【作者】 吴昊；

【导师】 林拥军；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2017， 博士

【摘要】 水稻是重要的粮食作物之一,世界一半以上的人口都以它为主粮。稳定的水稻产量维系着国家粮食安全与社会安定。但是水稻几乎在其生长发育的各个时期都会受到相应害虫的侵害,而二化螟则是其中影响最为严重的害虫之一。近几十年来,BT转基因抗虫技术的应用有效地控制了部分主要害虫对作物的侵害,减少了经济损失,同时减轻了农药对生态环境的破坏。然而我们也应该注意到,由于BT杀虫蛋白的选择压力,在实验室已然出现了 BT抗性个体。尽管科研工作者们已被动地采取了一些措施来控制这些抗性个体的发展,但是我们更应该采取更加积极主动的态度,开发有效控制害虫的新策略。RNAi技术在近二十年来,从发现到认识再到应用,逐渐成为害虫防治领域最有希望能够比肩BT转基因抗虫技术的潜力策略。本研究基于以上初衷,尝试将该策略应用到水稻的害虫防治上。主要结果如下:1.测序并收录水稻8种重要害虫的转录组数据,整理并构建水稻害虫转录组数据库(http://rptdb.hzau.edu.cn),旨在为世界范围内的昆虫功能基因组、昆虫治理等学科科研工作者提供相关数据,以弥补这些遗传信息的缺乏。统计90日内数据库的日均独立访客数为25次。2.选取22条候选片段设计并合成人工dsRNA,体外喂食二化螟并获得有抗虫潜力的片段 6 条(dsCSE、dsGLC、dsACD、dsTAS、dsVAS 和 xse7),加上其他候选片段共37条通过转基因稳定表达的方式进一步验证其抗虫效应,未观察到明显的抗虫效应。3.对miRNA介导的抗虫转基因材料csu-15进行抗二化螟鉴定,短期与全生育期抗虫鉴定均表明该材料可以显著影响二化螟的正常生长发育。我们分离茎秆喂虫7d后的二化螟中肠组织的总RNA进行RNA-seq并完成差异表达分析,共有33个unigenes被显著上/下调,其中一条unigene被miRNA靶位点预测软件预测具有miRNA csu-novel-15的靶标位点。4.为快速注释一个新物种的基因信息,并为后续昆虫基因组测序注释工作,我们开发一套全长cDNAPET文库构建及分析方法,并以水稻品种日本晴叶片RNA构建文库。利用水稻全长cDNA数据库KOME和日本晴基因组注释数据库RGAP 7的比对分析评估该策略的效率。结果表明至少75.1%的非冗余reads能够正确匹配到KOME克隆上,并且对于和谐匹配的reads对,73.3%的TSS的5’端与KOME克隆5’端的距离小于6个碱基。分别93.65%和70.40%的TSSC和PASC能够匹配到RAPG 7注释基因区前后1kb的范围内。均表明本方法具有较高的真实性与准确性。更多还原

【Abstract】 As the staple food for over half of the world population,rice is one of the most important food crops.Stablitiy of rice production maintains the food safety and social peace.During almost the whole growth period of rice,the yield is affected by various pests,and the striped stemborer(SSB,Chilo suppressalis)is one of the most important insect pests.In last decades,the application of BT transgenic crops reduces the economic losses and protects the ecological environment by effectively managing some major pests.It is worthwhile to note that,the BT-resistance pest populations have appeared under the laboratory conditions.Therefore,it is important to explore new strategies for pest controlling.In the last two decades,RNAi technology has gradually become the most promising strategy for pest controlling,that is potentially comparable with BT technology.In this study,we attempted to apply the RNAi strategy for rice pest management.The main results of this study are as follows:1.A comphresive transcriptome database(RPTDB,http://rptdb.hzau.edu.cn)consisting of the transcriptome data of eight main rice pests was organized and constructed.RPTDB aims at providing well-organized information for worldwide researchers who require genetic materials for insect functional research,pest management or others,and thus filled the genetic information lacks.And it has average 25 unique visitors per day in last 90 days.2.Artificial dsRNAs of 22 candidate targets were designed and synthesized,and then tested by the diet-based feeding test.Six dsRNAs(dsCSE,dsGLC,dsACD,dsTAS,dsVAS and xse7)showed application potential in pest management based on the feeding test.A total of 31 dsRNAs expression vectors were constructed and introduced into Japonica rice,ZH11,however none of the transgenic rice showed stable insect resistance.3.We assessed the SSB resistance ability of miRNA mediated transgenic rice line csu-15.Both short-term and consecutive feeding tests indicated that csu-15 exerted influences on growth of SSB.Larvae fed on stemcutting of csu-15 line for 7d,and their total RNAs of midgut issue were isolated for differential expression analysis by RNA-seq.We found 33 significant differential expression unigenes,and one of them was predicted to have a target side of miRNA csu-novel-15.4.A full-length cDNA PET library construction and data analysis pepline was developed for rapid gene annotation of a newly sequenced species and further insects’ genome annotations.Total RNA from the leaf of the rice variety Nipponbare was used for the library construction and the evaluation of the full-length cDNA PET library construction and data analysis pepline.Rice full-length cDNA database KOME and wide-used annotation database RARG 7 were used to evaluate the efficiency of our approach.At least 75.1%non-redundant reads could map to KOME clones,for those concordance mappings,the distances of 73.3%TSSs’ 0-bases with corresponding KOME clones’ 0-base below 6.The results reveal that our method shows high authenticity and accuracy.更多还原