CN110564702B - 二化螟生长发育相关蛋白ND及其编码基因、dsRNA干扰序列和应用 - Google Patents
二化螟生长发育相关蛋白ND及其编码基因、dsRNA干扰序列和应用 Download PDFInfo
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- CN110564702B CN110564702B CN201910725847.9A CN201910725847A CN110564702B CN 110564702 B CN110564702 B CN 110564702B CN 201910725847 A CN201910725847 A CN 201910725847A CN 110564702 B CN110564702 B CN 110564702B
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Abstract
本发明属于农业生物技术领域,具体涉及二化螟生长发育相关蛋白ND及其编码基因、dsRNA干扰序列和应用。本发明的二化螟生长发育相关蛋白ND,其氨基酸序列如SEQ ID NO.1所示。本发明首次发现ND基因参与二化螟的生长发育过程,其表达缺失可显著提高二化螟幼虫的死亡率,最终抑制种群的发展,可用于开发防治螟蛾科二化螟昆虫产品。
Description
技术领域
本发明属于农业生物技术领域,具体涉及二化螟生长发育相关蛋白ND及其编码基因、dsRNA干扰序列和应用。
背景技术
二化螟(Chilo suppressalis)属鳞翅目螟蛾科,是一种重要的多食性农业害虫,主要危害水稻、茭白以及玉米、小麦等多种植物,尤其对早栽、生长茂密的田块及杂交水稻田危害较重。目前,对于二化螟的防治以施用化学杀虫剂为主,然而长期大量使用化学农药引发了环境压力以及抗药性等问题。为转变此类情况,开发和利用符合环保、健康、绿色生态建设、发展可持续理念的水稻害虫无公害防治措施成为当前的防治热点。
还原型烟酰胺腺嘌呤二核苷酸(Nicotinamide adenine dinucleotide,NADH)脱氢酶(NADH dehydrogenase,ND)),又称辅酶Ⅰ脱氢酶(coenzymeⅠ-dependentdehydrogenases),是细胞能量代谢所必需的辅酶,对生物的有氧呼吸起重要作用。NADH是线粒体电子传递链(ETC)中的主要电子载体之一。线粒体是细胞能量代谢、生物合成、细胞程序性死亡和氧化应激的关键参与者。因此,在生物体的生长发育过程中,ND起着重要的作用。
现有技术对于昆虫中Nicotinamide adenine dinucleotide dehydrogenase(ND)的研究主要是分子机制,对于其功能机理和应用研究较少。
发明内容
本发明的目的在于提供一种二化螟生长发育相关基因ND。
本发明的再一目的在于提供上述基因所编码的蛋白。
本发明的再一目的在于提供上述基因的dsRNA干扰序列。
本发明的再一目的在于提供含有上述基因的重组表达载体。
本发明的再一目的在于提供含有上述基因的重组菌株。
本发明的再一目的在于提供二化螟ND基因的应用。
本发明的再一目的在于提供二化螟ND基因的dsRNA干扰序列的应用。
本发明的二化螟生长发育相关蛋白ND,其氨基酸序列如SEQ ID NO.1所示:
MVSWSALGRSFGINLLKTNNKLPNSIHNVTFSTSKAMSAGHGHKTMALQP
SRWQWHKFKDMFHYYAMVGLIPVGLIIFYTNVFIGPAQLAPIPEDYNPKH
WEYHRHPITRFIARYIHTNPQQEYEKFMHFIDEEAQRGKLRALEKAIIEK
MGERSDYQAYYYRPMVNKYLRLDKKAGDELYNRLGENLDE
本发明提供的二化螟生长发育相关基因ND的核苷酸序列如SEQ ID NO.2所示:
