| Squash (Cucurbita moschata Duch.) possesses strong resistance abilities to coldness, drought, many disease pathogens, barreness and especially Fusarium wilt, and so it is good grafting stock for the melon of the family Cucurbitaceae. Fusarium wilt, which is caused by Fusarium oxysporum f.sp.melonis, is one kind of world diseases in melon. The main method to prevent Fusarium wilt is using chemical pesticide which brings about potential danger to health of people and environment. To breed resistant lines which are fit for planting takes up agriculture has such many drawbacks as many years and low efficiency. So cloning and marking the resistance gene of squash are fundaments for disease resistance breeding of melon.The study adopted the strategy-based cloning resistance gene analog(RGA).The procedures are as follows, firstly the polymerase chain reaction(PCR) for amplifying RGAs were carried out by using the degenerate primer that designed according to the conserved domains of the cloned R genes-nucleotide binding site (NBS) and the plant genomic DNA as template; Secondly the RGAs were sequenced and homologous and phylogenetic relation among the RGAs and the cloned R genes were analyzed; Thirdly some of RGAs were selected as the candidate disease resistance genes;and finally new plant resistant genes were discovered from these candidate.The genome DNA as template from cultivar Junjinglong were extracted by using CTAB method, and twelve degenerate primers were designed based on the conserved amino acid domains (P-loop and GLPL) that exist in disease-resistance genes (R-genes), and amplifying products, which spanned approximately 500bp, were cloned into pMD18-T vector and transformed E. coli DH 5α. After blue and white plaque preliminary screening, recombinant clones were identified by PCR and were classified by restriction endonuclease digesting method. Twenty clones were sequenced and their homologous sequences were searched on GenBank with Blast. Finally,thirteen clones were distinguished as RGA of squash and named SQRGA1～6 , SQRGA8～14, which were submitted to GenBank and provided accession numbers as EF101660～EF101665, EF101667, EF199755～EF199760．The 13 SQRGAs had ORFs and recognizable NBS domains, and showed high similarities with the announced RGAs of melo, which displayed a close relationship as both species are in the same family.The sequences analysis of the 13 RGAs structures and NBS domains found that all contained the conserved motif,such as "P-loop", "Kinase-2", "Kinase-3a"and"GLPL". The phylogenetic analysis show 13 RGAs were divided into two distinct types,TTR-NBS-LRR type and non-TIR-NBS-LRR type, which confirmed the early reported that NBS-LRR gene has twe major groups in dicot species. Alignment of amino acid sequence of these 13 RGAs with the reported R genes showed high homologous(18．4%-77．4 % ).The deduced amino acids of RGA sequences SQRGA1-SQRGA6, SQRGA9-SQRGA12 are homologic (71．9 %-77．4 %) with MRGH21 in melon.These RGAs isolated from the squash used in present study would make the base for the further cloning of disease-resistance genes in squash and provided a clue for the origin and evolution of squash germ plasma. Based known RGAs from squash , the full sequences of R gene could be isolated by RACE and screened from squash genomic libraries. |
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