CN114438105B - 一种烟草NtMLO6-1基因及其敲除方法与应用 - Google Patents
一种烟草NtMLO6-1基因及其敲除方法与应用 Download PDFInfo
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Abstract
本发明公开了一种烟草NtMLO6‑1基因及其敲除方法与应用,烟草NtMLO6‑1基因的碱基序列如SEQ ID No.1所示。本发明利用CRISPR/Cas9介导的基因编辑技术敲除NtMLO6‑1基因的基因编辑植株;NtMLO6‑1基因敲除编辑植株叶片中总氮和氨基酸含量显著增加,为烟草MLO基因家族调控机制研究、烟叶总氮及氨基酸含量调控提供遗传材料和参考借鉴。
Description
技术领域
本发明涉及植物基因工程技术领域,特别涉及一种烟草NtMLO6-1基因及其敲除方法与应用。
背景技术
Mildew resistance locus O(MLO)基因家族为植物所特有,已经被从多种植物中分离并克隆。MLO基因的功能各异,存在功能冗余现象,例如番茄、拟南芥、烟草、茄、小麦等植物中某个或多个特定MLO基因的突变能够引发对白粉病的广谱抗性;沉默番茄SlMLO1及其同源物SlMLO5和S1MLO8对白粉病的抗性水平高于SlMLO1突变,提示MLO基因存在功能冗余;拟南芥AtMLO7基因突变导致植株育性降低且助细胞中花粉管过度生长,AtMLO4和AtMLO11基因突变会导致植株根部形态异常;此外,MLO基因还广泛参与生物和非生物的胁迫反应且存在功能互作。MLO家族及其亚家族的基因具有很多功能。
本发明的烟草MLO6-1基因在烟草叶片中总氮和氨基酸含量调控中的功能目前没有报道。
发明内容
本发明所要解决的技术问题是提供一种烟草NtMLO6-1基因及其敲除方法与应用,为烟草MLO基因家族调控机制、烟草叶片总氮及氨基酸含量调控提供遗传材料和借鉴参考。
本发明所要解决的技术问题是通过以下技术方案来实现的:
一种烟草NtMLO6-1基因,所述烟草NtMLO6-1基因的碱基序列如SEQ ID No.1所示。
优选地,上述技术方案中,烟草NtMLO6-1基因编码的蛋白质,其氨基酸序列如SEQIDNO.2。
一种烟草NtMLO6-1基因的敲除方法,包括以下步骤:
(1)选择NtMLO6-1基因中较特异的23nt核苷酸序列为CRISPR/Cas9的引导序列,并将该序列片段与CRISPR/Cas9载体进行连接、转化和PCR扩增检测,PCR阳性克隆,得CRISPR/Cas9-NtMLO6-1编辑载体;
(2)利用所构建的CRISPR/Cas9-NtMLO6-1编辑载体,进行遗传转化和组培,获得烟草NtMLO6-1发生敲除编辑的植株;
(3)利用分子检测确定为NtMLO6-1基因纯合敲除的植株,进行收种获得基因纯合编辑素材;以流动分析仪进行NtMLO6-1基因纯合敲除素材的叶片中总氮的检测试验;
(4)利用分子检测确定为NtMLO6-1基因纯合敲除的植株,进行收种获得基因纯合编辑素材;以GC-MS进行NtMLO6-1基因纯合敲除素材的叶片的氨基酸含量的检测试验。
优选地,上述技术方案中,步骤(1)中,所述NtMLO6-1基因中较特异的23nt核苷酸序列如SEQ ID No.5所示。
优选地,上述技术方案中,步骤(1)中,所述扩增NtMLO6-1基因的引物序列为:
F:5'-TAGATGGCTAAAGAACGG-3'(SEQ ID No.3);
R:5'-ACGCTGAGACAAACAAAG-3'(SEQ ID No.4)。
一种烟草NtMLO6-1基因的敲除方法创制的烟草突变体。
一种烟草NtMLO6-1基因或根据NtMLO6-1基因的敲除方法创制的烟草突变体在调控烟草叶片总氮及氨基酸含量中的应用。
一种烟草NtMLO6-1基因或根据NtMLO6-1基因的敲除方法创制的烟草突变体在制备调控烟草叶片总氮及氨基酸含量的材料中的应用。
本发明上述技术方案,具有如下有益效果:
(1)、本发明通过CRISPR/Cas9介导的基因编辑技术,构建了用于敲除,NtMLO6-1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NtMLO6-1基因敲除的烟草植株。
(2)、本发明利用CRISPR/Cas9介导的基因编辑技术敲除NtMLO6-1基因获得了叶片中总氮和氨基酸含量显著增加的编辑烟草素材,这为进一步阐明烟草MLO基因家族调控机制提供了参考补充,为培育烟叶中总氮和氨基酸含量改变的烟草品种提供了新的遗传材料。
附图说明
被结合在说明书中并构成说明书的一部分的附图示出了本发明的实施例,并且连同其说明一起用于解释本发明的原理。
图1为基于NtMLO6-1基因编码的氨基酸序列的系统发育树图。
图2为NtMLO6-1基因编辑材料的叶片总氮含量增加示意图。
图3为NtMLO6-1基因编辑材料的叶片氨基酸含量增加示意图。
具体实施方式
现在将参照附图来详细描述本发明的各种示例性实施例。应注意到:除非另外具体说明,否则在这些实施例中阐述的部件和步骤的相对布置、数字表达式和数值不限制本发明的范围。
以下实施例中所有使用的实验方法如无特殊说明,均为常规方法。以下实施例中所用的材料、试剂等,如无特殊说明,均可通过商业途径获得。
实施例1NtMLO6-1基因的获得
本实施例主要就烟草NtMLO6-1基因的获得过程,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:
