Construction Of Microclone Library Of The Alien Additional Chromosome Of Wheat-Thinopyrum Alien Additional Line TAI-27 And Studies On Disease-resistant Genes

Thinopyrum intermedium possesses the properties of disease resistance, drought resistance, cold resistance and other strict environmental stresses. Wheat-Thinopyrum alien additional line TAI-27 created by hybridization between common wheat and Thinopyrum intermedium has a pair of chromosomes derived from Th. Intermedium. The additional chromosomes carry the resistant gene to BYDV. In this paper several strategies were used for research on resistant genes located on the additional chromosomes.Firstly, one alien additional chromosome was isolated and collected successfully under an inverted phasecontrast microscope using a microneedle. Chromosome DNA of the alien additional chromosome was amplified by two rounds of PCR. Consequently, a chromosome specific DNA library with a higher cloning efficiency and relatively large insert size was constructed, which is very useful to help us to set up chromosome physical maps and genetic maps, screen molecular markers linked with important economical traits, clone key genes encoding important proteins and study the evolution of the genome.Secondly, a method to clone expressed sequences of specific chromosome was created by combining chromosome microdissection and microcloning and HSA (hybrid specific amplification) techniques. The expressed sequences of the alien additional chromosome of Wheat-Thinopyrum alien additional line TAI-27 were cloned and sequenced. The method established in this research can help us to construct EST database of the specific chromosome, clone important genes and provide molecular markers for wheat breeding. Additionally, it starts a new area of application of the plant chromosome microdissection and microcloning technique.Thirdly. A subtractive library was constructed using the leaves of Wheat-Thinopyrum alien additional line TAI-27 infected by aphid carried GAV strain of BYDV in the developmental stage of three leaves and the control. Some of differentially expressed gene sequences, which matched with ESTs expressed in wheat under certain stress, were screened out. It is significant to clone resistant genes located on the alien additional chromosome derived from Thinopyrum intermedium and understand resistant mechanism of TAI-27.Fourthly, Resistant gene analogs (RGAs) were amplified by PCR with the degenerated primers designed based on the NBS domain of R genes from the DNA of the alien additional chromosome of TAI-27, DNA and cDNA of Thinopyrum intermedium, TAI-27 and 3B-2. Sequence homology analyzing in GenBank showed that those sequences amplified with degenerated primers were high homologous with known resistant genes or resistant gene analogs. The results of Southern hybridization and Northern hybridization showed that one of the RGAs, which come from the alien additional chromosome, was the specific probe of the alien additional chromosome of TAI-27 which could be used for cloning of the complete resistant gene located on the alien additional chromosome. This research supplied a convenient method for resistant gene cloning by combining chromosome microdissection and microcloning and homology-based cloning techniques.This research based on chromosome microdissection and microcloning provided much molecular information about the alien additional chromosome of TAI-27, which would beuseful for further cloning resistant genes located on the alien additional chromosome derived from Thinopyrum intermedium and creating EST database of the specific chromosome.