JIAN Gui-Liang, ZHAO Lei, ZHANG Wen-Wei, QI Fang-Jun, WANG Sheng-Zheng
Cotton Science. 2011, 23(6): 490-499.
A specific fragment of about 500 bp was cloned from the genomic DNA of the nine identified cultivars of upland cotton(Gossypium hirsutum) by PCR degenerate primer designed according to the conserved domains P-loop and GLPL in the NBS(nucleotide-binding sites) region of reported R genes. The fragments are recycled and inserted into pGM-T vector, and then transformed into E. coli DH5α. 350 recombination bands were obtained through cloning and restriction digestion identification. The clones are sequenced and 74 RGAs with complete open reading frames (ORF) from eight varieties of cotton are finally obtained. Comparing with the reported R genes, the 74 RGAs all contain P-loop (Kinase1a), Kinase2, GLPL region, and motif RNBS-A, B, C defined by Meyers. Cluster analysis of their putative amino acid shows that the RGAs could be sorted into two distinct types, TIR-NBS type and nonTIR-NBS type, and nonTIR-NBS could be subdivided into three types, Ⅰ, Ⅱ, Ⅲ. The TIR type RGAs all contain the RNBS-A-TIR motif, while the nonTIR type RGAs all contain RNBS-A-nonTIR motif. This result consists with the early report that NBS-LRR gene has two major groups in dicotyledon. The deduced amino acid of the RGAs have high similarity with the reported R genes. NonTIR type RGAs have a similarity of 32％～50％ with L6, M, Gro1-4, N, while TIR type RGAs have a similarity of 23.2％～56.5％ with I2C-1, Mi-1.1, RPM1, RPP8, RPS2, RPS5, XA1, Prf. The similarity among deduced amino acids of the different type RGAs is lower, but that among deduced amino acids of the same type RGAs is very high, from which we deduced that the RGAs with high similarity belong to the same gene family, and maybe locate at the same gene cluster.