GACACACGTACATATAAAGTTAGGACATAAAACTATTTCTATTATGTTGGTGTCATTGAATTGATAAGAACCACAGATTACAAAAAGATCGAAAAAAGATTTAGAATAAAACTGTCACTGTCAGAAAAGTCAAAACAGGAATGGTTTCTTGGAGTGCTTTGGGCCGATCTTTCGGCATTAATTTATTAAAAACTAATAACAAATTACCTAATTCTATACACAATGTGACTTTTTCTACTAGCAAGGCCATGAGCGCCGGCCATGGACACAAAACAATGGCTTTACAACCCTCCAGATGGCAATGGCACAAATTCAAGGATATGTTCCATTACTACGCCATGGTAGGCTTAATCCCAGTGGGGCTTATTATATTCTACACCAACGTGTTCATTGGGCCTGCTCAACTGGCCCCAATTCCAGAGGACTACAACCCGAAGCACTGGGAGTATCATCGCCATCCAATAACTCGTTTTATCGCCCGCTATATTCATACCAATCCACAGCAGGAGTATGAAAAATTCATGCACTTCATTGATGAAGAAGCCCAAAGGGGTAAACTAAGAGCTCTGGAGAAGGCAATTATAGAAAAGATGGGAGAACGCAGCGACTATCAAGCATATTATTATCGACCAATGGTTAACAAATACCTCCGTTTGGATAAGAAGGCCGGCGATGAACTTTATAATCGCCTTGGTGAGAATTTAGATGAATAAAAAAAATTATTGCGTTTTTATAAACACTGTTAGTATATTTTTAGTAATCAAGCATATTGTTACATTAATTCAATGTGGATAAATAATCTTTTGATTGTTGCTTGAAAATAGATTTATTTGAAGTATTTCATGTAATCATACCCTGATAAATGCAACAGGAAAATAAAATTATTCTACATTATGTTTTGTATTCATTTATTGTAACTTGCATGCTCTGATCAAAAATTGGCCATGATGTTAATTTTATAGTCATAGAAAATTGAAAAAAGATGAAGTTTGGGTGAAATAAAAATAGTTCTTATGGTAAAAAAAAAAAAA
上述序列全场1031bp。
本发明提供的二化螟生长发育相关基因ND的cDNA核苷酸序列如SEQ ID NO.3所示:
ATGGTTTCTTGGAGTGCTTTGGGCCGATCTTTCGGCATTAATTTATTAAAAACTAATAACAAATTACCTAATTCTATACACAATGTGACTTTTTCTACTAGCAAGGCCATGAGCGCCGGCCATGGACACAAAACAATGGCTTTACAACCCTCCAGATGGCAATGGCACAAATTCAAGGATATGTTCCATTACTACGCCATGGTAGGCTTAATCCCAGTGGGGCTTATTATATTCTACACCAACGTGTTCATTGGGCCTGCTCAACTGGCCCCAATTCCAGAGGACTACAACCCGAAGCACTGGGAGTATCATCGCCATCCAATAACTCGTTTTATCGCCCGCTATATTCATACCAATCCACAGCAGGAGTATGAAAAATTCATGCACTTCATTGATGAAGAAGCCCAAAGGGGTAAACTAAGAGCTCTGGAGAAGGCAATTATAGAAAAGATGGGAGAACGCAGCGACTATCAAGCATATTATTATCGACCAATGGTTAACAAATACCTCCGTTTGGATAAGAAGGCCGGCGATGAACTTTATAATCGCCTTGGTGAGAATTTAGATGAATAA
上述序列全长573bp。
本发明还提供含有二化螟ND基因或其dsRNA干扰序列的重组表达载体和重组菌株。
根据本发明具体实施方式的二化螟ND基因的dsRNA干扰序列,其通过以下特异性引物序列扩增而得:
上游引物ds ND-F:tgGAATTCCAACGTGTTCATTGGGCCTG;
下游引物ds ND-R:tgGGTACCTTTTGATCAGAGCATGCAAGTT。
