CN116426567B - 一种n基因及其蛋白在抗番茄黄化曲叶病毒中的应用 - Google Patents
一种n基因及其蛋白在抗番茄黄化曲叶病毒中的应用 Download PDFInfo
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Abstract
本发明提供了一种N基因及其蛋白在抗番茄黄化曲叶病毒中的应用,属于作物抗病技术领域。本发明为番茄黄化曲叶病毒病的防治提供了新的有效手段。本发明利用同源重组的方法分别构建N基因、NSm基因、NSs基因和GP基因的植物表达载体,然后分别与TYLCV侵染性克隆瞬时共侵染本氏烟,发现N基因引起TYLCV相对表达量下调的程度最为明显,下调幅度达39%。另将其3种蛋白和TYLCV以TYLCV+N+GP+NSm、TYLCV+N+GP、TYLCV+N+NSm、TYLCV+GP+NSm等组合分别瞬侵本氏烟后发现所有含N的处理对TYLCV均产生了明显的影响,为提高作物对番茄黄化曲叶病毒的抗性提供了一条有效途径。
Description
技术领域
本发明涉及作物抗病技术领域,尤其涉及一种N基因及其蛋白在抗番茄黄化曲叶病毒中的应用。
背景技术
植物病毒病在全球范围内大面积流行,并呈逐年加重的趋势,已对农业生产、环境和经济安全构成了严重威胁。其中番茄黄化曲叶病毒(tomato yellow leafcurl virus,TYLCV)是蔬菜生产中危害最为严重的病毒之一。
番茄黄化曲叶病毒病是由番茄黄化曲叶病毒引起的、发生在番茄的病害。番茄植株感病初期主要表现为植株生长迟缓或停滞,节间变短,明显矮化,叶片变小、变厚,叶质脆硬,有褶皱,向上卷曲,变形,叶片边缘至叶脉区域黄化,以植株上部叶片症状为典型,下部老叶症状不明显;植株感病后期坐果很少,果实变小,膨大速度极慢;成熟期的果实不能正常转色。
目前番茄黄化曲叶病毒病的防治还是以“农业防治、物理防治为主、化学防治为辅”,未见分子领域的防治手段。
发明内容
本发明的目的在于提供一种N基因及其蛋白在抗番茄黄化曲叶病毒中的应用,为番茄黄化曲叶病毒病的防治提供新的有效手段。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种N基因在抗番茄黄化曲叶病毒中的应用,所述N基因的核苷酸序列如SEQ ID NO.1所示。
优选的,通过转基因技术将N基因转入作物中,提升作物对番茄黄化曲叶病毒的抗性。
本发明还提供了一种N蛋白质在抗番茄黄化曲叶病毒中的应用,所述N蛋白质由上述N基因编码得到,所述蛋白质的氨基酸序列如SEQ ID NO.2所示。
本发明还提供了一种提高作物对番茄黄化曲叶病毒抗性的方法,包括以下步骤:
将N基因构建至初始载体中,得到重组载体;
将所得重组载体转化至农杆菌中;
将农杆菌接种至作物,实现作物对番茄黄化曲叶病毒抗性的提高。
优选的,所述初始载体为pYBA-1132-GFP。
优选的,所述农杆菌为农杆菌GV3101。
本发明的技术效果和优点:
本发明利用同源重组的方法分别构建N基因、NSm基因、NSs基因和GP基因的植物表达载体,然后分别与TYLCV侵染性克隆瞬时共侵染本氏烟,发现N基因引起TYLCV相对表达量下调的程度最为明显,下调幅度达39%。将其3种蛋白和TYLCV以TYLCV+N+GP+NSm、TYLCV+N+GP、TYLCV+N+NSm、TYLCV+GP+NSm等组合分别瞬侵本氏烟后发现所有含N的处理对TYLCV均产生了明显的影响,为提高作物对番茄黄化曲叶病毒的抗性提供了一条有效途径。
附图说明
图1为各基因(RdRp除外)与载体pCB301构建示意图;
图2为qPCR分析TYLCV与单个蛋白共同接种本氏烟3d后的TYLCV相对表达量;
图3为Westernblot分析TYLCV与单个蛋白共同接种本氏烟3d后的TYLCV相对表达量;
图4为qPCR分析TYLCV与多个蛋白共同接种本氏烟3d后的TYLCV相对表达量;
图5为Westernblot分析TYLCV与多个蛋白共同接种本氏烟3d后的TYLCV相对表达量
