CN114807081B - 一种烟草谷胱甘肽转移相关的基因及其应用 - Google Patents
一种烟草谷胱甘肽转移相关的基因及其应用 Download PDFInfo
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Abstract
本发明涉及一种烟草谷胱甘肽转移酶相关的基因及其应用,该基因的核苷酸序列如SEQ ID NO.1所示。本发明提供的烟草谷胱甘肽转移酶相关的基因NtGST23,利用CRISPR/Cas9介导的基因编辑技术敲除NtGST23基因获得在抗逆胁迫下表达量降低的基因编辑植株;在干旱胁迫与盐胁迫条件下,基因编辑植株的长势弱于对照植株,这为烟草谷胱甘肽转移酶研究及烟草抗逆性研究提供遗传材料和理论依据。
Description
技术领域
本发明涉及一种烟草谷胱甘肽转移酶相关的基因及其应用,属于植物基因工程技术领域。
背景技术
非生物胁迫,如干旱胁迫、盐胁迫、低温胁迫、重金属离子等,能促进植物体内产生大量的氧自由基,引发氧化损伤,严重影响植物的生长发育,造成作物减产。抗氧化系统是植物抵御非生物胁迫的重要机制,由一些能清除活性氧的酶系和抗氧化物质组成,保护细胞免受氧化胁迫的伤害,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽硫转移酶(GST)、抗坏血酸过氧化物酶(APX)是抗氧化系统的重要组成部分。谷胱甘肽硫转移酶(Glutathione S-transferases, GSTs)是谷胱甘肽代谢途径中的关键酶,广泛存在于植物的细胞质基质、线粒体、微粒体中,多项研究表明,GSTs 在植物抵抗非生物胁迫引起的氧化胁迫的过程中,发挥了重要的作用。GST 不仅被真菌和其他一些病原物诱导表达,植物激素如生长素、水杨酸、乙烯和ABA等也能诱导它的表达。研究表明,GST在植物遭受各种生物和非生物胁迫时,通过解毒和抗氧化作用保护植物细胞免受伤害。利用农杆菌侵染法将盐碱地碱蓬GST 基因转入低温敏感的水稻幼苗品种‘中花11’,转基因植株增强了对低温胁迫的抗性。过表达野生大豆GsGST 基因可以提高烟草的耐盐性。GST 在水稻抗重金属镉胁迫中起到一定作用。盐地碱蓬GST 基因在拟南芥中过量表达后,拟南芥转基因植株的抗旱能力与对照相比有所增强。
烟草作为我国重要的经济作物和模式作物,研究烟草谷胱甘肽转移酶基因响应非生物逆境胁迫的分子机制具有重要意义。
发明内容
本发明的目的是为了解决现有技术的不足,提供一种烟草谷胱甘肽转移酶相关的基因及其应用,为研究烟草抗逆能力与基因功能提供遗传材料和理论依据。
为实现上述目的,本发明采用的技术方案如下:
一种烟草谷胱甘肽转移酶相关的基因,核苷酸序列如SEQ ID NO.1所示,包括675bp个碱基,该基因来源于烟草(Nicotiana tabacum,命名为NtGST23)。
SEQ ID NO.1:
ATGGAAGACCAAGTGAAACTGCTAGGAGCTTTTCCAAGTCCCTTTAGTTATAGGGTAATTTGGGCTCTGAAACACAAGGGTATCAACTATGAATACATAGAGGAAGATCTTTCAAATAAGAGCCATGATCTTTTGACATACAACCCTATCTATAAGATGATTCCTGTTCTTGTACATGCTGGAAAACCAATAGCAGAGTCCACAGTCATCCTTGAATACATCGAAGAGACATGGCCTCAGAATCCTTTGCTACCAAAGGATCCTCATGAAAGGGCTCAGGCTAGATTCTGGATCAAGTTCGGAGAAGATAAGAGCCCAGAATTTTTCGCAATATTTCACAAGATAGGGGAAGAGCAAGTCAAGGCAACTGAAAAAGCAAAGGAAGTGTTGAAAATTATAGAAGAGCAAGGTCTTGGAGAGAAGAAGTTTTTTAGCGGGGACACAATTGGATTAAGTGACATAGTCTTTGGATGGATAGCGTTATGGCTGGAAGTCATACAAGAAGCTGCTGAAGTAAAGGTCTTCGACTCAGTTAGTACTTTTCCTCGTTTACATGCTTGGATACATAACTTTAAGCAACTCCCTGTAATCAAACAAAATACCCCACATCGGGATGCAATGCTAGCTTATTTCAAACGTCGTCGAGAAATGGTTGTAGCAGCGGCACAAGGTTGA。
优选的,所述烟草谷胱甘肽转移酶相关的基因的编码蛋白,氨基酸序列如SEQ IDNO.2所示,包括224个氨基酸。
SEQ ID NO.2:
MEDQVKLLGAFPSPFSYRVIWALKHKGINYEYIEEDLSNKSHDLLTYNPIYKMIPVLVHAGKPIAESTVILEYIEETWPQNPLLPKDPHERAQARFWIKFGEDKSPEFFAIFHKIGEEQVKATEKAKEVLKIIEEQGLGEKKFFSGDTIGLSDIVFGWIALWLEVIQEAAEVKVFDSVSTFPRLHAWIHNFKQLPVIKQNTPHRDAMLAYFKRRREMVVAAAQG。
本发明还提供了一种基因编辑植株的制备方法:
通过CRISPR/Cas9介导的基因编辑技术,构建了用于敲除NtGST23基因的CRISPR/Cas9编辑载体,经遗传转化后获得了NtGST23基因发生编辑的烟草植株;所述NtGST23基因如权利要求1中SEQ ID NO.1所示。
本发明还提供了烟草谷胱甘肽转移酶相关的基因在烟草抗逆方面的应用。
所述烟草谷胱甘肽转移酶相关的基因在四叶一心时期在抗干旱胁迫方面的应用。
本发明的有益效果是:
1、本发明通过CRISPR/Cas9介导的基因编辑技术,构建了用于敲除NtGST23基因的CRISPR/Cas9编辑载体,经遗传转化后获得了NtGST23基因发生编辑的红花大金元植株。本发明通过在四叶一心的时期利用20%浓度PEG-6000模拟干旱处理编辑植株与对照植株,发现NtGST23基因编辑植株萎蔫而对照(未转化)植株正常;
