leu Leu His Pro Ser Pro Thr Val Leu Ile
290 295 300
Pro Ala Leu Arg Thr Ala Gly Asn Ile Val Thr Gly Asp Asp Ala Gln
305 310 315 320
Thr Gln Tyr Met Ile Asp Asn Gln Val Leu Pro Cys Leu Tyr Gln Leu
325 330 335
Leu Ser Glu Asn His Lys Lys Ser Ile Lys Lys Glu Ala Cys Trp Thr
340 345 350
Ile Ser Asn Ile Thr Ala Gly Asn Arg Ala Gln Ile Gln Ala Val Ile
355 360 365
Glu Ala Asn Ile Ile Leu Pro Leu Val His Leu Leu Gln His Ala Glu
370 375 380
Phe Asp Ile Lys Lys Glu Ala Ala Trp Ala Ile Ser Asn Ala Ala Ser
385 390 395 400
Gly Gly Ser His Asp Gln Ile Arg Phe Leu Ala Ser Gln Gly Cys Ile
405 410 415
Lys Pro Leu Cys Asp Leu Leu Ile Cys Pro Asp Pro Arg Ile Val Ile
420 425 430
Val Cys Leu Glu Gly Leu Glu Asn Ile Leu Lys Val Gly Glu Ala Asp
435 440 445
Arg Glu Ala Gly Met Asn Gly Gly Ile Asn Leu Tyr Ala Gln Arg Ile
450 455 460
Asp Glu Cys Glu Gly Leu Asp Lys Ile Glu Asn Leu Gln Thr His Asp
465 470 475 480
Asn Asn Glu Ile Tyr Glu Lys Val Val Lys Ile Leu Glu Lys Tyr Trp
485 490 495
Ala Glu Glu Asp Glu Glu Gln Asn Leu Ser Asp Gly Phe Asp Arg Asn
500 505 510
Gln Gln Gly Phe Gln Phe Gly Asn Asn Gln Pro Asn Val Pro Ala Gly
515 520 525
Gly Phe Lys Phe Gly
530
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 3
atgtcaagtc gccctcaa 18
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 4
aagtaaacca gccataacca 20
<210> 5
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 5
atgcggatga ggctcgtcgt agg 23
Claims (4)
1.一种烟草输导蛋白相关基因NtImpα4在调控烟草响应干旱胁迫中的应用。
2.一种烟草输导蛋白相关基因NtImpα4在调控烟草叶片中总氮和氨基酸含量中的应用。
3.一种烟草输导蛋白相关基因NtImpα4调控烟草响应干旱胁迫的方法,其特征在于:通过CRISPR/Cas9介导的基因编辑,构建用于敲除NtImpα4基因的CRISPR/Cas9编辑载体,经遗传转化后获得了NtImpα4基因发生编辑的烟草植株。
4.一种烟草输导蛋白相关基因NtImpα4调控烟草叶片中总氮和氨基酸含量的方法,其特征在于:通过CRISPR/Cas9介导的基因编辑,构建用于敲除NtImpα4基因的CRISPR/Cas9编辑载体,经遗传转化后获得了NtImpα4基因发生编辑的烟草植株。
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IMPa-4, an Arabidopsis importin alpha isoform, is preferentially involved in agrobacterium-mediated plant transformation;Saikat Bhattacharjee等;Plant Cell;第20卷(第10期);第2661-2680页 * |
PREDICTED: Nicotiana tabacum importin subunit alpha-4-like (LOC107830598), transcript variant X2, mRNA.《NCBI GenBank》.2016,第1-2页. * |
核转运蛋白的结构及功能;樊静等;生命的化学;第85-87页 * |
植物核质转运与抗病防卫反应信号传导交叉调控;吕贝贝等;《南京农业大学学报》;第34卷(第6期);摘要 * |
番茄IMPα/β和LYK家族基因的鉴定、表达模式分析和功能研究;田丽梅;《中国优秀硕士学位论文全文数据库 农业科技辑》;第31-32、42页 * |
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