Construction Of CDNA Library And Analysis Of Expressed Sequence Tags(ESTs) From Puccinia Striiformis F SP. Tritici

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici, is considered a major disease in China. The current strategy for management of wheat stripe rust is use of resistant cultivars. However, the resistance of cultivars were soon broken because of pathogen virulence variance. Cereal rusts have been researched for over a 100 years on the epidemiological, population and cell biological level because of their economic importance world-wide. However, molecular genetic analyses of these rusts have been difficult because of their obligate biotrophic nature and lack of transformation systems. In recent years, with the development of molecular genetics and molecular biology, more and more approaches were currently applied to the study of fungal functional gene during fungi interaction with host.The key stage of rust fungus infection host is the urediospores germination. In this study, optimal germination condition of urediospores of Puccinia striiformis was explored by incubation in water. Germinated urediospores were collected for construction of cDNA library by SMART thechnique. Study on expression profile of germinated urediospores was carried out by EST analysis, SqRT-PCR and bioinformatics. The result were obtained as follows:1. The urediospores of CY32 race were used to explore optimal condition of germination with incubation in water in a large scale. The results demonstrated that the optimal temperature is 9℃. The optimal inoculation dose is 6mg/200ml water. Sterilized distilled water is more beneficial than tap water to germinate.2. The nonnomalized cDNA library was constructed by SMART thechnique. The titer of the unamplfied library is 1.1×106 pfu/mL. The percentage of recombinant clones is 87.5 %. Indibidual clones of the library are 5.775×105. The average sequence length of inserts is 750bp. The qulity of the library meets all the conditions of standard library.3. sequencing randomly selected 6000 clones of the library from the 5'end resulted in the acquisition of 4798 sequences. 4798 sequences were merged into 315 contigs and 803 singletons, that is 1118 uniseqences. 451 uniseqs exhibited significant homology to genes in...