| As a traditional cultivation and medicine combination food of vegetative tuber crop,greater yam(Dioscorea alata)is planted in south of the Yangtze River of China.Anthracnose is one of the most widespread and serious disease of greater yam,without efffective method to control.Development and adoption of disease resistance breeding is the most effective measure.Basing on the previous work,as a dominant strain of anthracnose in Hainan,Colletotrichum asianum was used to infect the susceptible Da90 and the resistant Da94,then observed the ultrastructure alteration of greater yam leaves by transmission electron microscopy technique.On the basis of the transcriptome database,we identified 15 resistance-related genes,which were used to analysis the expression by fluorescence quantitative PCR.Cloning and bioinformatics analysis DaPRl and two WRKYs genes,then used yeast one-hybrid technique to judge the interaction between them.The results were as follows:1.After inoculation 72h,96h and 120h with C.asianum,surveyed the ultrastructure changes of leaves.In susceptible material Da90,cells were abnormal at 72h,with great damages of chloroplasts and mitochondria,chioroplast lamellar structure were distorted and swelled,the mitochondrial crest were swelled and appeared vague.Cells appeared primary hypha at 96h,with great degradation of chloroplasts and mitochondria.Secondary hypha appeared at 120h between cells which were garded as a conversion character from biotrophic to necrotrophic of C.asianum.In resistant material Da94,cells were normal at 72h and 96h some cells appeared plasmolysis at120h,and vesicles emergeed at the edge of the cell membrane,starch granules were increasing in chloroplasts,without hyphae during the process of observation.This result suggested Da94 resistant germplasm may have some factors or structures to prevent the infection structure forming of Colletotrichum asianum.2.15 disease resistance related genes were selected,and analyzed the expression trend after inoculation.The resultsof qRT-PCR showed that these genes were up-regulated but different genes showing different expression patterns.GST,SOD,PERK2,CCHI2,CHI5,WRKY33,and PR1 were up-regulated in resistant Da94,it showed that these genes specifically expressed may play an important role in the resistance to C.asianum.3.A full-length cDNA sequence of DaPR1,DaWRKY28 and DaWRKY33 was cloned by RT-PCR.Bioinformatic analysis of protein DaPR1 revealed the amino acids possessed SCP-PR1-like conserved domain.Homology analysis of DaPRl gene showed that the amino acid sequence of Dioscorea alata shared 79%,76%,75%,74%homology to Elaeis guineensis,Phoenix dactylifera,Musa acuminata and Asparagus officinalis.DaWRKY28 and DaWRKY33 encoding amino acids contained WRKYGQK sequences and had a typical zinc-finger structure C2H2 at the C-termini,both belong to group II of WRKY transcription factor family.Phylogenetic tree displayed that DaWRKY28 was grouped with Elaeis guineensis(Eg WRKY28)and Phoenix dactylifera(PdWRKY28),their genetic relationship were closer.DaWRKY33 was grouped with Gossypium hirsutum(GhWRKY9)and their genetic relationship was closer.4.Promoter cloning library of t Da90 and Da94 were constructed by Adaptor-PCR method.The 5’ end of upstream transcriptional regulatory sequences of DaPRl was amplified by nested PCR,sequences analysis showed that the length of promoterin Da90 was 1526 bp,with named PPR1a,and the length of promoter in Da94 was 1336,with named PPR1b.The sequence analysis indicated that they contained not only the basic promoter elements CAAT-box and TATA-box,but also the hormone response components such as jasmonic acid(JA),gibberellin(GB)and the fungal induction response components(W-box).5.Point-to-point of yeast one hybrid analysis showed the interaction between DaWRKY33 and DaPRl promoter,mightbe control the expression of DaPRl.There was no interaction between DaWRKY28 and DaPRl promoter. |
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