CN116004646A - 一种烟草NtSWEET11基因及其应用 - Google Patents
一种烟草NtSWEET11基因及其应用 Download PDFInfo
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- CN116004646A CN116004646A CN202111230542.4A CN202111230542A CN116004646A CN 116004646 A CN116004646 A CN 116004646A CN 202111230542 A CN202111230542 A CN 202111230542A CN 116004646 A CN116004646 A CN 116004646A
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Abstract
本发明公开了一种烟草NtSWEET11基因及其应用。烟草NtSWEET11基因受青枯病病原物青枯雷尔式菌接种诱导表达，参与烟草青枯病胁迫响应。人工接种青枯病后，与K326相比，NtSWEET11基因过表达烟草植株表现出较强的青枯病抗性。烟草NtSWEET11基因在烟草青枯病抗性调控过程中发挥重要作用，可应用于烟草青枯病抗性改良的基因功能研究和基因工程育种。
Description
技术领域
本发明属于烟草基因工程技术领域，具体涉及一种与植物青枯病抗性相关的烟草NtSWEET11基因及其在调控烟草青枯病抗性中的应用。
背景技术
烟草青枯病是青枯雷尔式菌(Ralstonia solanancearum)引起的一种细菌性土传病害，在烟草苗期和大田期发生，严重影响了烟草的生长发育，对烟叶生产造成严重经济损失。烟草青枯病的防治包括化学、农业和生物防治等方法，目前这些方法仍不能有效解决问题。在烟草等作物中，挖掘并利用青枯病抗性基因是防治青枯病的根本的途径。利用基因工程相关技术，从烟草中分离出青枯病抗性相关基因并将其应用到青枯病抗性烟草分子育种中，对于保证烟草的品质和产量具有重要的现实意义。
本发明首次发现了一个与烟草青枯病抗性相关的SWEET蛋白，即烟草NtSWEET11基因，为烟草抗青枯病提供新的途径。
发明内容
本发明的首要目的在于提供一个与烟草青枯病抗性相关的烟草NtSWEET11基因，利用烟草青枯病抗性相关的NtSWEET11基因所构建重组过表达载体转化烟草，提高NtSWEET11基因表达量进而能够增强烟草对青枯病的抗性，从而为提高烟草青枯病抗性以及烟草青枯病抗性育种提供育种中间材料。
一种烟草NtSWEET11基因，基因CDS序列如SEQ ID NO.1所示。
进一步的，所述的基因序列还包括相似性不低于95％的具有类似功能的基因序列；所述的类似功能包括表达抗青枯病的蛋白。
进一步的，所述的抗青枯病的具体对象为植物，进一步优选为烟草。
本发明的第二个目的是提供一种NtSWEET11蛋白，蛋白序列如SEQ ID NO.2所示。
进一步的，所述的蛋白序列还包括相似性不低于95％的具有类似功能的蛋白序列；所述的类似功能包括抗青枯病。
进一步的，所述的抗青枯病的具体对象为植物，进一步优选为烟草。
本发明的第三个目的是提供所述的烟草NtSWEET11基因在提高青枯病抗性中的应用。
进一步的，用于提高植物抗青枯病抗性，尤其是烟草抗青枯病抗性。
进一步的，通过过表达烟草NtSWEET11基因，获得烟草青枯病抗性提高的烟草转化植株。
进一步的，过表达烟草NtSWEET11基因的载体为pCHF3。
所述重组过表达载体pCHF3为双元农杆菌载体。构建重组表达载体时，将NtSWEET11基因CDS碱基序列插入花椰菜花叶病毒(CAMV)35S启动子之后。
本发明使用的载体包括但不限于pCHF3，只要满足在烟草中正常表达NtSWEET11基因的载体均可。
所述转化烟草为使用含有重组过表达载体的农杆菌侵染烟草愈伤组织，利用农杆菌介导的转化方法，将花椰菜花叶病毒35S启动子和烟草NtSWEET11基因整合进烟草基因组。在烟草内，通过对NtSWEET11基因过表达，提高烟草的青枯病抗性。
本发明首次在烟草中完成了NtSWEET11基因的克隆、表达和功能分析，分析结果表明该基因与烟草的青枯病抗性应答相关。此外，在烟草中过表达NtSWEET11基因可明显提高烟草的青枯病抗性，从而为提高烟草青枯病抗性以及烟草青枯病抗性育种提供材料。
附图说明
图1烟草NtSWEET11基因克隆PCR扩增产物电泳图；
图1中M为2000bp DNA maker；1为阴性对照；2为NtSWEET11基因扩增结果。
图2烟草接种青枯病后NtSWEET11基因表达模式分析；
图2中mock表示对照；
图3烟草NtSWEET11基因过表达烟草植株中NtSWEET11的表达量分析；
图4烟草NtSWEET11基因过表达烟草植株和K326对照的青枯病抗性评价；