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'-TAGATGGCTAAAGAACGG-3'(SEQ ID No.3);
R:5'-ACGCTGAGACAAACAAAG-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):
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NtMLO6-1基因的序列,其碱基序列如SEQID No.1所示,共包括1563bp个碱基。对该基因序列进行翻译后,其所编码蛋白序列如SEQID No.2所示,共包括520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。
实施例2载体的构建
利用实施例1中所获得烟草NtMLO6-1基因,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NtMLO6-1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-NtMLO6-1编辑载体。
实施例3转化农杆菌
利用上一步所构建的CRISPR/Cas9-NtMLO6-1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NtMLO6-1基因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(15-20d),移入含MS固体培养基的培养瓶中,于25±1℃、光照强度30-50μmol/(m2·s),光照时间为16h/d条件继续培养35-40d,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-NtMLO6-1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。
实施例4侵染愈伤组织
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-NtMLO6-1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NtMLO6-1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NtMLO6-1基因发生纯合编辑的植株,之后进行收种获得NtMLO6-1基因纯合编辑的T1代种子。
本发明所述的烟草NtMLO6-1基因的应用为在烟草植株体内降低所述NtMLO6-1基因的表达,可调控烟草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5分子检测
利用实施例2中分子检测确定为NtMLO6-1基因纯合敲除的植株,进行收种获得基因纯合编辑素材。然后以流动分析仪进行NtMLO6-1基因纯合敲除素材的叶片中总氮的检测试验。
样品采集及处理:选择成熟期的烟株,分别采集5株对照(未编辑)和NtMLO6-1基因纯合编辑的烟草植株样本,采集同一叶位的叶片;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。然后进行样品前处理后,采用流动分析仪进行检测。
NtMLO6-1基因纯合编辑烟草及对照(未编辑)植株叶片中总氮含量比较(结果如图2所示)。与对照相比,NtMLO6-1基因纯合编辑烟草植株叶片中总氮含量显著增加。
实施例6GC-MS检测
利用实施例2中分子检测确定为NtMLO6-1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NtMLO6-1基因纯合敲除素材的叶片的氨基酸含量的检测试验。
NtMLO6-1基因纯合编辑烟草及对照(未编辑)植株叶片中氨基酸含量比较(结果如图3所示)。与对照相比,NtMLO6-1基因纯合编辑烟草植株叶片中脯氨酸和总氨基酸等含量显著增加。
虽然本发明已以实施例公开如上,然其并非用于限定本发明,任何本领域技术人员,在不脱离本发明的精神和范围内,均可作各种不同的选择和修改,因此本发明的保护范围由权利要求书及其等同形式所限定。
序列表
<110> 云南中烟工业有限责任公司
<120> 一种烟草NtMLO6-1基因及其敲除方法与应用
<130> WPC220851
<141> 2022-03-23
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cctctttatg aagcacttga aaagatcaaa gcagaactga tgttgttggg attcatatca 180
ctgctgttga cagtggtgca aagcccagtg tctaacttat gcgtgccaaa gagtgttggt 240
tattcttggc atccttgtaa gtctgatgaa gctgccaaga ataaatatga tgacccttgt 300
ctaccaaagg gaaaagtcca atttgcatct tcatatgcaa tacaccagct ccacattttc 360
atctttgtct tggcagttgc tcatgtatta tactctatag caacttttgc tttaggcagg 420
