Cloning And Characteristic Analysis Of NBS-LRR Like And STK Like Disease Resistance Gene Analogs From Banana, Papaya And Mango

The method that finds Resistance Gene Analogs (RGA) using the degenerate primers designed based on the conserved motifs of plant disease resistance genes (R) was named as RGA method. There were many RGAs obtained from a variety of plant species. Some of them were proved the candidate R genes, some else were even R genes. Therefore, this was a new kind of method of cloning R genes. And what's more, these RGAs may used be as more pinpoint molecular markers of R genes and used to clone the relative R genes from plants, including tropical fruit trees. It was significant meaning that the RGAs from tropical fruit trees were applied to Molecular marker-assisted breeding. It was also the foundation of isolating R genes from the tropical fruit trees and comprehending deeply their structure and function.For cloning the NBS-LRR like RGAs and STK like RGAs from banana, papaya and mango, four pairs of degenerate primers were designed separately based on the NBS-LRR (Nucleotide Binding Site-Leucine-Rich Repeats) and STK (Serine/Threonine Kinase) conserved motifs of plant R proteins. Many fragments were amplified based on the PCR and RT-PCR from genomic DNA extracted from leaf of Musa acuminat spp.cv.'Brazilian', Carica Papaya cv.'Suoluoyihao' and Mangifera indica cv.'Zihuamang' by CTAB method, and cDNA reversedly transcipted from total RNA extracted from the above metioned fruits by CTAB and SDS method. Many desired RGAs were cloned and sequenced. The main results are as follows:1. The major fragments of 500bp were amplified using PCR and RT-PCR approach with degenerate primers based on the conserved domains of plant R proteins from leaf of M.acumina spp.cv.'Brazilian', C.papaya cv.'Suoluoyihao' and M.indica cv.'Zihuaman-g'. Several hundred clones were obtained and sequenced.2. One NBS-LRR-like RGA from cDNA of M.acuminata spp.cv.'Brazilian' and two from genomic DNA of C.papaya cv.'Suoluoyihao' were obtained. Sequence analysis of the deduced amino acids from the cloned RGAs showed that all RGAs contained conserved motifs of NBS-LRR, which include kinase-la domain, kinase-2a domain,...