图5烟草NtSWEET11基因过表达烟草植株和K326对照的烟草根部青枯病病原菌繁殖情况。
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，而不会形成对本发明的限制；在介绍具体实施例前，就下述实施例中所涉及部分生物材料、实验试剂、实验仪器等基本情况简要介绍如下。
生物材料：
烟草材料，栽培烟草(Nicotiana tabacum)品种K326(常见烟草主栽品种)保存于本实验室。过量表达烟草基因所用到的载体质粒pCHF3购买自武汉普因特生物工程有限公司。
实验试剂：
本实验开展过程中所使用的试剂及试剂盒如下：限制性内切酶购于NEB(北京)有限公司；RNA提取TRIzol试剂盒购于康为世纪生物科技有限公司；高保真DNA扩增酶、反转录试剂盒和荧光定量试剂盒购于南京诺唯赞生物科技股份有限公司；DNA凝胶回收试剂盒MiniBEST Agarose Gel DNA Extraction Kit、质粒DNA小量纯化试剂盒MiniBEST Plasmidpurification Kit购买于Invitrogen公司；卡那霉素、利福平等抗生素购于上海生工生物工程有限公司。
实施例1
本实施例主要就NtSWEET11基因的获得过程简要介绍说明如下。
1、总RNA提取
采用康为世纪公司的TRIzol试剂提取法提取RNA，具体步骤如下：在正常生长条件下，生长4周的栽培烟草K326取材后迅速在液氮中研磨成粉末状，每30-50mg组织加入1mlTRIzon Reagent(cwbiotech)，混匀；室温放置5min后，加入200μl氯仿，剧烈振荡15秒，室温放置2min。4℃12,000rpm离心10分钟，小心取出上层水相，转入另一离心管中，加入等体积的70％乙醇。将混合液全部转移到吸附柱中，12,000rpm离心20秒，倒掉收集管中的废液，加入700μl Buffer RW1，12,000rpm离心20秒，倒掉收集管中的废液。加入500μl Buffer RW2，12,000rpm离心20秒，倒掉收集管中的废液。空离2min，倒掉收集管中的废液，室温放置5min，加入30μl水，室温放置1分钟，12,000rpm离心1分钟，收集RNA溶液，-70℃保存RNA，防止降解。所提RNA经DNA酶(Fermentas)处理。
2、反转录反应
反转录步骤采取诺唯赞公司R323试剂盒，具体步骤如下：取1μg K326总RNA进行反转录，加入4×gDNA wiper Mix 4μl，加入去离子水补足到16μl；42℃保温2min后，加入4μl的5×HiScript III qRT SuperMix，反应终体积为20μl；37℃反应15min，85℃加热5s终止反应。获得的cDNA在-20℃保存。
3、NtSWEET11基因的鉴定与克隆
通过分析烟草青枯病接种前后的转录组数据，发现一个在接种青枯病后表达量显著上调的烟草基因NtSWEET11。烟草NtSWEET11基因在茄科基因组网站中烟草基因组数据库
(ftp://ftp.solgenomics.net/genomes/Nicotiana_tabacum/edwards_et_al_2017)登录号为Nitab4.5_0001332g0130.1，该基因被注释编码一种糖转运蛋白，具体功能未知。
为扩增NtSWEET11基因序列，人工合成其上游引物及下游引物，引物序列如下：
NtSWEET11-F：5′-ATGGCGGGTATTTCTGGTCACT 3′，如SEQ ID NO.3所示，
NtSWEET11-R：5′-CTAGGCTTCGGCAGTTTGCAGT 3′，如SEQ ID NO.4所示；
以上述K326品种整株烟草幼苗cDNA作为模板，以NtSWEET11-F和NtSWEET11-R为引物，采用高保真2×Phanta Max Master Mix(Dye Plus)(Vazyme)进行PCR扩增；50μl反应体系设计如下：
反应程序:95℃，3min预变性；95℃，15s；56℃，15s；72℃，1min；35个循环，最后72℃延伸5min；反应结束后，电泳检测PCR结果(图1)。电泳后，在紫外光照射下进行切胶回收，利用凝胶回收试剂盒(TAKARA)回收目的基因片段。对所回收扩增产物进行测序，即为NtSWEET11基因，其包括891bp碱基，其碱基序列如SEQ ID NO.1所示。
实施例2
烟草接种青枯病后NtSWEET11基因表达模式分析
