【作者】 吴蓓蕾；

【导师】 王锡锋；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2007， 博士

【摘要】 小麦矮缩病毒（Wheat dwarf virus，WDV）是联体病毒科玉米线条病毒属的成员，属于第1亚组联体病毒。寄主包括小麦、大麦、燕麦和多种杂草，引起植株的严重矮缩、黄化、条斑和分蘖增多等症状，由条沙叶蝉（Psammotettix striatus L．）以持久性增殖方式传播。全基因组由2739-2750个核苷酸组成，编码4个蛋白，包括运动蛋白（MP／V1）、外壳蛋白（CP／V2）、两个复制酶相关蛋白酶（Rep A和Rep）及两个基因间隔区（LIR和sIR）。目前已公布了19个WDV分离物的序列，其中8个全序列，其余11个为部分序列，多集中在LIR-RepA-MP区间。序列分析发现小麦和大麦上的WDV基因组存在明显差异，全基因组同源性仅69.8％左右。2007年本实验室首次报道了WDV在中国的发生。本研究完成了采集自我国不同地区28个WDV小麦分离物的全序列测定。通过对中国小麦分离物与非中国小麦分离物群体遗传结构的分析，发现中国是侵染小麦的WDV起源地之一，石家庄分离物（HBSJZ06-7）是目前在遗传距离中进化最为古老WDV。依据进化的距离分析推测了WDV小麦分离物进化和出现顺序。结合34个WDV小麦分离物（29个中国分离物，5个非中国分离物）群体遗传多样性和选择压力下的进化模式分析，发现目前的WDV群体结构还处在净化选择状态，基因在进化的过程中比较保守，其中MP在进化上最保守，基本符合DNA病毒进化保守的的特点。通过对WDV编码区和非编码区种间单模标样的多样性、核苷酸的多样性和负的Tajima’D值分析发现WDV群体正在扩张。对29个WDV中国分离物基因组的重组分析，发现属于同一个进化分支，拥有不同进化时间的三个分离物，YNKM06-2、GSGG05-2和HBSJZ06-11（以进化早晚顺序排列），发生了明显重组。分析结果显示，进化古老的YNKM06-2与GSGG05-2的MP基囚区域核酸序列的相似性高，而与后进化的HBSJZ06-11核酸序列的相似性低，这符合WDV的MP基因进化保守的规律，与遗传多样性分析的结果相符。另外在重组分析中发现，后期进化分离物（GSGG05-2和HBSJZ06-11）的CP基因、Rep A基因、基因间隔区（SIR、LIR）、以及非编码区（Intron）等区域相似性高，而这些区域都是在群体遗传多样性分析中多样性好的区段，也就是突变位点多的区段，这些位点的高突变率对于WDV适应小麦栽培制度与传播、地理环境变化以及寄主与介体多样性起着重要作用，也体现了联体病毒的进化过程是不同组份的病毒、不同植物寄主及相应介体三者间独特的进化机制作用下的共进化过程。大麦黄矮病毒（Barley yellow dwarf viruses，BYDVs）是一类+ssRNA病毒，分布广泛，是世界上危害最严重、流行最广泛的植物病毒之一。RNAi由dsRNA介导的干涉现象，作为细胞的一种防御机制可针对性地降解细胞内与之具有同源性的核酸如转座子、外源病原体（如病毒）核酸，同时还调节细胞内编码基因的表达。本研究依此原理设计了BYDV-GAV外壳蛋白（CP）介导的RNA干涉的dsRNA（600bp）前体，构建了适合禾本科植物转化的干涉载体，利用基因枪方法进行了小麦遗传转化，期望利用RNA干涉机制获得高抗BYDV-GAV的转基因小麦植株，探索利用分子生物学技术获得抗BYDV的基因工程新策略。通过基因枪的转化，实现了四个载体：pSA、pS、pA、pMCG161对小麦品种扬麦158的遗传转化，PCR鉴定分别获得14株、4株、3株、3株TO代阳性转基因小麦。TO代的阳性转化率是分别是0.46％、0.15％、0.12％、0.11％。其中干涉载体的转化率最高（0.46％），获得了14株TO代转基因小麦。对于TO代干涉载体的转基因小麦植株的发夹结构进行了鉴定，发现Intron左边界发生了部分片段的缺失，基因枪轰击转化植物细胞时，不仅会使受体植物细胞内染色体的断裂，也会使转基因片段断裂缺失。对T1、T2代转基因小麦分别进行了PCR鉴定。发现T1代阳性率在45-50％之间，不遵循孟德尔遗传定律。T2代是平均是30％左右，遵循孟德尔遗传定律。接种试验完成后获得了抗BYDV-GAV干涉载体的转基因株系pSA-6₀、9₀、11₀、12₀，对照载体的转基因株系不具有抗病性。通过T3代干涉载体转基因小麦的田间抗病性鉴定的得到了10％的抗病性植株。在T1和T2代转基因植物中发现体细胞克隆变异的现象，如矮缩、不抽穗、分蘖增多等，这是外源基因的整合干扰了受体生物体基因组中其它基因的表达，引起了植物表型变化的结果。更多还原

