| Apple (Malus x domestica) has become one of the largest fruit crops in the world, also ithas become a model plant for study of commercial traits such as disease and pest resistance inthe fruit crops. Marssonina coronaria causes apple blotches. It is a widely-spread applefungus disease in the North America, Oceania and Asia, leading to a heavy loss in the appleindustry, especially in Eastern Asian countries including China and Japan, since it can lead tosevere defoliation. Research on the interactions between apple and M. coronaria is veryimportant to further studies of resistance mechanisms of apple to M. coronaria and diseaseresistance breeding. In this study, a suppression subtractive hybridization (SSH) cDNAlibrary was constructed with 'Qinguan' apple leaves induced by M. coronaria. Andexpression sequence tags (ESTs) were analyzed characters of the expression of defencerelating genes. These are major results from this study:1. Total RNA of inoculated and inoculated leaves of different periods of 'Qinguan' applehighly resistant to M. coronaria were extracted with modified SDS/phenol method, andmRNA were isolated successfully. Leaves were sampled at3,6,12,24,48,72, and96hourspost-inoculation (hpi) as the Tester, and the leaves treated with sterile water were used ascontrol materials (Driver). Suppression subtractive hybridization library was constructedsuccessfully with the high resistance to M. coronaria of 'Qinguan' apple leaves.2.2349clones were selected randomly from the SSH library and then sequenced,175high quality ESTs were obtained after removing pollution,redundant,low quality(﹤100bp)sequences. These ESTs were successfully deposited in GenBank, and their accession numbers(JK263619, JK263620, JK263621, JK263622, JK263623, JK263624, JK263625, JK263626,JK263627, JK263628, JK263629, JK263630, JK263631, JK263632, JK263633, JK263634,JK263635, JK263636, JK263637, JK263638, JK263639, JK263640, JK263641, JK263642,JK263643, JK263644, JK263645, JK263646, JK263647, JK263648, JK263649, JK263650,JK263651, JK263652, JK263653, JK263654, JK263655, JK263656, JK263657, JK263658,JK263659, JK263660, JK263661, JK263662, JK263663, JK263664, JK263665, JK263666,JK263667, JK263668, JK263669, JK263670, JK263671, JK263672, JK263673, JK263674,JK263675, JK263676, JK263677, JK263678, JK263679, JK263680, JK263681, JK263682, JK263683, JK263684, JK263685, JK263686, JK263687, JK263688, JK263689, JK263690,JK263691, JK263692, JK263693, JK263694, JK263695, JK263696, JK263697, JK263698,JK263699, JK263700, JK263701, JK263702, JK263703, JK263704, JK263705, JK263706,JK263707, JK263708, JK263709, JK263710, JK263711, JK263712, JK263713, JK263714,JK263715, JK263716, JK263717, JK263718, JK263719, JK263720, JK263721, JK263722,JK263723, JK263724, JK263725, JK263726, JK263727, JK263728, JK263729, JK263730,JK263731, JK263732, JK263733, JK263734, JK263735, JK263736, JK263737, JK263738,JK263739, JK263740, JK263741, JK263742, JK263743, JK263744, JK263745, JK263746,JK263747, JK263748, JK263749, JK263750, JK263751, JK263752, JK263753, JK263754,JK263755, JK263756, JK263757, JK263758, JK263759, JK263760, JK263761, JK263762,JK263763, JK263764, JK263765, JK263766, JK263767, JK263768, JK263769, JK263770,JK263771, JK263772, JK263773, JK263774, JK263775, JK263776, JK263777, JK263778,JK263779, JK263780, JK263781, JK263782, JK263783, JK263784, JK263785, JK263786,JK263787, JK263788, JK263789, JK263790, JK263791, JK263792and JK263793) havebeen released., All the sequences were checked for homologies using the BLASTalgorithms based on the NCBI database.Those best matched protein were from many plantspecies, such as Malus domestica, Vitis vinifera, Prunus persica, Pyrus pyrifolia, Arabidopsisthaliana, Ricinus communis, Populus trichocarpa.3. Based on BLAST analysis, functions of these175ESTs were classified into tencategories: defense/stress functions (28%). protein metabolism (15%), photosynthesis (11%),secondary metabolism (10%), signal transduction (8%), transporters (7%), energy metabolism(7%) and cell growth/division (7%), transcription (6%), and unknown function (1%). Itindicated that many cell metabolic process and response to stress process involved in plantdefense process.4. Quantitative Real Time-PCR (qRT-PCR) was used to characterize the relativeexpression of catalase (CAT), hypersensitive-induced response protein (HIR), calmodulin(CaM) and distinctive expression patterns of three genes were found in 'Qinguan' and 'Fuji'hitted by spores of M. coronaria. Expression levels of CAT gene was down-regulated at3hpiin 'Qinguan' apple, then up-regulated and reached the peak at72hpi, in contrast, catalaseup-regulated at3and72hpi in 'Fuji' apple, but down-regulated at6to48hpi. Expression ofHIR was up-regulated from24hpi, and reached a peak at72hpi in 'Qinguan', however, itsresponse postponed at48hpi in 'Fuji', and lasted less than24hrs. Levels of calmodulin (CaM)gene expression with insignificantly changes in 'Qinguan',however, the activity of the CaMgradually increased after3hpi in 'Fuji', and peaked at24hpi.5. we also found there were common genes in apples in response to the fungal diseases between our work and other study in response to challenge by V. inaequalis, includingchitinase, thaumatin, cysteine protease inhibitor, proline-rich protein.In conclusion, the interaction between the apple plant host and the M. coronariapathogens is a complex process. This process may be regulated via interactions that involvedmultiple genes. |
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