图6为转基因本氏烟接种TYLCV和非转基因本氏烟接种TYLCV后7d、14d后的症状;
图7为qPCR分析各处理接种病毒7d时的TYLCV相对表达量;
图8为qPCR分析各处理接种病毒14d时的TYLCV相对表达量;
图9为Westernblot分析各处理接种病毒7d时的TYLCV相对表达量;
图10为Westernblot分析各处理接种病毒14d时的TYLCV相对表达量。
具体实施方式
N基因的核苷酸序列:ATGTCTAAGGTTAAGCTCACTAAGGAAAACA TTGTTGCTTTGTTGACACAAGGCAAAGATCTTGAATTTGAAGAAGATCAGAATCTGGTAGCATTTAACTTCAAGACTTTTTGTCTGGAAAACCTTGACCAGATCAAGAAGATGAGCATTATTTCATGTCTGACATTCCTGAAGAATCGTCAGAGTATAGTGAAGGTTATTAAGCAAAGTGATTTTACTTTTGGCAAAATCACTATAAAGAAAACTTCAGACAGGATTGGAGCCACTGACATGACCTTCAGAAGGCTTGATAGCTTGATCAGGGTCAGGCTTGTCGAGGAAACTGGGAATTCTGAGAACCTCAATACTATCAAATCTAAGATTGCTTCTCACCCTCTGATTCAAGCCTATGGATTACCTCTTGATGATGCAAAGTCTGTGAGGCTTGCCATAATGCTGGGAGGTAGCTTACCTCTTATTGCTTCAGTTGATAGCTTTGAGATGATCAGTGTTGTCTTGGCTATATATCAGGATGCAAAATACAAAGACCTCGGGATCGATCCAAAGAAGTATGACACCAGGGAAGCCTTAGGAAAAGTTTGCACTGTGCTAAAAAGCAAAGCATTTGAAATGAATGAAGATCAGGTGAAGAAAGGGAAAGAGTATGCTGCTATACTTAGCTCCAGCAATCCTAATGCTAAAGGAAGTATTGCTATGGAACATTACAGTGAAACTCTTAACAAGTTCTATGAAATGTTCGGGGTTAAAAAACAGACAAAACTTGCAGAACTTGCTTAA,如SEQ ID NO.1所示。
N蛋白的氨基酸序列:MSKVKLTKENIVALLTQGKDLEFEEDQNLVAF NFKTFCLENLDQIKKMSIISCLTFLKNRQSIVKVIKQSDFTFGKITIKKTSDRIGATDMTFRRLDSLIRVRLVEETGNSENLNTIKSKIASHPLIQAYGLPLDDAKSVRLAIMLGGSLPLIASVDSFEMISVVLAIYQDAKYKDLGIDPKKYDTREALGKVCTVLKSKAFEMNEDQVKKGKEYAAILSSSNPNAKGSIAMEHYSETLNKFYEMFGVKKQTKLAELA,如SEQ ID NO.2所示。
NSs基因的核苷酸序列:ATGTCTTCAAGTGTTTATGAGTCGATCATTC AGACAAAAGCTTCAGTCTGGGGATCAACTGCATCTGGTAAAGCTGTTGTAGATTCTTACTGGATTCATGAACTTGGTACTGGTTCTCCACTAGTTCAAACCCAGTTGTATTCTGATTCAAGAAGCAAAAGTAGCTTTGGCTATACTGCAAAGGTGGGGAATCTTCCCTGTGAAGAAGAAGAGATTCTTTTTCAGCATGTGTATATCCCTATTTTTGATGATATTGATTTTAGCATCAATATTAATGACTCTGTTCTGGCACTATCTGTTTGCTCAAATACAGTTAATACTAACGGAGTGAAACATCAAGGTCATTTGAAAGTTTTGTCTCCTGCTCAGCTCCATTCTATTGGATCTACCATGAACAGATCTGATATTACAGACCGATTCCAGCTCCAAGAAAAAGACATAATTCCCAATGACAGATACATTGAAGCTGCAAACAAGGGCTCTTTGTCTTGTGTTAAAGAGCATACCTATAAGGTCGAGATGTGCTACAATCAAGCTTTGGGCAAAGTGAATGTTCTATCCCCTAACAGAAATGTCCATGAATGGCTGTACTGTTTCAAGCCAAATTTCAATCAAGTTGAAAGCAACAACAGAACTGTAAATTCTCTTGCAGTGAAATCTCTTCTCATGTCAGCAGAAAACAACATCATGCCTAACTCTCAGGCTTTTGTAAAAGCTTCCACTGATTCTCATTTCAAGCTGAACCTCTGGCTAAGAGTTCCAAAGGTTTTGAAGCAGATTTCCATTCAGAAATTGTTCAAAGTTGCAGGAGATAAAACAAACAAAACATTTTATTTATCTATTGCTTGCATTCCAAACCATAACAGTGTTGAGACAGCTTTAAACATTTCTGTTATTTGCAAGCATCAGCTTCCAATCCGTAAATGTCAAGCTCCTTTTGAATTATCAATGATGTTTTCTGATTTAAAGGAGCCTTACAACATTGTTCATGATCCTTCATACCCTCAGAGGATTGTTCATGCTCTGCTTGAAACTCACACATCTTTTGCACAAGTTCTTTGCAACAACTTGCAAGAAGATGTGATCATCTACACTTTGAACAACTATGAGCTAACTCCTGGAAAGTTAGATCTAGGTGAAAGAACCTTGAATTACAGTGAAGATATCTGCAAAAGGAAATATTTCCTTTCAAAAACACTTGAATGTCTTCCATCTAACACACAAACTATGTCTTACTTAGACAGTATCCAAATCCCTTCCTGGAAGATAGACTTTGCCAGGGGAGAAATTAAAATTTCTCCACAATCTGTTTCAGTTGCAAAATCTTTGTTAAAGCTTGATTTAAGCGGGATCAAAAAGAAAGAATCTAAGATCTCGGAAGCATATGCTTCAGGATCAAAATAA,如SEQ IDNO.3所示。
GP基因的核苷酸序列:ATGAGAATTTTAAAACTACTAGAACTAGTG GTAAAAGTGAGTCTTTTCACAATTGCCCTGAGTTCTGTCTTGTTGGCATTCTTGATCTTCAGAGCCACAGATGCCAAAGTAGAAATAATTCGTGGAGATCATCCTGAGGTTTATGATGATTCTGCTGAGAATGAAGTACCCACTGC
TGCATCGATTCAACGCAAGGCTATCTTGGAGACTTTAACTAATCTAATG
CTAGAATCTCAGACTCCTGGAACCCGTCAGATACGAGAAGAAGAATCA
ACCATCCCTATTTTTGCTGGGTCAACTACGCAAAAAATAATCTCTGTCT
CGGATCTTCCTAACAACTGCTTGAATGCTTCTTCATTAAAATGCGAGAT
AAAAGGGATATCCACTTATAATGTTTATTATCAAGTGGAAAATAATGGTG
TCATATATTCCTGTGTTTCTGATTCAGCAGAAGGGTTAGAAAAATGTGA
CAATTCTTTAAATTTGCCAAAGAGATTCTCCAAAGTCCCGGTTATTCCC
ATTACCAAACTTGACAACAAAAGGCATTTTTCAGTTGGAACAAAATTC
TTCATTTCAGAAAGCCTGACACAAGATAATTATCCTATAACTTATAACTC
GTACCCTACTAATGGAACAGTATCATTACAAACCGTAAAGTTATCTGGA
GACTGCAAAATAACTAAATCAAACTTTGCCAATCCCTATACTGTTAGCA
TCACTAGCCCCGAGAAGATCATGGGTTATTTGATAAAAAAACCTGGTG
