Special Origin Of PVY CP Influences The Resistance Of Hairpin RNA And Artificial MiRNA Expressing Plants Against PVY

RNA silencing is a host defence against invasive nucleic acid, such as viruses or transposable retro elements, preserving the integrality of biology. Small interfering and microRNA which is the most important parts of RNA silencing system are the inducing factor of post transcript gene silencing. RNA mediated virus resistance (RMVR) is regarded as an effective strategy in plant resistance against viruses. The expression of virus specific dsRNA which has homology to the invasive virus will active the post transcriptional gene silencing response of the host plant. The RNA silencing system will recognize complementary invasive viral RNAs and triggers degradation of both the transgene RNA and the corresponding viral RNA. RMVR has been widely used in plant antiviral research for its advantage, such as nearly immunity; sustaining resistance; no biosafety problem. In the application of gene silencing, the depending of the postion of target sequence has been found. There is no systematically study in the RNA mediated virus resistance. In our studies, we designed hairpin RNA constructs and artificial miRNA consructs based on different regions of the CP gene of PVY and generated transgenic plants. We aim at disserting if these kinds of tansgene construction could make transgenic plant exhibit different resistant ratio. The result will contribute to the selection of the target sequence and successfully apply the strategy of RMVR, which has an important theory and application value. The main results and conclusions presented in this thesis are as follows:(1) Different origins of PVY CP gene influence the resistance of hairpin expressing plants against PVYThe PVY CP gene was divided into 16 regions. For every region, two PCR fragments were amplified from the whole length of the CP gene. Two PCR fragments were inverted inserted into binary vector pROKⅡ. The ligated products were transferred into E.coli DH5αby heat shock. The plant expression vectors obtained were dubbed pROK CPs (pROK CP9~pROK CP16).The 16 hpRNA constructs (the other 8 hpRNA expression vectors were constructed by Wu Bin) were transferred into N. benthamiana through Agrobacterium tumfaciens mediated transient infection. Northern blot analysis revealed siRNAs were detected in all pROK CPs in?ltrated leaves in transient assay. And all 16 biologically active siRNAs that could effectively down regulate the expression of target mRNA.The 16 hpRNA constructs were introduced into NC89 mediated by LBA4404. T1 50, 62, 56, 55, 45, 63, 72, 55 transgenic lines exhibiting kanamycin resistance and positive results in PCR tests for each vector were regenerated after T0 selfing. Virus resistance assay revealed that the 16 transgenic tobacco groups exhibited varying degrees of virus resistance in preventing PVY infection. All the transgenic plants of pROK CP1, pROK CP2, pROK CP3, and pROK CP4 are susceptive to the PVY infection; The resistance ratios of pROK CP5~ pROK CP16 are 27.15%, 6.27%, 67.65%, 70.83%, 66.01%, 61.34%, 66.04%, 23.59%, 11.11%, 55.56%, 77.78% and 67.25%. We demonstrate that 50bp length hpRNA construct is effective for RMVR; however, the hairpin constructs harboring different cDNA regions of the CP gene engendered different silencing efficiencies, the hpRNA targeting the 3′end (nt701~nt750) region exhibited the most highly virus resistance. The plants which harboring the hpRNAi constructs targeting 3′of PVY CP can induced higher virus resistance than those targeting 5′of PVY CP.Northern blot analysis revealed that the trangene have been expressed in level of RNA, and the transcripts accumulation level of resistant plants was lower than that of the susceptible transgenic plants. There was an inverse correlation between the resistance and the amount of RNA accumulation in the transgenic plants. The results proved this resistance was RNA mediated. siRNAs were present in all selected resistant and susceptible transgenic plants; No obvious correlation was observed between the expression level of sequence speciflc siRNAs and virus resistance.Virus assary in the T2 generation showed that most of the progeny transgenic plants of resistant plant still exhibited high resistance. The results indicated that hpRNA mediated virus resistance can be stably inherited in T2 generation.(2) Different origins of PVY CP gene influence the resistance of artificial miRNA expressing plants against PVY We designed eight amiRNAs M1 (nt96~nt115), M2 (nt140~nt159), M3 (nt322~nt341), M4 (nt380~nt399), M5 (nt475~nt494), M6 (nt567~nt586), M7 (nt679~nt698) and M8 (nt735~nt754) derived from PVY Coat Protein RNA sequences. Arabidopsis thaliana miR319a precursor was used as backbone. Through oligonucleotide directed mutagenesis, the natural miR319 and miR319* sequences were replaced with synthetic sequences, each corresponding to a designed amiRNA that targets one region of the PVY CP gene. The generated fragment was digested and inserted into plant binary expression vector pROKⅡto generate pROK amiRcps.The 8 amiRNA constructs were transferred into N. benthamiana. In transient assay, Northern blot analysis revealed amiRNAs were detected in all pROK amiRcps in?ltrated leaves. All plants with amiRNA expression exhibited a significantly decreased CP accumulationThe eight amiRcp expression constructs were introduced into NC89 mediated by LBA4404. After the kanamycin resistance and PCR test, T1 plants 98, 96, 114, 116, 117, 111, 110, 116 were screened. The resistance ratios of amiRcps expressing plants were 37.68%, 50.29%, 53.76%, 37.75%, 29.86%, 17.05%, 26.27%, 64.69%. Virus resistance assay revealed that expression of amiRNA can effective in preventing PVY infection; but not all amiRcps targeting viral CP sequence are equally effective. The amiRcp 8 targeting the 3′end (nt735~nt754) region exhibited high virus resistance.Northern blots showed that the trangene have been expressed in level of RNA. There was an inverse correlation between the resistance and the amount of RNA accumulation in the transgenic plants. The results proved this resistance was RNA mediated. AmiRNAs were present in all selected resistant transgenic plants and there is a positive correlation between virus resistance and expression level of amiRNA.Virus resistance assay in the T2 generation revealed that most of the progeny transgenic plants of resistant plant still exhibited high resistance. It indicated that amiRNA mediated virus resistance can be stably inherited in T2 generation.