对烟草栽培品种K326植株进行青枯病接病处理。青枯病病原菌接种前进行伤根处理。伤根处理的具体实施方法为，室内培育烟苗生长至三叶一心大小时，移栽至口径为9cm的育苗盆中，然后在培养至四叶一心时用于接种青枯病。在制剂和青枯菌处理烟草植株前，进行伤根预处理，即用锋利的剪刀剪掉烟草根部的三分之一后重新移栽至花盆中。青枯病具体接种方法和过程为，从-80℃超低温冰箱中取出保存的青枯病菌株CQPS-1，配置NB培养基，在超净工作台接入青枯病菌，28℃/220rpm过夜培养。采用灌根接种的方法在每株烟苗的根部四周接种10mL约108cfu/mL的菌悬液。调节环境温度至30℃，环境湿度至80％培养。随后进行病情调查，并分别在0天、2天、4天、6天后检测青枯菌在烟草根部中的繁殖情况。采用实施例1中方法提取发病烟草根部RNA，并同时提取未接病烟草根部RNA作为对照。提取的RNA经反转录得到cDNA，并以cDNA为模板(反转录步骤与实施例1相同)，利用NtSWEET11的特异引物进行实时定量PCR检测，引物序列如下：
实时定量引物F:AAACACGACCCTTATCATCACT，如SEQ ID NO.5所示，实时定量引物R:CATCTTCACAGTTTGAACCCTG，如SEQ ID NO.6所示
结果显示，NtSWEET11基因在接种青枯病6天后表达显著上调(图2)，约为对照的53.6倍。
实施例3
利用实施例1所获得NtSWEET11基因，发明人进一步构建了转化用的过表达载体pCHF3-NtSWEET11，相关过程简要介绍如下。
参照Clontech公司的In-fusion无缝连接说明书，将NtSWEET11基因的扩增引物与pCHF3质粒的Sac I酶切位点接头序列连接在一起，并人工合成。序列如下，其中下划线为载体的接头序列：
F:5′-AGAACACGGGGGACGAGCTCATGGCGGGTATTTCTGGTCACT-3′，如SEQ ID NO.7所示；
R:5′-GATCCCCGGGTACCGAGCTCCTAGGCTTCGGCAGTTTGCAGT-3′，如SEQ ID NO.8所示。
首先将实施例1所获得NtSWEET11基因与经Sac I酶切后的pCHF3质粒进行连接，按试剂盒要求建立连接10μL的反应体系如下：5x in-fusion 2μl；pCHF3(Sac I酶切)4μl；NtSWEET11基因扩增产物4μL；50℃,15min，放冰上，用于下一步的转化。采用热激法，将上述连接产物转化大肠杆菌感受态细胞，具体过程为：无菌条件下取2μl连接产物加到感受态细胞中，轻轻混匀后冰浴30min；42℃热激90s，将离心管迅速转到冰浴中放置2-3min；加无抗生素的LB培养基800μl，37℃摇床温和摇振1h左右；取200μl培养液涂于含壮观霉素100μg/ml的LB固体培养基上，37℃倒置培养12-16h。
挑取培养基中长出白色菌斑，分别接种于含有100μg/ml壮观霉素的LB液体培养基中振荡培养12-16h，用自身引物和载体引物进行PCR验证，引物序列如下：
自身引物：TGTGGAATACATGCCACTTC，如SEQ ID NO.9所示；
载体引物：GTGTGTGCGCAATGAAACTG，如SEQ ID NO.10所示；
结果正确的阳性克隆进一步送公司测序，以确保重组质粒构建正确。
实施例4
利用热激法，将实施例3所制备pCHF3-NtSWEET11载体转化农杆菌GV3101(购于北京全式金生物技术有限公司)，挑选单菌落进行PCR验证，以确定表达载体pCHF3-NtSWEET11成功转化农杆菌。
转化烟草植株：
采用农杆菌介导叶盘转化法获得转基因烟草植株，具体步骤如下：
1.无菌苗培养：取适量K326种子，用75％酒精表面消毒30秒，无菌水清洗3次，再用15％双氧水浸泡8分钟，无菌水清洗3次后浸泡在无菌水中24小时。经消毒的种子点播在MS培养皿上，待长出3片小叶后将幼苗转移到含有MS的组培瓶中培养，在人工气候室培养45天左右，选择生长健壮的叶片进行农杆菌侵染。
2.农杆菌侵染：取出经鉴定正确保存的农杆菌菌液，待完全融化后吸取500uL于相应抗性的50mL YEP液体培养基中，于28℃/220rpm培养至OD600为0.6。取50mL菌液，4000rpm离心10分钟，去上清。收集菌体，并重悬至OD600为0.6，然后加入终浓度为20mg/L的AS，用于侵染。将无菌苗的叶片边缘剪掉，沿主叶脉将叶片剪成1cm2的叶盘放入农杆菌侵染液中侵染5分钟。
3.共培养与继代培养：将侵染的叶片捞出，用滤纸吸干农杆菌，叶面朝下平铺在共培养基上，放入人工气候室，黑暗培养3天。S1继代：将共培养3天的叶片叶面朝上转移到S1分化培养基中，在人工气候室黑暗培养1周左右转移至光照下继续培养，至叶片边缘长出0.5cm左右的丛芽。S2继代：将S1上长出的丛芽转移到S2分化培养基上，去除未长出丛芽的叶片部分，光照培养2周待丛芽长成幼苗。S3继代：将S2上的小苗转移到S3分化培养基上，光照培养2周。生根培养：去除小苗底部膨大部分和发黄的叶片，转移小苗至含有生根培养基组培瓶中，光照培养2周。
4.转基因烟草的获得：待小苗生出8条左右、长约3cm的根时，打开培养瓶的盖子进行炼苗。3天后将幼苗转移到装有无菌土的花盆中，盖上塑料膜保温。一周后去除保鲜膜，使其在自然条件下快速生长。
