Molecular Population Genetics Of Wheat Dwarf Virus (WDV) And Research On Transgenic Wheat Resisitance To BYDV-GAV Mediated By RNA-Interference

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 0.46ï¼…, the other vector transgenic rate were 0.15ï¼…, 0.12ï¼…and 0.11ï¼…,respectively. The transgenic rate of T1 and T2 generation were 45-50ï¼…and 30ï¼…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 10ï¼…. The occurrence of somato-clonalvariation was found in the generation of the transgenic wheat.