Construction Of Astragalus Sinicus AD-cDNA Library And Identification Of Proteins Interact With The Leghemoglobin

The legume Astragalus sinicus and Mesorhizobium huakuii 7653R symbiotic nitrogen-fixing system was diachronic research and characteristics symbiotic nitrogen-fixing resource in our country.The research aimed to study biological nitrogen fixation system on molecular mechanism between the legume A. sinicus and M. huakuii 7653R symbiotic nitrogen-fixing system. An AD-cDNA library was constructed by using materials from the root of legume A. sinicus infected by M. huakuii7653R at different times, meanwhile the library was screened with the Leghemoglobin AsB2510 as bait by yeast two-hybrid system.Harvested the root of A. sinicus after inoculated by the M. huakuii 7653R in different times, including 2d、4d、6d、8d、10d、12d、15d、18d、21d、24d、28d、32d、36d、40d、45d、50d and 55d. Combined the tissues in two groups depending the limit of 12 days. The total RNA of the tissues roots in different group was isolated, and the double-stranded cDNA was obtained by RT-PCR and LD-PCR.After the double-stranded cDNA purified and concentrated, was transformed into the yeast AH 109 with the linear pGADT7-Rec plasmids.In yeast cells the cDNA fragment inserted into GAL4AD,and the recombination pasmid will express the cDNA insert as GAL4 AD fusion protein. when the transformant came out, washed them out with the liquide culture and save in different botters. Cultivate the transformant from different botters on the SD/-Leu/-Trp/-His/-Ade plate. If there were no wild-type yeast and other bacteria, mixed the liquid in different botters and store the library as 1 ml aliquo at-80℃.The efficiency of transformation whatever library achieved 1.02×106/3μg pGADT7-Rec DNA, and the inserts size were all about 1.5kb checked by the random chosen library colonies. In all,the libraries were high-quality and without pollutionThe leghemoglobin gene AsB2510 was cloned to the pGBKT7 as the bait plasmid.After restriction enzyme digestion and sequencing analysis,transformed it into Y187. When transformants subsequently tested without the self-activating activity and toxicity AD-cDNA library screening,used them to screen the library. The library was screened with the Leghemoglobin AsB2510 as bait by two hybrid system and 26 positive clones were selected on SD/-Leu/-Trp/-His/-Ade containing X-gal. At last 10 clones were further confirmed by resuing the plasmid from yeast,amplifying the DNA insert by PCR and retesting the phenotype.The positive clones were sequenced and searched for NCBI database by blast analysis, and the amino acid sequence encoded clone LY-53 containing tify domain and divergent CCT motif. the tify domain contained zinc finger was found in a variety of plant transcription factors that contain GATA domains as well as other motifs,and The CCT motif contained a putative nuclear localisation signal.Based on the gene functional characteristics of the domains, we concluded that LY-53 was predicted transcription factor.