AAAATGCGGAACACAAGGTCATATCTTTTTCTGGATCAGCAAGCATCA
CTTTTACTGAAGAAATGTTGGATGGTGAGCACAATCTCTTGTGCGGTG
ACAAATCAGCTAAAATACCAAAAACAAACAAAAGAGTTAGAGATTGCA
TAATCAAATATTCAAAGAGCATTTATAAACAAACAGCCTGCATCAATTT
CTCTTGGATAAGATTAATATTGATAGCTTTGTTGATCTATTTCCCTATCCG
GTGGTTAGTAAACAAGACAACTAAACCTCTCTTTCTCTGGTATGATCTT
ATAGGCTTGATCACATACCCTATTTTGTTGCTCATAAATTGCTTATGGAA
ATATTTTCCATTCAAATGTTCTAACTGTGGCAATTTGTGCATAATCACAC
ATGAGTGTACTAAAATTTGCATCTGCAACAAAAGCAAAGCCTCAAAAG
AACACTCTTCAGAGTGTCCCATACTGTCCAAAGAAACAGATCATGACT
ACAACAAACATAAGTGGACTAGCATGGAATGGTTCCATCTAATAGTGA
ACACTAAGCTAAGCTTTAGTTTGCTGAAATTTGTGACTGAAATCTTAAT
AGGTTTGGTCATTTTGTCTCAAATGCCCATGTCTATGGCTCAAACTACT
CAATGTTTGAGTGGATGTTTTTATGTTCCAGGCTGTCCAGTTTTGGTTAC
AAGCAAATTTGAAAAATGCCCTGAAAAAGATCAATGTTACTGTAATGT
AAAAGAAGATAAGATTATAGAAAGTATCTTTGGCACTAACATTGTTATA
GAAGGTCCTAATGATTGCATAGAGAACCAGAATTGTGTTGCACACCCAT
CTATTGATAATCTTATAAAATGTAGATTAGGTTGCGAATACTTAGATTTAT
TTCGCAACAAACCTTTGTATAATGGGTTTTCAGATTATACAGGAAGCTC
TTTGGGTTTAACATCGGTTGGTCTGTATGAGGCTAAGAGATTGAGGAAT
GGTATAATAGATTCCTATAACCGTACAGACAAGATTTCCGGAATGATTGC
AGGAGATTCTCTAGACAAAAATGAAACAAGCATACCAGAGAACATTCT
GCCTAGACAATCATTGATCTTTGATTCTGTTGTGGATGGGAAATATAGAT
ATATGGCAGAACAATCTCTTTTAGGAGGAGGAGGGACTGTATTCATGTT
AAATGATAAGACCTCAGAAAAAGCAAAAAAATTCGTGATTTATATCAA
AAGTGTGGGAATTCATTATGAAGTGTCTGAAAAATACACAACAGCTCC
TATCCAAAGCACTCACACAGATTTTTATTCCACTTGTACAGGAAACTGT
GACACTTGCAGAAAAAATCAAGCTTTAACAGGTTTCCAAGATTTTTGTA
TAACACCAACTTCTTATTGGGGATGTGAAGAAGCTTGGTGTTTTGCAAT
TAATGAAGGTGCTACATGCGGGTTCTGTCGAAATATTTATGACATGGAT
AAATCATATAGAATTTATTCAGTGCTAAAATCAACTATAGTGGCAGATGT
TTGCATTTCTGGTATTTTAGGAGGTCAATGCTCAAGGATTACTGAAGAG
GTTCCTTATGAAAATGCATTGTTTCAAGCTGATATACAAGCAGATCTGC
ACAATGATGGTATCACTATAGGTGAACTGATAGCTCATGGACCTGACAG
CCATATTTATTCTGGAAATATTGCAAACTTGAATGATCCTGTCAAAATGT