ctaaagatga gaaaatggag agcctgggag gaagaaacaa aaacaattga gatttttatt 480
atttttacag atccagagag gttcagattt gcaagggaga cgtcatttgg acgtaggcac 540
ttgcattatt ggagcaagtc tccagtgctg ctctggatag tttgtttctt caggcaattc 600
ttctcatcag tagcaaaagt tgactatcta acccttagac atgggttcat gatggcacat 660
ttaactccac agaatcagga aaattttgat ttccagatat acatcaatag agcagttgaa 720
aaagacttca aatttgttgt ggaaataagt ccagcattat ggctcttcac agtactatat 780
tttctaacca ctaccaatgg attgtactcg tacctttggg tgccatttat cccgttagta 840
ataatattgc tagttggcac aaaacttgaa atgataatag cagaaatggg agtaaggatt 900
tcaaagaggg gagacatagt gagaggtgta ccagtggtgg agacaggtga ccatcttttc 960
tggttcaacc gacctggctt tgtccttttc ttgattaact ttgtgctctt tcagaatgca 1020
ttccaagttg ctttcttcgt ttggagttgg tggaaattta gttacccatc ttgcttccac 1080
cagaatgctg cagatatagc cataaggctg accatggggg tgatcataca ggtccattgc 1140
agctatgtga ctctccctct ttatgccttg gtcacacaga tgggaacatc aatgaaacct 1200
ataatctttg gtgataatgt ggcaacagct cttagaagct ggcacaacac ggcgaaaaag 1260
cgggtgaaac acggccggct atcggaaaac accacccctg tctctagcag accggccaca 1320
ccgttgcatg gtacctcgcc ggttcactta ttacgcagtt acccacaata tagtaatgag 1380
gagagtcgga catccaatgc ggaaaatgaa ggctgggcta atgaaatacc aacctctcct 1440
cgtagacaaa ttgagaatat taaagatgat gatcatcagg agggagaaat ccatgcctcc 1500
agctctgtgc atcaagttga gattgcaatg tcagaattca catttggcaa caaaatgagt 1560
tga 1563
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Claims (6)
1.一种烟草NtMLO6-1基因在调控烟草叶片总氮及氨基酸含量中的应用,其特征在于,所述烟草NtMLO6-1基因的碱基序列如SEQ ID No.1所示。
2.根据权利要求1所述的应用,其特征在于,NtMLO6-1基因编码的蛋白质,其氨基酸序列如SEQ IDNO.2。
3.根据权利要求1所述的应用,其特征在于,所述烟草NtMLO6-1基因的敲除方法包括以下步骤:
(1)选择NtMLO6-1基因中较特异的23nt核苷酸序列为CRISPR/Cas9的引导序列,并将该序列片段与CRISPR/Cas9载体进行连接、转化和PCR扩增检测,PCR阳性克隆,得CRISPR/Cas9-NtMLO6-1编辑载体;
(2)利用所构建的CRISPR/Cas9-NtMLO6-1编辑载体,进行遗传转化和组培,获得烟草NtMLO6-1发生敲除编辑的植株;
(3)利用分子检测确定为NtMLO6-1基因纯合敲除的植株,进行收种获得基因纯合编辑素材;以流动分析仪进行NtMLO6-1基因纯合敲除素材的叶片中总氮的检测试验;
(4)利用分子检测确定为NtMLO6-1基因纯合敲除的植株,进行收种获得基因纯合编辑素材;以GC-MS进行NtMLO6-1基因纯合敲除素材的叶片的氨基酸含量的检测试验;
步骤(1)中,所述NtMLO6-1基因中较特异的23nt核苷酸序列如SEQ ID No.5所示。
4.根据权利要求3所述的应用,其特征在于,步骤(1)中,扩增NtMLO6-1基因的引物序列为:
F:5'- TAGATGGCTAAAGAACGG -3';
R:5'- ACGCTGAGACAAACAAAG -3'。
5.一种利用CRISPR/Cas9介导的基因编辑技术敲除如权利要求1所述的烟草NtMLO6-1基因创制的烟草突变体在提高烟草叶片总氮及氨基酸含量中的应用。
6.一种利用CRISPR/Cas9介导的基因编辑技术敲除如权利要求1所述的烟草NtMLO6-1基因创制的烟草突变体在制备调控烟草叶片总氮及氨基酸含量的材料中的应用。
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