本发明提供上述基因在防治螟蛾科二化螟昆虫中的应用,包括将二化螟生长发育相关基因ND的dsRNA干扰序列饲喂二化螟的步骤。该干扰序列为双链RNA分子,利用特异性引物体外合成该序列的dsRNA,通过涂抹饲喂二化螟初孵幼虫。结果显示该序列可显著抑制Nicotinamide adenine dinucleotide dehydrogenase(ND)基因的表达。沉默Nicotinamide adenine dinucleotide dehydrogenase(ND)基因对二化螟幼虫死亡率的影响,结果发现抑制Nicotinamide adenine dinucleotide dehydrogenase(ND)基因的表达显著提高了幼虫的死亡率,可抑制种群的发展。
本发明的有益效果:
本发明首次发现Nicotinamide adenine dinucleotide dehydrogenase(ND)基因参与二化螟的生长发育过程,Nicotinamide adenine dinucleotide dehydrogenase(ND)表达的缺失显著提高二化螟幼虫的死亡率,最终抑制种群的发展。该基因可用于开发防治螟蛾科二化螟昆虫产品。
本发明提供了一种二化螟Nicotinamide adenine dinucleotide dehydrogenase(ND)基因的干扰序列,该序列可显著抑制Nicotinamide adenine dinucleotidedehydrogenase(ND)基因的表达,可利用该序列干扰Nicotinamide adenine dinucleotidedehydrogenase(ND)基因的表达,抑制二化螟的生长,最终控制其种群的发展。可用于开发绿色环保的抗虫植物,最终达到绿色防治害虫的目的。
附图说明
图1显示饲喂ND基因的dsRNA后,二化螟ND基因的沉默效率;其中,dsND表示饲喂Nicotinamide adenine dinucleotide dehydrogenase(ND)基因dsRNA的处理组;dsEGFP表示饲喂EGFP基因dsRNA的对照组;H2O表示饲喂ddH2O的对照组;
图2显示饲喂Nicotinamide adenine dinucleotide dehydrogenase(ND)基因的dsRNA对二化螟生长发育的影响;其中,dsND表示饲喂Nicotinamide adeninedinucleotide dehydrogenase(ND)基因dsRNA的处理组;dsEGFP表示饲喂EGFP基因dsRNA的对照组;H2O表示饲喂ddH2O的对照组。
具体实施方式
实施例1二化螟Nicotinamide adenine dinucleotide dehydrogenase(ND)基因的克隆与分析
称取20mg的二化螟样品置于1.5ml无酶管中,利用一次性无酶研磨棒及液氮充分研磨后,使用提取试剂盒提取总RNA。
使用反转录试剂盒将提取的总RNA合成cDNA模板。
设计并合成如下引物:
上游引物序列ND-F:5'-ATGGTTTCTTGGAGTGCTTTGGG-3';
下游引物序列ND-R:5'-TTATTCATCTAAATTCTCACCAAGGCG-3'。
利用上述引物ND-F和ND-R进行PCR扩增,扩增完毕后,用1%的琼脂糖凝胶电泳鉴定后,切胶并利用DNA凝胶回收试剂盒纯化回收目的片段。
将PCR产物连接到pEASY-T1载体上,转化大肠杆菌DH5α感受态细胞中,筛选阳性克隆子,进行测序。
本发明的Nicotinamide adenine dinucleotide dehydrogenase(ND)基因开放阅读框架全长573bp,编码190个氨基酸残基,氨基酸序列如SEQ ID NO.1所示,分子质量为22.4kDa,理论等电点为9.35。
实施例2饲喂ND基因的dsRNA后基因的沉默效率及其对二化螟生长发育的影响
2.1制备dsRNA模板
根据实施例1中所获得的Nicotinamide adenine dinucleotide dehydrogenase(ND)基因序列,设计特异性扩增引物(5'-端加上适当的酶切位点),用于Nicotinamideadenine dinucleotide dehydrogenase(ND)基因的dsRNA片段的扩增,设计的特异性引物如下:
上游引物序列ds ND-F:tgGAATTCCAACGTGTTCATTGGGCCTG
下游引物序列ds ND-R:tgGGTACCTTTTGATCAGAGCATGCAAGTT。
