Isolation And Functional Analysis Of Early Response Genes In BYDV Resistance In Wheat (Triticum Aestivum)

Wheat yellow dwarf is one of the most serious virus diseases caused by barley yellow dwarf virus, which was spreaded by aphids. In resent years, the disease has been more serious and wider in our country because the aphid becomes harmful serious. Thinopyrum intermedium's resistances to BYDV have been introduced into wheat through biotechnology and distant crossing methods by different groups. Although some Wheat-Thinopyrum intermedium translocation Lines with BYDV resistance, for example HW642, have been developed and identified, it is difficult to select a superior cultivar with both high yield and resistance, because there are linkage drags derived from the alien fragment in those translocation lines. It is necessary to isolate BYDV resistance genes and some important resistance signal regulation components for unraveling BYDV resistance mechanism and solving above-mentioned problems. Results about resistance mechanisms of model plants show that some early response genes against diseases are important regulation components on resistance signal pathways. So we isolated and identified some wheat early response genes against BYDV in this study.This study made the following progresses:1. A cDNA library of a wheat line HW642 with BYDV resistance was constructed. HW642 mRNA was isolated from seedling leaves infected with aphids carrying barley yellow dwarf virus GAV strain(BYDV-GAV) for 6hrs. The titer of the cDNA library is 6×10~5pfu/ml. The recombinant rate of the cDNA library is 95%. The insert size of the cDNA library ranges from 250bp to 6kb with an average of 2kb.The quality of the cDNA library should meet the demands of screening for low abundance genes. The cDNA library laid a foundation of further screening for BYDV resistance genes and early response genes.2. A new early response gene in BYDV resistance mediate by Bdv2 was isolated, which encode a transmembrane protein with a'DUF543' domain. Fifty-five positive cDNA clones were isolated from HW642 cDNA library by hybridization in situ using TAC1 insert fragment as probe. TAC1 is a candidate clone for BYDV resistance gene. A early response gene in BYDV resistance, temporary named Triticum aestivum BYDV respond gene 1(TaBR1), was isolated from those positive cDNA clones, which shows no obvious homology to cloned resistance relative genes.3. The transcript patterns, distribution and gene structure of TaBR1 was characterized. There is obvious difference in TaBR1 expression patterns in the interactions between the resistant HW642 and a susceptible parent Zhong8601 after infected by BYDV. TaBR1 expression can only be induced quickly in HW642 after BYDV infection, whereas TaBR1 does not shows any response in Zhong8601 after BYDV infection.The results suggested that TaBR1 is an early response gene in BYDV resistance mediate by Bdv2.Two TaBR1 homologous was isolated from wheat and Thinopyrum intermedium, temporary named TaBR1a and TaBR1b.Their cDNA sequences shared homology with 95.45% identity. TaBR1 DNA sequence is 3.47kb with two introns, in which one is 1.9kb and another is 760bp. TaBR1a and TaBR1b has obvious difference in intron sequence, but the intron's positions are identical. There are 3 copies of TaBR1 in genomes of Thinopyrum intermedium, wheat lines Zhong8601 and CS. However, HW642 has 4 copies of TaBR1, in which 3 copies shoulde derive from its wheat parent but 1 copy shoulde derive from Thinopyrum intermedium.4. The cDNA sequences of SGT1 containing full-length open—reading-frame was isolated from wheat and Thinopyrum intermedium, and the expression patterns were chacterized for the frist time. The expression character and overexpression in wheat of SGT1 was analyzed in this study. SGT1 cDNA with full length ORF was isolated from wheat and Thinopyrum intermedium, temporary named TaSGT1 and TiSGT1.Their amino acids sequence showed homology for 97.79% identity. There are 3 copies SGT1 in wheat and Thinopyrum intermedium genome. The expression of SGT1 can be induced on different level in 4 kinds of wheat disease resistance response, including wheat yellow dwarf, powdery mildew, fusarium head blight and sharp eyespot. The expression of SGT1 can be induced sharply and early in HW642 after infected by BYDV, so SGT1 maybe an early response gene in BYDV resistance mediate by Bdv2.5. Overexpression of SGT1 enhancing wheat resistance to BYDV and powdery mildew was elucidated for first time. TiSGT1 was transformed into a wheat variety Yangmai 12 by biolistic particle method. After the transgenic plants was detected and screened by molecular analyses, including PCR Southern and RT-PCR analyses, their resistances of BYDV and powdery mildew were investigated. The results showed that the overexpression of TiSGT1 enhanced the wheat resistance to BYDV and powdery mildew. Therefore, SGT1 could be used as a potential gene for improving broad resistance spectrum in wheat breeding programs.