5.转基因烟草的阳性鉴定：按照实施例1中的方法提取所获得的转基因烟草总RNA，经反转录得到cDNA。利用实施例2中NtSWEET11基因的特异引物进行实时定量PCR检测。结果显示，和对照相比(对照是指未进行农杆菌转化的植株)，OE2和OE3两株转基因烟草中NtSWEET11基因的表达量显著增加，分别为对照的57倍和60倍(图3)。
青枯病抗性鉴定
将上述转pCHF3-NtSWEET11烟草的阳性植株OE2和OE3转移至温室培养，自交，收集转基因种子。同时以转pCHF3空载体的烟草K326为对照，用于后续的青枯病抗性鉴定。
在植株生长到45天时，以实施例2中所述的青枯病接种和调查方法，分别对NtSWEET11基因过表达烟草植株和转pCHF3空载体的烟草对照植株进行青枯病接病处理，6天后观察植株的发病情况。如图4所示，和对照相比，NtSWEET11基因过表达烟草植株具有明显的抗青枯病表现。同时，从青枯菌在烟草根部的繁殖情况来看，NtSWEET11基因过表达烟草植株根部青枯菌繁殖速率明显慢于对照(图5)。综上表明，在烟草中过表达NtSWEET11基因对烟草青枯病抗性有明显的增强作用。
序列表
<110> 湖南中烟工业有限责任公司
<120> 一种烟草NtSWEET11基因及其应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 891
<212> DNA
<213> 烟草(Nicotiana tabacum)
<400> 1
atggcgggta tttctggtca ctgggctttt gcttttggtg tccttggcaa catcgtctca 60
ttcattgtgt tcctttctcc gctgcctacg ttttataaaa tttacaagaa gaaatcaact 120
gaaggctatc aatcaattcc atatgtggtt gctctcttta gttccatgct ttggatttat 180
tatgcatttc tcaagacaaa cacgaccctt atcatcacta taaactcctt tggttgtttc 240
attgaaacta tctacgtcag tttctatctt ttctacgcac caaagaaagc cagggttcaa 300
actgtgaaga tgctcgtatt atcagtagtg ggtggctttg gtgctattgt tcttgttacc 360
caatttctat tcaaaggtgc cgctcggggc caagcggttg gatggatttg ccttgtgttt 420
tccttgtgtg tgtttgtagc acccttaggc attgtgagac aagttataaa aacaaagagt 480
gtggaataca tgccacttct cctatcagtt tttctcacct taagtgctgt aatgtggttc 540
ttctatggcc ttctcgtaaa agacattaac atagctattc caaacgtatt gggattcatc 600
ctaggaattc tccaaatagt gctctacgta atatacaaca aaaaggagaa ggccatttta 660
aaggagcaga aactaccggc gaagatacaa aaccatgtca ttatcgtaga tgagaacaat 720
aatcagaagc ttccagaact cacagaggaa cagattattg acattgtaaa gcttggttcc 780
ctaatttcct caggaaaaat tcacgtggct tcgtgtttgc ataattctac atgtggagtt 840
gaagcagcta aagttgagaa tatgcccaaa ctgcaaactg ccgaagccta g 891
<210> 2
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<212> PRT
<213> 烟草(Nicotiana tabacum)
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Met Ala Gly Ile Ser Gly His Trp Ala Phe Ala Phe Gly Val Leu Gly
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Asn Ile Val Ser Phe Ile Val Phe Leu Ser Pro Leu Pro Thr Phe Tyr
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Lys Ile Tyr Lys Lys Lys Ser Thr Glu Gly Tyr Gln Ser Ile Pro Tyr
