Construction Of A CDNA Library Of Wangshuibai Induced By DON And Gene Expression Analysis

Fusarium head blight (FHB), also called scab, caused mainly by Fusarium gramearwn, is a worldwide disease of wheat in the warm, semi-humid or humid regions, resulted in yield loss and deterioration of seed quality. In addition, the infected grains were usually contained with trichothecenes toxin, which is harmful to the health of human and animals. Of them, deoxynivalenol (DON) is one of the most harmful toxins. The genetic mechanism of DON resistance of wheat is unclear. In this paper, a new full cDNA library of scab-resistant wheat Wangshuibai induced by DON was constructed and some ESTs from the library were sequenced. Combined with the semi-quantitative RT-PCR and chromosome mapping, this research aimed at revealing the gene expressing patterns of wheat induced by DON and providing new information for breeding wheat with DON resistance. The results were as the following.Using 100 mg. kg^-1 DON, the spikes 2 d after heading of Wangshuibai were inoculated by single floret inoculation(SFI). Total RNA was isolated from the spikes inoculated after 0, 6, 12, 24, 48, 72, 96 and 120 h, respectively, by TRIZOL method. Total RNA from the spikes inoculated after 24 and 48 h, were mixed equally and used to construct theλphage cDNA library as described by SMART cDNA library construction kit. The titer of the primary library was 3×10⁶ pfu/mL.The recombinant percent was 95%. The titer of the amplified library reached at 2×10⁹ pfu/mL. The primary library was directly introduced into the E. coli strain BM25.8 without amplification. The inserted fragments size of the positive clones varied from 0.5 kb to 2.0 kb with an average at 1053 bp.Ninty-six random cDNA clones were sequenced at 3' end. The sequenced size ranged from 60 bp to 891 bp with an average of 393 bp. 48 ESTs over 300 bp were selected and analyzed by BLAST software in NCBI GenBank database. These sequences were divided into 6 groups. (1)6 ribosomal protein genes(13%); (2)8 primary and secondary metabolism relative genes(18%); (3) 4 DNA binding and RNA processing associated genes (9%); (4) 5 immune associated and stress induced genes(11%); (5) 9 unknown genes (20%); (6)13 no BLAST hit ESTs (11%).Seventeen pairs of EST-derived primers were developed based on these sequences. Analyzed by semi-quantitative RT-PCR using these markers, the expressing patterns of 17 EST genes at 0, 6,12, 24, 48, 72, 96 and 120 hs after inoculation with DON were revealed.14 genes were up-regulated, one gene was down-regulated and two genes were constitutive. Three EST genes were mapped on 4 chromosomes of wheat after amplified in a set of nulli-tetrasomic lines of Chinese Spring. Of them, Clonel-1(SNF2 domain-containing protein gene) was located on 7A. Clonel-9(a ribosomal protein L19 gene) was on 2D. Clone2-5, (a metallothionein-like protein gene) was on 1A and 1B.