Study On Molecular Basis Of Resistence To Ralstonia Solanacearum In Peanut

Peanu(tArachis hypogaea L.) is an important economic and oil crop in the world. Bacterial wilt (Bw) caused by Radstonia solanacearum (Rs) is one of devastating bacterial diseases affecting peanut (Arachis hypogaea L.) production and quality in the tropical,sub-tropical and some regions of temperate zone of the world and it is more serious in the Yangtze basin and south area of China. As a soil-borne disease, there are not any efficient chemical methods to control it so far. Although rotation, intercropping of peanut with other non-host crops and biological control could be useful, they are less feasible. Therefore the most promising way is to develop resistant cultivars. There are some reasons affecting breeding resistent cultivars: the lack of exellent resistance genes, resistantance easily subjected to environment change, and the complexity of the strain classification and so on. So it is very important to find a convinient also accurate method for resistance evaluation to be carried out in greenhouse. At present, researches of Bw is little in China and other courtries. The genetics of host resistance and the molecular mechanism underlining peanut resistance are still poorly understood. In this study, we will search an ideal method for resitent identification of Bw, construct a mixed root full-length cDNA library induced by Rs, and construct a vector harboring a known arabidopsis resistant gene, RRS1, for its efficient in other crop. The main results of this investigation are as follows:1. We inoculated different cultivars with various resistance to Bw, such as Minhua 6, via wounded root steeped in different strains, and a virulent stain 431 appeared better than others, We have compared different performance after inoculating peanuts at seedling stage using several different inoculation methods. Although the method of inoculation via wounded root steeping can reflect cultivars resistence, more inoculation volume, longer duration of disease presence brought a lot of inconvenience to experiment; Axillary injection does not reflect resistence or susceptibility so well with quite difficulty in operation, but leaf-cutting inoculation method can reflect the resistance of variety,with merits of short evaluation cycle, easy operation and less inoculation volume. In short, inoculation via root steeping and cutting leaf could tell the resistance of varieties, and the cutting leaf method is better.2.We inoculated bacteria liquid of Rs to resistent cultivar Minhua 8 via wounded root,steeping inoculation and sampled roots of different stage after inoculation, then construsted a mixed root full-length cDNA library induced by Rs or not by SMART technique, with a primary titer of 1.902×106pfu/mL and 99% of recombinant clones in unamplified library. The inserts varied from 500 to 2500bp in size with an average size about 1200bp. Twenty four clones were randomly selected for sequencing from both sides of the inserts. After bioinformatics analysis, about 45% sequences have no function, only with predicted proteins. It means functional genomics research in peanut is so poor. Seventy four clones were randomly chosen for sequencing from both sides of inserts. We do annotation and functional anylysis using the BLAST2G0 online software. Based on the function analysis, the fuction annotation fall into three categories: (1) cellular component, (2) molecular function, and (3) biological process. Most genes in first functional category involved in mitochondrion and plasma membrane;in the molecular function class, the majority proteins have binding and catalytic activity,and in the biological process category of function, more genes participate in cellular process and metabolic process . Distribution analysis of species similarity showed the sequences are closer with rice, grapes, and Arabidopsis thaliana.3.We designed primers according to the sequence of RRS1-R in the Genebank, then amplified RRS1-R in Arabidopsis Nd-1by RT-PCR. Sequencing results showed that RRS1-R gene is 4137 bps with an identity of 99% to the original sequence on GenBank. Then we constructed plant expressing vector pSC1301-RRS1 with 35S promoter and Nos terminal on both ends, and transformed innto Agrobacterium tumefaciens strain EHA105. Then Transformed into Tobaco for evaluation of its resistance.