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Val Val Ala Leu Phe Ser Ser Met Leu Trp Ile Tyr Tyr Ala Phe Leu
50 55 60
Lys Thr Asn Thr Thr Leu Ile Ile Thr Ile Asn Ser Phe Gly Cys Phe
65 70 75 80
Ile Glu Thr Ile Tyr Val Ser Phe Tyr Leu Phe Tyr Ala Pro Lys Lys
85 90 95
Ala Arg Val Gln Thr Val Lys Met Leu Val Leu Ser Val Val Gly Gly
100 105 110
Phe Gly Ala Ile Val Leu Val Thr Gln Phe Leu Phe Lys Gly Ala Ala
115 120 125
Arg Gly Gln Ala Val Gly Trp Ile Cys Leu Val Phe Ser Leu Cys Val
130 135 140
Phe Val Ala Pro Leu Gly Ile Val Arg Gln Val Ile Lys Thr Lys Ser
145 150 155 160
Val Glu Tyr Met Pro Leu Leu Leu Ser Val Phe Leu Thr Leu Ser Ala
165 170 175
Val Met Trp Phe Phe Tyr Gly Leu Leu Val Lys Asp Ile Asn Ile Ala
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Ile Pro Asn Val Leu Gly Phe Ile Leu Gly Ile Leu Gln Ile Val Leu
195 200 205
Tyr Val Ile Tyr Asn Lys Lys Glu Lys Ala Ile Leu Lys Glu Gln Lys
210 215 220
Leu Pro Ala Lys Ile Gln Asn His Val Ile Ile Val Asp Glu Asn Asn
225 230 235 240
Asn Gln Lys Leu Pro Glu Leu Thr Glu Glu Gln Ile Ile Asp Ile Val
245 250 255
Lys Leu Gly Ser Leu Ile Ser Ser Gly Lys Ile His Val Ala Ser Cys
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Leu His Asn Ser Thr Cys Gly Val Glu Ala Ala Lys Val Glu Asn Met
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Pro Lys Leu Gln Thr Ala Glu Ala
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<210> 3
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggcgggta tttctggtca ct 22
<210> 4
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
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ctaggcttcg gcagtttgca gt 22
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<213> 人工序列(Artificial Sequence)
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aaacacgacc cttatcatca ct 22
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<213> 人工序列(Artificial Sequence)
<400> 6
catcttcaca gtttgaaccc tg 22
<210> 7
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
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agaacacggg ggacgagctc atggcgggta tttctggtca ct 42