TTGGTCATCCACAATTGACTCATGATGGAGTGCCTATTTTTACTAAGAA
AACTCTAGAAGGACATGACATGTCTTGGGATTGTGCAGCAATAGGGAA
AAAATCAATCACTATTAAAACATGCGGATATGACACATACAGATTTAGA
TCTGGTTTAGAGCAAATATCAGATATTCCCATTAGTTTCAAAGATTTCTC
TAGTTTTTTCTTGGAAAAATCTTTTAGTTTAGGGAAACTCAAGATTGTC
GTTGATCTTCCATCTGATCTTTTTAAAGTTGCTCCTAAGAGACCTTCCAT
AACTTCGACAAGATTGAATTGCAACGGCTGTCTTCTATGCGGTCAAGG
TTTATCTTGCATTTTGGAATTTTTCTCAGATTTGACATTTTCTACTGCAAT
TTCTATAGATGCTTGCTCTCTATCTACTTATCAGCTGGCTGTTAAAAAAG
GGTCTAACAAATACAACATAACAATGTTTTGTTCTGCTAATCCGGATAA
GAAGAAAATGACATTGTATCCAGAAGGCAATCCGGATATTTCTGTGGA
AGTTCTGGTCAATAATGTTATTGTAGAAGAACCGGAGAACATAATAGAT
CAAAATGATGAGTATGCTCATGAAGAACAACAATATAATTCTGATTCCT
CAGCATGGGGCTTCTGGGATTATATTAAAAGCCCATTCAATTTCATTGCA
AGTTACTTTGGCTCATTTTTTGATACTATCAGAGTGATATTGCTTATTGCA
TTCATTTTCCTTGTGATTTATTTCTGTTCTATTCTAACAACAATTTGTAAA
GGATATGTCAAGAATAAATCTTATAAATCTAGATCCAAGATAGAGGATGA
TGATGATTCTGAGATCAAAGCCCCTATGTTAATGAAAGATACAATGACAAGACGAAGGCCACCTATGGATTTCTCTCACCTTGTCTGA,如SEQ ID NO.4所示。
NSm基因的核苷酸序列:ATGTTGACTTTTTTTGGTAATAAGGGGTCT TCTAAGTCTGCCAGAAAGGATGAAGGGCCTTTAGTTTCACTTGCTAAACATAACGGTAATGTTGAAGTCTCAAAACCATGGTCTTCTTCTGATGAAAAGCTTGCTTTAACCAAAGCTATGGATACATCCAAAGGAAAGATACTGTTGAACACAGAGGGAACATCTTCCTTTGGAACATATGAATCTGATTCTATCACAGAATCAGAGGGTTATGATCTTTCTGCGAGAATGATAGTAGATACAAACCACCATATCTCAAACTGGAAAAATGATCTTTTTGTCGGCAACGGGAAGCAAAACGCTAATAAGGTCATCAAGATCTGTCCAACTTGGGACAGCAGAAAACAATACATGATGATTTCCAGGATTGTGATATGGGTCTGCCCCACTATACCAAACCCTACAGGGAAACTTGTGGTTGCTCTGGTCGATCCCAACATGCCATCTGAAAAGCAAATCATTCTGAAGGGTCAGGGGACAATAACTGATCCTATCTGTTTTGTTTTTTATCTGAACTGGTCTATTCCGAAAATGAATAACACTCCAGAAAACTGCTGTCAGCTGCACTTGATGTGCAGTCAAGAATACAAGAAGGGGGTTTCTTTTGGTAGTGTCATGTATTCTTGGACAAAGGAGTTTTGTGATTCACCCAGAGCTGATAAAGACAAAAGTTGCATGGTCATACCTCTAAACAGGGCTATTAGAGCTAGATCTCAAGCATTCATTGAGGCTTGCAAGCTGATAATTCCTAAAGGAAACAGTGAGAAGCAGATTAAAAAACAGCTTAAAGAACTGAGCTCAAATCTTGAGAGATCAGTTGAAGAAGAGGAGGAAGGGGTTTATGATAATGTTGCTCAGTTATCTTTTGATGAGATATAG,如SEQ ID NO.5所示。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
