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Transgenic Wheat Expressing Virus-derived Hairpin RNA and pac1 Gene Respectively Showed the Resistance to Barley Yellow Dwarf Virus-GPV

Author: YanFei
Tutor: ChengZhuoMin
School: Chinese Academy of Agricultural Sciences
Course: Plant Pathology
Keywords: Barley yellow dwarf virus Wheat Transgenic hairpin RNA RNA interference pac1 gene Agrobacterium-mediated transformation Particle bombardment Pollen tube path way
CLC: S512.1
Type: PhD thesis
Year: 2006
Downloads: 232
Quote: 2
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Abstract


Resistance derived by virus gene had been applied in the transgenic wheat against Barley yellow dwarf virus (BYDV) research. All transgenic plants obtained by such way showed the reduced and delayed symptoms, however, did not reveal the high level of resistance. Furthermore, this resistance was special only against virus which conferred transgene in transformation. In this paper, we carried out the transformation of wheat research in order to obtain the high level of resistance wheat plants against BYDV and the multi-virus resistance wheat plants.To improve the resistance of transgenic wheat against BYDV, Replicase gene fragment(Rep) and Coat protein gene fragment(CP) of BYDV-GPV were used to construct an expression frame, which could express the composite hairpin RNA with the dsRNA stem of Rep(CP) and the antisense RNA loop of CP(Rep). The expression frame was then inserted into plant expression vector under 35S promoter or Emu promoter control. hpRNA expressed in plant was hoped to induce RNA interference, then interfere the replication of virus genome, and so confer transformed wheat plant the high level resistance to BYDV-GPV. Agrobacterium-mediated transformation (transform the vector with 35S:CP+/Rep-/CP-expression frame), particle bombardment(transform the vector with Emu:Rep+/CP-/Rep- expression frame) and pollen tube path way(transform the vector with Emu:CP+/Rep-/CP- expression frame and Emu:Rep+/CP-/Rep- expression frame respectively) were applied to transfer the vector into plant cell. The results showed as follow. ①75 of 82 G418-resistant plants obtained by Agrobacterium-mediated transformation, were PCR positive lines. In first antivirus test, 59 of 75 lines did not reveal viral symptoms. And 46 of 59 lines were proved containing the transgene in their genomes by Dot blot analysis. Southern blot result suggested transgene consisted in plant as double copy. ELISA result of nptII showed 37 of 46 dot blot positive lines could express the transgene normally. In 37 ELISA positive lines, 14 plants showed the low level of resistance to BYDV-GPV, 12 plants showed the middle level resistance, and 10 plants showed the high level resistance throughout the second antivirus test. ②In particle bombardment transformation, because no marker gene was used, a rapid PCR system though the treated leaves, was carried out to select the putative positive plants at the stage of transferring the regenerated plantlets into root-growing media. Total 64 plants which were positive in rapid PCR analysis were transplanted into soil and alive. No symptoms were observed on 30 of 64 plants in the first antivirus test, among which 21 plants were confirmed by Dot blot analysis consisting the transgene in their genomes. In the second antivirus test, 5 of 21 plants showed the low level of resistance to BYDV-GPV, 8 plants showed the middle level resistance, and 8 plants showed the high level resistance. ③ 367 seeds(transformed with Emu:Rep+/CP-/Rep- vector) and 545 seeds(transformed with Emu: CP+/Rep-/CP- vector) were obtained by pollen tube path way. T1 plants growing in fields were inoculated with virus to detect the resistance phenotype. The result showed that 2 lines (transformed with Emu: CP+/Rep-/CP-) did not reveal the obvious viral symptom after 40 days past inoculation.In transgenic wheat research for multi-virus resistance, pad gene was transformed into wheat by Agrobacterium-medisted transformation. As an RNaselll like protein, pad could degrade the double-strand RNA which would be formed when plant RNA viruses replicate in plant cell. Total 41 plants were obtained with resistance to G418, in which 30 plants showed resistance to BYDV-GPV in the first antivirus test. PCR and Dot blot analysis proved that 27 of these 30 plants contained the transgene in their genomes. However, ELISA and RT-PCR results revealed 25 of 27 positive plants could express the foreign expression frame, and the 2 others could not. Inoculated with the higher dose of virus, 12 plants showed the low level of resistance to BYDV-GPV, 12 plants showed the middle level of resistance to BYDV-GPV, and 1 plant showed the high level of resistance to BYDV-GPV.To solve the problem of multi-ligation reaction needed to construct the hpRNA expression vector, a new expression vector was designed. Construction of hpRNA expression vector now could be finished in only one ligation reaction. To confirm the availability of this vector to induce the RNA silencing, gfp in transgenic tobacco 16c was targeted. Result showed that green fluorescence could not be observed on the leaves infected by Agrobacterium containing this vector which should express the gfp hpRNA. It proved this vector could be used to silence target gene.

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