<210> 8
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gatccccggg taccgagctc ctaggcttcg gcagtttgca gt 42
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tgtggaatac atgccacttc 20
<210> 10
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gtgtgtgcgc aatgaaactg 20
Claims (10)
1.一种烟草NtSWEET11基因，其特征在于，基因CDS序列如SEQ ID NO.1所示。
2.根据权利要求1所述的烟草NtSWEET11基因，其特征在于，所述的基因序列还包括相似性不低于95％的具有类似功能的基因序列；所述的类似功能包括表达抗青枯病的蛋白。
3.根据权利要求2所述的烟草NtSWEET11基因，其特征在于，所述的抗青枯病的具体对象为植物，进一步优选为烟草。
4.一种NtSWEET11蛋白，其特征在于，蛋白序列如SEQ ID NO.2所示。
5.根据权利要求4所述的NtSWEET11蛋白，其特征在于，所述的蛋白序列还包括相似性不低于95％的具有类似功能的蛋白序列；所述的类似功能包括抗青枯病。
6.根据权利要求5所述的NtSWEET11蛋白，其特征在于，所述的抗青枯病的具体对象为植物，进一步优选为烟草。
7.权利要求1或2或3所述的烟草NtSWEET11基因在提高青枯病抗性中的应用。
8.根据权利要求7所述的应用，其特征在于，用于提高烟草抗青枯病抗性，尤其是烟草抗青枯病抗性。
9.根据权利要求8所述的应用，其特征在于，通过过表达烟草NtSWEET11基因，获得烟草青枯病抗性提高的烟草转化植株。
10.根据权利要求9所述的应用，其特征在于，过表达烟草NtSWEET11基因的载体为pCHF3。
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Citations (2)
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|---|---|---|---|---|
| US20140359899A1 (en) * | 2011-12-08 | 2014-12-04 | Carnegie Institution Of Washington | Sucrose Transporters and Methods of Generating Pathogen-Resistant Plants |
| WO2020208017A1 (en) * | 2019-04-11 | 2020-10-15 | Wolf Frommer | Diagnostic kit and method for sweet-based rice blight resistance and resistant breeding lines |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140359899A1 (en) * | 2011-12-08 | 2014-12-04 | Carnegie Institution Of Washington | Sucrose Transporters and Methods of Generating Pathogen-Resistant Plants |
| WO2020208017A1 (en) * | 2019-04-11 | 2020-10-15 | Wolf Frommer | Diagnostic kit and method for sweet-based rice blight resistance and resistant breeding lines |
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| XIAO TONG GAI等: "NtSWEET1 promotes tobacco resistance to Fusarium oxysporum-induced root rot disease", PLANT SIGNAL BEHAV, vol. 16, no. 11, pages 1 - 3 * |
| 孙荣华;李融梅;何翔;宋从凤;: "水稻白叶枯病菌毒力基因tal17.5病理学功能的研究", 南京农业大学学报, no. 06, pages 63 - 69 * |
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