使用的质粒小量提取试剂盒、琼脂糖凝胶回收纯化试剂盒、普通DNA聚合酶(2×Taq MasterMix)、第一链cDNA合成试剂盒、高保真DNA聚合酶(2×Phanta Flash MasterMix)、同源重组快速克隆试剂盒等均购自南京诺唯赞生物科技有限公司;
测序及引物合成相关工作由深圳华大基因股份有限公司完成;
使用的植物基因组DNA提取试剂盒、Trizol提取试剂、DNA分子量标准Marker、2×SG Fast qPCR MasterMix、各种限制性核酸内切酶、AP标记山羊抗小鼠IgG、PVDF印迹膜等均购自上海生工生物科技有限公司。
实施例1
将NSs、N、GP、NSm基因分别克隆到植物双元表达载体pCB301中获得重组克隆pNSs301、pN301、pGP301、pNSm301,构建示意图如图1所示。采用方法如下:
首先选取目的基因序列,根据引物实验需求,通过在线网站(https://crm.vazyme.com/cetool/simple.html)获得插入片段的扩增引物。提取所需载体的质粒,在合适的反应体系中加入相应的限制性内切酶,37℃孵育25min,5ˊ-限制性内切酶采用StuI,3ˊ-限制性内切酶采用Sal I,为了减少扩增突变的引入,使用高保真DNA聚合酶(2×Phanta Flash MasterMix)进行PCR扩增,反应体系为:cDNA溶液0.75μL、2×PhantaFlashMasterMix12.5μL、上下游引物各1μL(10μM)、DEPC水补足至25μL,视插入片段大小采用一段或分段扩增的方法。
采用同源重组的方法进行质粒构建,于冰盒上将线性化的载体与纯化的目的片段以一定的摩尔比加入到离心管中进行重组反应,各组分的加入量可通过网站(https://www.novoprotein.com.cn/product-detail?productNumber=NR 005)的在线计算器获得。反应体系如下:线性化载体XμL、插入片段YμL、2×ClonExpress Mix 5μL、ddH2O补足至10μL。使用移液器轻轻吸打混匀,短暂离心将反应液收集至管底。单片段重组反应,50℃孵育5min,放于4℃或立即置于冰上冷却;多片段重组反应,50℃孵育15min,放于4℃或立即置于冰上冷却;连接产物转化至大肠杆菌DH5α中。
分别将构建正确的重组质粒转化至农杆菌GV3101,再分别与TYLCV侵染性克隆复合接种于本氏烟,所用方法如下:
(1)将目的质粒转化至农杆菌GV3101中,涂布于有50μg/mL卡那霉素(kanamycin,Kan)的固体LB平板上,48h后待长出单菌落将其挑至含50μg/mL卡那霉素和100μg/mL利福平的液体LB培养基中,28℃培养48h;
(2)将培养好的菌液分装到10mL或者15mL的圆底离心管中,10,000rpm离心2min收集菌体,用MMA缓冲液[10mM MES(pH 5.6),10mM MgCl2和200mM乙酰丁香酮(acetosyringone,As)]重悬菌体,根据实验需要调至OD 600≈0.5,于室温黑暗静置4h后用于接种;
(3)选取长势基本一致的番茄或烟草幼苗,用1mL的注射器吸取菌液在叶子的背面进行注射,待菌液扩至整个叶片,每株注射3片;
(4)将接种后的植株暗处理过夜后于正常条件下进行培养。
其处理设计为TYLCV+空载(CK)、TYLCV+NSs、TYLCV+N、TYLCV+GP、TYLCV+NSm。待接种3d后采集接种部位的叶片,分别对TYLCV进行qPCR和westernblot相对定量检测,结果如图2~3所示(数据利用Duncan新复极差法进行多重比较分析,误差线代表每个处理三个生物学重复的标准差,不同字母代表差异显著水平,P<0.05。@代表TYLCVV1的特异性抗体。考马斯亮蓝R-250染色凝胶用于表示样品上样量)。
