| Peanut (Arachis hypogaea L.) is an important oil and cash crop worldwide and an important resource of plant oil and protein. Bacterial wilt is a major bacterial disease in peanut which greatly decreased peanut production. The general incidence of bacterial wilt in southern part of China is between 10-30%, resulting in more than 20% peanut yield reduction. In the heavily infected areas, more than 50% of peanut are affected by bacterial wilt disease, leading to severe yield reduction or no production at all. In the past three decades, significant progress in bacterial wilt resistance has been made in peanut genetic improvement in China. A number of resistant varieties have been released and their application in the field greatly decreased wilt incidence. However, because of the narrow genetic base of resistant parents and the close linkage between resistance, low yield and poor quality, it is difficult to use conventional breeding methods to select an elite resistant peanut line. Therefore, it is necessary to identify and to use the bacterial wilt resistant gene in peanut via biotechnology. In this study, a SSH(suppression subtractive hybridization) library from bacterial wilt resistant peanut line J4 is constructed to identify the potential resistant genes, and real time PCR is carried out for gene expression analysis. And the full length of potential resistant genes, lectin and ARF(ADP-ribosylation factor, ARF) were identified. The main results are as follows:1. Both resistant (Yuanza 9102) and susceptible (Zhonghua 12) peanut varieties growing to 3-leaf age were used to evaluate the inoculation method in the water culture system. The results indicated that root dipping with 3.0×107 cfu/ml bacterial and 30 min inoculation is optimal to differentiate the resistant from the susceptible ones, and the condition was used in the following experiment system.2. Resistant peanut line J4 was challenged with R. solanacearum with root dipping inoculation method. Roots collected 0.5 hpi (hour post inoculation), 2 hpi, 4 hpi, 6 hpi, 12 hpi, 24 hpi, 48 hpi were used as starting material to extract RNA. The RNA were then mixed and transcribed for cDNA synthesis. The same was done with the control plantlet. And the double stranded cDNA from both treatment and control were used for the subsequent hybridization procedure to construct the SSH library. The library contains 3072 clones harboring potential resistant genes. The recombination rate of the plasmid is 73%, with the inserted fragments length ranged from 200-500bp. A total of 1036 clones were sequenced and they were clustered into 183 unigenes. 162 out of 183 unigenes are from peanut and the rest are deemed to be virus coating protein encoding genes. Comparison of the unigenes with the public available database indicated that 58 unigenes can be annotated with their biological function, including 44 genes that belong to the host response to pathogen attacks, such as cell structure, signal transduction, defense response and transcription related.3. Based on bioinfomatic analysis, 14-3-3gene, Glutaredoxin-C2, lectin, pathogenesis-related class 10 protein, ubiquitin conjugating enzyme 1(RCE1), C3HC4-type RING finger protein and ADP-ribosylation factor (ARF) were selected for gene expression analysis. The results indicated that the selected genes were all upregulated in peanut materials with resistance to BW, while in susceptible ones, they were downregulated or delayed.4. We have obtained two potential BW resistant full-length genes, lectin and ADP-ribosylation factor via cDNA library screening. The gene length is 1069 bp and 841 bp respectively. Lectin gene encodes 280 amino acid, which has 96% similarity with the original peanut gene deposited in genebank. ADP-ribosylation factor encodes 181 putative amino acid, which shares 100% similarity with rice ARF gene. |
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