【Abstract】 Wheat dwarf virus （WDV）, which is transmitted by leafhopper （Psammotettixstriatus L.）, is a member of the genus Mastrevirus, Geminiviridae. Its host rangeincludes wheat, barley, oats and many cereal grasses. Typical symptoms of the diseaseare dwarfing, mottling, yellowing and reduced heading in wheat and barley. WDVgenome （2739-2750nt in length） was a circular single-standed （ss）-DNA, containingfour ORFs and two intergenic regions （LIR and SIR）, which encoded four differentproteins: movement protein （MP/V1）, coat protein （CP/V2） and tworeplication-associated proteins （Rep and RepA）. Full genomic sequences of the 19isolates have been published on GenBank. Among them, only eight are full genomicsequences, others are partial sequences, mainly in LIR-Rep A-MP regions. Theidentities among the sequences from wheat and barley were about 69.8%. It issuggested that the WDV isolates formed two distinct clades.In this research, the complete genomic sequences of 28 wheat isolates collectedfrom China were sequenced. Though the analysis of phylogenetic trees on WDV byNeighbor-joining （NJ）, the oldest isolate （HBSJZ06-7） was found from Shijiazhuang,Hebei province. The route of transmission of WDV in the world was proposed basedevolution distances. The results of the intraspecific polymorphism between thepopulation （including China and non-China） suggested the population of WDV isexpanding. Among the genome of WDV, diversity is fluctuant with stable evolutionof MP gene sequence.The recombination among the isolates was detected by SiScan soft.Recombination was found among YNKM06-2, GSGG05-2 and HBSJZ06-11 isolates.From the regions of recombination, the characteristics of evolution on WDV sequencewere obtained; the regions on the CP, RepA, SIR, LIR and Intron were connected withthe co-evolution by changing the nucleotide site to adapte the cheng of environmentand hosts.Barley yellow dwarf viruses （BYDVs）, transmitted naturally by at least 25 aphidspecies in a highly specific, circulative, non-propagative manner, are a kind of+ssRNA viruses. BYDV-GAV is a Chinese isolate which caused severely yellowingand dwarfing on wheat, barley and oat. RNAi is an important gene silencingphenomenon, in which experimentally introduced double-stranded RNA （dsRNA）leads to loss of expression of the corresponding cellular gene by sequence-specialRNA degradation. Almost all transformation mediated by RNA virus or mRNA couldlead the resistance of plant to virus, according to the mechanism of RNAi.The coat protein （CP） gene of BYDV-GAV was obtained by reverse transcription polymerase chain reaction （RT-PCR）. The interference vector pSA wasconstructed using the special interference vector pMCG161 for transformation ofgraminaceous plants. At the same time, three control vectors were also designed,namely pS, pA and pMCG161. The plants of T0, T1, T2 and T3 generations of the eachvector were obtained using biolistic particle method for the transformation on wheat.By PCR analysis in TO generation, 14 transgenic plants of pSA and 4, 3 and 3transgenic plants of pS, pA and pMCG161 respectively. The high transgenic rate ofthepSAwas 0.46％, the other vector transgenic rate were 0.15％, 0.12％and 0.11％,respectively. The transgenic rate of T1 and T2 generation were 45-50％and 30％respectively. In the detection of hairpin in TO generation, the loss of left borderregion was found. Though the inoculation, the transgenic wheat with resistance toBYDV-GAV in four TO generation lines of pSA-6₀, -9₀, -11₀ and -12₀ were found inT2 generation, and the rate of the resistance is 10％. The occurrence of somato-clonalvariation was found in the generation of the transgenic wheat.更多还原