由结果可以看出,N与TYLCV共同侵染本氏烟后能够引起TYLCV相对表达量的下调,且以N抑制TYLCV的程度最为明显,其下调幅度达39%。
本发明还设计了TYLCV+CK、TYLCV+N+GP+NSm、TYLCV+N+GP、TYLCV+N+NSm、TYLCV+GP+NSm等5个处理,将其分别接种于本氏烟,3d后采集接种部位的叶片进行相对定量检测,结果如图4~5所示(数据利用Duncan新复极差法进行多重比较分析,误差线代表每个处理三个生物学重复的标准差,不同字母代表差异显著水平,P<0.05。@代表TYLCVV1的特异性抗体。考马斯亮蓝R-250染色凝胶用于表示样品上样量)。
由结果可知,N+GP+NSm与TYLCV复合侵染本氏烟后能显著降低TYLCV的相对表达量,而TYLCV+N+GP和TYLCV+N+NSm两处理中的TYLCV相对表达量较TYLCV+GP+NSm的处理更低,其中所有含N的处理对TYLCV均产生了明显的影响,进一步说明N基因对TYLCV的影响最为显著。
实施例2
构建重组质粒N-GFP和空载质粒pYBA-1132-GFP,方法同实施例1,5ˊ-限制性内切酶采用BamH I,3ˊ-限制性内切酶采用EcoR I,将验证正确的重组质粒分别转至农杆菌GV3101。利用农杆菌介导的叶盘法转化至本氏烟,经过无菌苗的获得、农杆菌侵染、共培养、分化培养、生根培养、驯化移栽等阶段,最后分别获得T0代转基因(genetically modified,GM)植株,即转N基因(GM-N)和转空载(GM-CK)基因的幼苗。方法如下:
(1)播种:数取一定数量的本氏烟种子,先用75%的酒精浸泡60s,再用20%的次氯酸钠浸泡45s,然后用无菌水冲洗5次,每次需充分洗涤,干净后将种子平铺在固体MS培养基上。暗培养4d后,放于光照培养箱(25℃,16h明/8h暗)培养25d;
(2)种子萌发:待长出两片子叶后,将无菌苗转入固体MS培养基中,培养至8叶期用于后续实验;
(3)预培养阶段:取出大小适宜的无菌苗,选取中上部叶位的叶片用剪刀剪成1cm2的方块,将叶片正面朝下平铺于固体RM培养基(MS+6-BA2.0mg/L+NAA 0.2mg/L)上,暗培养2d,一般一皿放6片;
(4)共培养阶段:收集过夜培养的带有目的质粒的农杆菌菌体,用液体RM培养基清洗两次,用液体RM培养基和25mg/L As重悬并调至OD600≈1.0,形成侵染液。将预培养2d后的外植体浸泡于侵染液中,不断震荡,侵染10min后倒掉侵染液,滤纸吸干,置于固体RM培养基上,暗室共培养2d,直至有微菌斑形成;
(5)洗菌:取出共培养的外植体,用液体RM培养基和1000mg/L特美汀(Timentin)洗涤外植体5min,再用液体RM培养基冲洗外植体2次,冲洗表面残留的农杆菌;
(6)芽诱导期:叶片取出后用滤纸吸干,转移到固体选择RM培养基(MS+6-BA2.0mg/L+NAA 0.2mg/L+300mg/LTimentin+100mg/LKan)上,外植体的背面与培养基接触,于黑暗中25℃下培养外植体2周,然后在光照培养箱进行培养,每隔3周将外植体更换至新鲜的固体选择RM培养基上(一般3片/瓶),直至外植体发芽;
(7)幼苗形成:将整个外植体与芽一起转移至固体选择生根培养基(MS+300mg/LTimentin+100mg/LKan)上,在光照培养箱中培养3周后,对生根的植株进行驯化移栽;
(8)驯化移栽:将转基因苗从培养瓶中取出后,冲洗掉残留的培养基,移栽到消毒的营养土中,在湿度可控的条件下进行培养,待成活后再移至温室内继续培养。
以获得的转基因阳性植株为材料,通过接种TYLCV来进一步验证N基因对TYLCV致病力的影响,方法如下:
收集保存所有转基因阳性植株的种子。选取籽粒饱满的T0代种子撒播于营养钵中,得到T1代转基因植株用于后续实验。