注:划线部分系列为EcoR I酶切位点
利用上述引物ds ND-F和ds ND-R进行PCR扩增,扩增完毕后,用1%的琼脂糖凝胶电泳鉴定后,切胶并利用DNA凝胶回收试剂盒纯化回收目的片段。最后通过TA克隆将其连接到pEASY-T1载体上。
2.2构建表达载体
利用AxyPrep Plasmid Miniprep kit试剂盒提取含有目的片段的质粒并酶切。酶切的目的片段与pET-2P载体连接。
重组载体转化入感受态细胞HT115的过程:将HT115从-80℃超低温冰箱中取出至于冰上融化5min,然后将连接产物用移液枪加入到HT115感受态细胞中,轻轻吸打混匀,冰上放置30min,然后于42℃水浴热激1min,再于冰上复苏2-3min,加入已灭菌的LB液体培养基至1ml,然后于37℃、150rpm摇床培养1h。取200μl菌液均匀涂布在卡那霉素抗性的LB培养基平板上,37℃倒置过夜培养。隔天挑取圆润饱满的单菌落,在卡那霉素抗性的LB液体培养基中扩大培养,将新鲜菌液于-80℃保存在30%已灭菌的甘油中,备用。
2.3饲喂ND基因
取100ml大肠杆菌中表达好的菌液,5000rpm离心5min,弃上清,用5ml(20:1)ddH2O悬浮菌液沉淀,4℃冰箱保存待用或直接用于饲喂实验。
提前两天用脱脂棉加水浸泡水稻种子MH63,饲喂实验时种子发芽。
用浓缩后的菌液均匀涂抹水稻种子,将三粒MH63水稻种子置于U形管中并保持环境湿润,取40头二化螟初孵幼虫接种于每个U形管中,饲喂二化螟幼虫72h。每个饲喂实验设置5个重复,分别用ddH2O、dsRNA(GFP、ND)饲喂。
饲喂72h后,取一部分干扰的幼虫收集作为定量样品来检测干扰效率,每个处理3个重复,每个重复15头幼虫。
7天后统计虫子的死亡率,并记录实验结果。
如图1所示,qRT-PCR检测结果显示,与对照组相比,饲喂ND基因的dsRNA显著抑制了ND在二化螟体内的表达。由此可见,该二化螟ND基因的干扰序列可显著抑制Nicotinamide adenine dinucleotide dehydrogenase(ND)基因的表达。
观察饲喂处理7天后二化螟生长情况,统计死亡率,如图2所示,与对照组H2O相比,饲喂ND基因dsRNA处理组的二化螟死亡率提高了35%;与对照组基因EGFP dsRNA相比,饲喂ND基因dsRNA处理组的二化螟死亡率提高了24.2%。因此,本发明提供的RNA干扰序列可应用于转基因抗二化螟植物的开发。
序列表
<110> 华中农业大学
<120> 二化螟生长发育相关蛋白ND及其编码基因、dsRNA干扰序列和应用
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cagcaggagt atgaaaaatt catgcacttc attgatgaag aagcccaaag gggtaaacta 420
agagctctgg agaaggcaat tatagaaaag atgggagaac gcagcgacta tcaagcatat 480
tattatcgac caatggttaa caaatacctc cgtttggata agaaggccgg cgatgaactt 540
tataatcgcc ttggtgagaa tttagatgaa taa 573
Claims (3)
1.一种提高二化螟死亡率的方法,其特征在于,所述方法包括将二化螟生长发育相关基因ND的dsRNA干扰序列饲喂二化螟的步骤,
所述二化螟生长发育相关基因ND编码氨基酸序列如SEQ ID No.1所示的蛋白。
2.根据权利要求1所述的提高二化螟死亡率的方法,其特征在于,所述dsRNA干扰序列通过以下特异性引物序列扩增编码氨基酸序列如SEQ ID No.1所示的蛋白质的基因得到:
上游引物ds ND -F:tgGAATTCCAACGTGTTCATTGGGCCTG;
下游引物ds ND -R:tgGGTACCTTTTGATCAGAGCATGCAAGTT。
3.根据权利要求1所述的提高二化螟死亡率的方法,其特征在于,所述二化螟生长发育相关基因ND的核苷酸序列如SEQ ID NO.2所示。
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