2、综上所述,利用CRISPR/Cas9介导的基因编辑技术敲除NtGST23基因获得在抗逆胁迫下长势较弱的基因编辑植株,这为烟草谷胱甘肽转移酶研究及烟草抗逆性研究提供遗传材料和理论依据。
附图说明
图1为对照(未转化)植株叶片和基因编辑植株叶片在四叶一心时期,20%浓度PEG-6000模拟干旱处理下的对照图。
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谷胱甘肽转移酶相关的基因NtGST23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,之后置于-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’- ATGGAAGACCAAGTGAAACT-3’,(SEQ ID No.3)
R:5’- TCAACCTTGTGCCGCTGCTA-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,获得烟草谷胱甘肽转移酶相关的基因NtGST23序列,其碱基序例如SEQ ID No.1所示,共包括675bp个碱基。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224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
利用实施例1中所获得烟草谷胱甘肽转移酶相关的基因NtGST23,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NtGST23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,获得转化的克隆子,进行PCR扩增检测,后将PCR阳性克隆送测序公司进行测序确认,最后得到CRISPR/Cas9-NtGST23编辑载体。
利用上一步所构建的CRISPR/Cas9- NtGST23编辑载体质粒,以红花大金元为例,进行遗传转化试验,以敲除植物体内的烟草谷胱甘肽转移酶相关的基因NtGST23,相关实验过程简要介绍如下。
将烟草种子点种于培养皿中,待长到4片子叶(15-20d),便可将其移入培养瓶中(含80mL MS液体培养基),每瓶2株,于25±1℃、光照强度30-50μmol/(m2·s),光照时间为16h/d条件继续培养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,加入含有编辑NtGST23基因的2μL质粒,轻轻弹混匀,置于冰上。后将混匀的液体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。8,000rpm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- NtGST23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/LNAA+0.5mg/L6-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5-6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7-10d,获得经LBA4404农杆菌介导转化NtGST23基因的再生植株,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NtGST23基因编辑植株。
实施例3
利用实施例3中同一份NtGST23基因敲除素材种子,进行播种到漂浮育苗盘上。随后将幼苗移栽到花盆中进行生长,待幼苗生长至4叶一心时期进行干旱胁迫处理,选择NtGST23基因编辑烟草种子与对照(未转化)种子,选取饱满无明显缺陷的种子进行播种,放置于温室进行培养;
待播种后生长25-30d时,将其移栽至花盆中继续生长,待生长至四叶一心时期,进行20%浓度PEG-6000浇灌处理,每3d浇灌一次,15d后拍照记录植株的萎蔫程度。
结果表明,烟草谷胱甘肽转移酶相关的基因NtGST23在干旱胁迫条件下与对照植株相比表现为萎蔫(结果如图1所示)。
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> 一种烟草谷胱甘肽转移相关的基因及其应用
<140> 2022105380228
<141> 2022-05-18
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 675
<212> DNA
<213> Nicotiana tabacum
<400> 1
atggaagacc aagtgaaact gctaggagct tttccaagtc cctttagtta tagggtaatt 60
tgggctctga aacacaaggg tatcaactat gaatacatag aggaagatct ttcaaataag 120
agccatgatc ttttgacata caaccctatc tataagatga ttcctgttct tgtacatgct 180
ggaaaaccaa tagcagagtc cacagtcatc cttgaataca tcgaagagac atggcctcag 240
aatcctttgc taccaaagga tcctcatgaa agggctcagg ctagattctg gatcaagttc 300
ggagaagata agagcccaga atttttcgca atatttcaca agatagggga agagcaagtc 360