(1)以获得的转N基因和转空载的T1代植株为材料,逐株提取植物总DNA,以35S-F(序列:GACGCACAATCCCACTATCC,如SEQ ID NO.6所示)和YBA-1132-R(序列:CGTAGGTCAGGGTGGTCA,如SEQ ID NO.7所示)为引物进行PCR扩增,琼脂糖凝胶电泳检测扩增结果;
(2)根据电泳检测结果分别选取转N基因和转空载的阳性植株,待长至8-10叶期时,分别接种TYLCV侵染性克隆,方法与实施例1相同,接种7d和14d时采集对应的上部叶片冷冻保存;
(3)提取植物总DNA,进行荧光定量PCR检测;
(4)提取植物总蛋白,以TYLCV的鼠单克隆抗体为一抗,以AP标记山羊抗小鼠IgG为二抗,以Rubisco蛋白为上样量对照进行westernblot半定量检测。
本发明设计了GM-CK单接TYLCV、GM-N单接TYLCV、non-GM单接TYLCV、健康对照共4个处理,结果如图6所示。
从图6可以看出,各处理接种7d和14d后,相比接种TYLCV的GM-CK和non-GM的植株,接种TYLCV的GM-N植株症状明显变轻,主要表现为上部叶片只是轻微卷曲。
分别采集上部叶片对其中的TYLCV积累量进行qPCR,结果如图7(病毒接种后7d)~8(病毒接种后14d)所示,以及进行westernblot检测,结果如图9(病毒接种后7d)~10(病毒接种后14d)所示。数据利用Duncan新复极差法进行多重比较分析,误差线代表每个处理三个生物学重复的标准差,不同字母代表差异显著水平(P<0.05)。@代表TYLCVV1蛋白的特异性抗体。考马斯亮蓝R-250(CBB)染色凝胶用于表示样品上样量。
由结果可知,GM-N接种TYLCV后其体内的TYLCV相对表达量显著低于GM-CK和non-GM接种TYLCV的处理,因此可以看出TYLCV在瞬时表达和稳定表达N基因的本氏烟中均受到明显抑制。
由以上实施例可知,本发明利用同源重组的方法分别构建N基因、NSm基因、NSs基因和GP基因的植物表达载体,然后分别与TYLCV侵染性克隆瞬时共侵染本氏烟,发现N基因引起TYLCV相对表达量下调的程度最为明显,下调幅度达39%。另将其3种蛋白和TYLCV以TYLCV+N+GP+NSm、TYLCV+N+GP、TYLCV+N+NSm、TYLCV+GP+NSm等组合分别瞬侵本氏烟后发现所有含N的处理对TYLCV均产生了明显的影响,为提高作物对番茄黄化曲叶病毒的抗性提供了一条有效途径。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (3)
1.一种N基因在抗番茄黄化曲叶病毒中的应用,其特征在于,所述N基因的核苷酸序列如SEQ ID NO.1所示;
通过转基因技术将N基因转入作物中,提高作物对番茄黄化曲叶病毒的抗性;
所述提高作物对番茄黄化曲叶病毒抗性的方法,包括以下步骤:
将N基因构建至初始载体中,得到重组载体;
将所得重组载体转化至农杆菌中;
将农杆菌接种至作物,实现作物对番茄黄化曲叶病毒抗性的提高;
所述初始载体为pYBA-1132-GFP;
所述农杆菌为农杆菌GV3101。
2.一种N蛋白质在抗番茄黄化曲叶病毒中的应用,其特征在于,所述N蛋白质由权利要求1中的N基因编码得到,所述蛋白质的氨基酸序列如SEQ ID NO.2所示;
通过转基因技术将N基因转入作物中,提高作物对番茄黄化曲叶病毒的抗性;
所述提高作物对番茄黄化曲叶病毒抗性的方法,包括以下步骤:
将N基因构建至初始载体中,得到重组载体;
将所得重组载体转化至农杆菌中;
将农杆菌接种至作物,实现作物对番茄黄化曲叶病毒抗性的提高;
所述初始载体为pYBA-1132-GFP;
所述农杆菌为农杆菌GV3101。
3.一种提高作物对番茄黄化曲叶病毒抗性的方法,其特征在于,包括以下步骤:
将N基因构建至初始载体中,得到重组载体;
将所得重组载体转化至农杆菌中;
将农杆菌接种至作物,实现作物对番茄黄化曲叶病毒抗性的提高;
所述N基因的核苷酸序列如SEQ ID NO.1所示;
所述初始载体为pYBA-1132-GFP;
所述农杆菌为农杆菌GV3101。
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