| Ramie (Boehmeria nivea L.), a perennial crop of bast fiber, also known as white leaf ramie belongs to Urticaceae Boehmeria.. Ramie anthracnose (Colletotrichum spp.) is one of the common and serious diseases occurred on ramie, which is caused a general incidence rate of 20%-40%. In the serious occurred period, the diseased leaves rate and disease index were reached 90% and 55.0%, respectively, causing 10% fiber loss. There are few researches on ramie anthracnose especially in abroad. In China the related reports the disease are mostly concentrated in 1980s and 1990s. It is of great significance on the analysis of biological characteristics of ramie anthracnose and the pathogen defense mechanism, for creating a broad-spectrum resistance to anthracnose and the clone of resistance genes from ramie.The main contents of this study are as follows:Identification, characterization of ramie anthracnose and analysis of polymorphism; build induced subtractive library of expression of pathogen-specific cDNA fragments using suppression subtractive hybridization (SSH) technology. Ramie disease-related expression and gene fragments enhanced the expression of specific were screened out from the SSH cDNA library; through GenBank database searches, access to different expression of fragments of possible biological information. Finally, these fragments for further analysis and suggested the role in disease resistance and the relationship with the disease. Understand the level of resistance to anthracnose ramie involved in signal transduction, disease-related gene expression in the type and quantity. The level of initial establishment of disease resistance to anthracnose Ramie related gene expression profiles; for subsequent cloning, functional studies and disease-depth study of the mechanism of the establishment of technology platforms.We obtained the main outcomes as follows:1. In our research, we collected the pathogen-infected tissues from several main producing regions of ramie (Wuhan and Xianning of Hubei province, Changsha of Hunan province, Yichun of Jiangxi province and Dazhou of Sichuan province), and isolated 89 ramie anthracnose strains (of which 20 strains from Wuhan,24 from Xianning,18 from Jiangxi,21 from Changsha and 6 from Dazhou) from these tissues. These strains were identified basing on morphological and pathogenicity test as well as analyses of a partial sequence of the ITS,β-tubulin gene and Actin gene. Based on colony, conidial and appressoria morphology, and the molecular phylogenetics, C. fructicola, C. siamense, C gloeosporioides and C. higginsianum were identified as the causal agents of ramie anthracnose.2. RAPD clustering analyses among 45 strains from the main producing regions of ramie were performed using UPGMA average clustering method, the results showed abundant genetic divercity among these strains with no surprise, and at the identity level of 60%, the 45 strains were separated into two classes:the first contained WH-4, XN2-1 and XN2-4, and the second of WH-1, WH-9, WH-A, WH-B, WH-C, WH-D, WH-H, WH-J, JB-1, XN1-1, XN1-6, XN2-1, XN2-4, XN3-3, XN3-4, XN4-1, XN5, JX-1, JX-4, JX-6, JX-12, JX-14, JX-15, JX-16, JX-17, JX-18, JX-19, DZ-2, DZ-3, DZ-4, DZ-5, DZ-6, HN-1, HN-2, HN-3, HN-7, HN-8, HN-9, HN-12 and HN-13.3. We studied and measured the biological characteristics of a typical strain named HN-3 (C. gloeosporioides). The related results were as follows:the mycelium grew well from 20 to 30℃, best at 25℃; the yield of conidiophores came to a higher value also at the temperature from 20 to 30℃, best at 28℃. When under different pH values, the mycelium grew at pH 4.0-10.0; and different media, faster growth rates were obtained on PDA, PSA and ramie leaves (RLs) media, while most conidiophores on beef extract media (BE). Otherwise, when cultured on different media to measure its utilization ratio of different carbon and nitrogen sources, it showed high utilization ratios of every carbon sources and best of maltose, but it was not that similar if turned to sporulation. In short, we got widely different yield of conidia under the combinations of different carbon sources with one nitrogen source, e.g., a higher yield of conidia could be obtained on the medium combined of potassium nitrate (KNO3) and D-glucose; diameters on calcium nitrate [Ca(NO3)2] was high; Diameter on C-yeast was only 52.8mm, but its yield of conidia was highest by 3.16×107/ml. When adding VB2, its yield of conidia and the dry biomass of mycelia were significant higher than CK. We got a fastest growth rate in L/D condition (or, the 12-h/12-h condition) with a lowest yield of conidia, while in all dark condition, the conidia got a larger number.4. We constructed a subtractive library that abundant of anthracnose-resistant interrelated gene sequences by using suppression subtractive hybridization (SSH), and the reverse Northern dot-blot method was also performed for further screening to this library. As a result of it,132 EST fragments that representative of different genes were identified by sequencing and combination the screening differential expression clones. After that, we compared these sequences in NCBI, and the outcome showed that among the matched ESTs, the higher scores mostly came from that of Populus L., Prunus, Ricinus communis and Vitis vinifera, so had Glycine max and Vinca rosea, etc., but no that from ramie. Among these ESTs,37 of them (28%) were classified as disease-resistant and defense-related; 23 (17%) for unclassified; 15 (11%) for unclear classification; 10 (8%) for transcription; 7 (5%) for both transporter and metabolism; 6 (4%) for each function of cell growth/division, protein synthesis and transposons; and some EST ralated to signal transduction, energy, protein destination and storage, cell structure, secondary metabolism eg. In these ESTs, some ESTs from Poplar cDNA sequences (SSH library infected with rust fungus), metallothionein-like protein, heat shock protein, PR-1.2 protein, pathogenesis related protein.
|
PDF Full Text Request