| Barley yellow dwarf virus (BYDV) is one of the most serious diseases of cereals worldwide. Isolation and characterization of resistance related genes to BYDV are of great significance.In this thesis a transformation-competent artificial chromosome (TAG) library was constructed from the genomic DNA of wheat-Th. intermidium translocation line HW642 harboring the BYDV resistance gene derived from Th. intermidium. The library consists of 2.3 X 106 clones with an average insert size of 22kb, representing approximately 2.5 haploid genome equivalents and single-copy DNA sequence is able to be isolated from it with a probability greater than 95.77%. The library was stored as frozen cultures in 24 96-well formats, each well containing approximately 1000 different clones. TAG clones containing interest gene could be identified by the pooled PCR technique. The TAG library is a useful resource for isolating genes.A cDNA library of HW642 was constructed using mRNA isolated from wheat seedling leaf infected with aphids carrying barley yellow dwarf virus (BYDV) for 48hrs. The liter of the cDNA library is 1.5X106pfu/ml. 103 randomly selected recombinant clones were digested with EcoR. I and Xho I to evaluate the cDNA library. The insert size of the cDNA library ranges from 150 to 4000bp with an average of 2000bp and the recombinant rate of the cDNA library is 95.15%.A sequence characterized amplified region (SCAR) marker SC-W37, which was converted from a simple sequence repeat (SSR) or microsatellite marker wms37 of 7DL arm of wheat and cosegregated with the BYDV resistance gene, was used to screen the TAG library. 12 positive clones were successfully selected by the pooled PCR method and their PCR products were identified by hybridizing with the SCAR marker band of Th. intermidium. The positive clones restricted by HindIII were hybridized with genomic DNA of Th. intermidium and Chinese spring, respectively. Only 2 of them showed difference in signal intensity. No obvious polymorphism was detected between Th. intermidium, HW642 and its susceptible parent Zhong8601 in RFLP analysis with 5 restriction enzymes.The SCAR marker band of Th.intermidium was also applied to the screening of HW642 cDNA library. Two positive cDNA clones were obtained. One contained a complete open reading frame (ORP) of 822bp encoding 273 amino acids. Blast search was performed against databases in NCBI and EMBL. This ORF showed high homology to prohibitin gene family bearing 82.28% identity with plant prohibitins, 90.07% identity with yeast prohibitins and 82.27% identity with human and rat prohibitin. It was considered as a prohibitin gene candidate in wheat and named w-ph. The results of Southern and Northern hybridization indicated that the sequence of w-ph derived from wheat and was single copy in the genome with low but constitutive expression. Two primers annealed to 5' and 3' ends of w-ph were used to amplify the genomic DNA of HW642, two collections of Thinopyrum intermedium from different origin and the susceptible parent Zhong8601. 2-5 bands were obtained. One band with a similar size (approximately 820bp) as the positive control was detected in HW642 by PCR-Southera hybridization using w-ph as probe, indicating that w-ph had no intron. The existence of homologous sequence to w-ph in Thinopyrum intermedium and Zhong8601 was also revealed by hybridizing w-ph with the 1.8kb band in their amplified DNA. The other positive cDNA clone was a partially coding frame homologous to abhydrolase2 with a length of 1050bp and named w-abh. It was low copy sequence in the wheat genome and expressed constitutively after BYDV infection and chilling exposure. As part of a gene w-abh was lack of intron too.Another two positive cDNA clones, Stl-7 and Stl-22, were found when screening the HW642 cDNA library using a RGA sequence ST1 as probe. They were 1095bp and 1221bp in lenghth, encoding 281 and 305 amino acids, respectively, and were separately homologous to phytochelatin synthetase and purple acid phosphatase.
|
PDF Full Text Request