aaggcaactg aaaaagcaaa ggaagtgttg aaaattatag aagagcaagg tcttggagag 420
aagaagtttt ttagcgggga cacaattgga ttaagtgaca tagtctttgg atggatagcg 480
ttatggctgg aagtcataca agaagctgct gaagtaaagg tcttcgactc agttagtact 540
tttcctcgtt tacatgcttg gatacataac tttaagcaac tccctgtaat caaacaaaat 600
accccacatc gggatgcaat gctagcttat ttcaaacgtc gtcgagaaat ggttgtagca 660
gcggcacaag gttga 675
<210> 2
<211> 224
<212> PRT
<213> Nicotiana tabacum
<400> 2
Met Glu Asp Gln Val Lys Leu Leu Gly Ala Phe Pro Ser Pro Phe Ser
1 5 10 15
Tyr Arg Val Ile Trp Ala Leu Lys His Lys Gly Ile Asn Tyr Glu Tyr
20 25 30
Ile Glu Glu Asp Leu Ser Asn Lys Ser His Asp Leu Leu Thr Tyr Asn
35 40 45
Pro Ile Tyr Lys Met Ile Pro Val Leu Val His Ala Gly Lys Pro Ile
50 55 60
Ala Glu Ser Thr Val Ile Leu Glu Tyr Ile Glu Glu Thr Trp Pro Gln
65 70 75 80
Asn Pro Leu Leu Pro Lys Asp Pro His Glu Arg Ala Gln Ala Arg Phe
85 90 95
Trp Ile Lys Phe Gly Glu Asp Lys Ser Pro Glu Phe Phe Ala Ile Phe
100 105 110
His Lys Ile Gly Glu Glu Gln Val Lys Ala Thr Glu Lys Ala Lys Glu
115 120 125
Val Leu Lys Ile Ile Glu Glu Gln Gly Leu Gly Glu Lys Lys Phe Phe
130 135 140
Ser Gly Asp Thr Ile Gly Leu Ser Asp Ile Val Phe Gly Trp Ile Ala
145 150 155 160
Leu Trp Leu Glu Val Ile Gln Glu Ala Ala Glu Val Lys Val Phe Asp
165 170 175
Ser Val Ser Thr Phe Pro Arg Leu His Ala Trp Ile His Asn Phe Lys
180 185 190
Gln Leu Pro Val Ile Lys Gln Asn Thr Pro His Arg Asp Ala Met Leu
195 200 205
Ala Tyr Phe Lys Arg Arg Arg Glu Met Val Val Ala Ala Ala Gln Gly
210 215 220
<210> 3
<211> 20
<212> DNA
<213> synthetic construct
<400> 3
atggaagacc aagtgaaact 20
<210> 4
<211> 20
<212> DNA
<213> synthetic construct
<400> 4
tcaaccttgt gccgctgcta 20
<210> 5
<211> 23
<212> DNA
<213> Nicotiana tabacum
<400> 5
aagctcctag cagtttcact tgg 23
Claims (1)
1.一种烟草谷胱甘肽转移酶相关的基因NtGST23在研究烟草四叶一心时期抗干旱胁迫方面的应用,所述基因NtGST23如SEQ ID NO.1所示;
SEQ ID NO.1:
ATGGAAGACCAAGTGAAACTGCTAGGAGCTTTTCCAAGTCCCTTTAGTTATAGGGTAATTTGGGCTCTGAAACACAAGGGTATCAACTATGAATACATAGAGGAAGATCTTTCAAATAAGAGCCATGATCTTTTGACATACAACCCTATCTATAAGATGATTCCTGTTCTTGTACATGCTGGAAAACCAATAGCAGAGTCCACAGTCATCCTTGAATACATCGAAGAGACATGGCCTCAGAATCCTTTGCTACCAAAGGATCCTCATGAAAGGGCTCAGGCTAGATTCTGGATCAAGTTCGGAGAAGATAAGAGCCCAGAATTTTTCGCAATATTTCACAAGATAGGGGAAGAGCAAGTCAAGGCAACTGAAAAAGCAAAGGAAGTGTTGAAAATTATAGAAGAGCAAGGTCTTGGAGAGAAGAAGTTTTTTAGCGGGGACACAATTGGATTAAGTGACATAGTCTTTGGATGGATAGCGTTATGGCTGGAAGTCATACAAGAAGCTGCTGAAGTAAAGGTCTTCGACTCAGTTAGTACTTTTCCTCGTTTACATGCTTGGATACATAACTTTAAGCAACTCCCTGTAATCAAACAAAATACCCCACATCGGGATGCAATGCTAGCTTATTTCAAACGTCGTCGAGAAATGGTTGTAGCAGCGGCACAAGGTTGA。
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