Construction of Astragalus sinicus AD-cDNA library and identification of proteins interact with the leghemoglobin

Abstract:

Abstract: [Objective] The objectives of this work were (i) to construct a yeast two-hybrid AD-cDNA library of Astragalus sinicus and provide a fundamental system to screen target proteins involved symbiotic nitrogen fixation, and (ii) to isolate the target proteins interacting with the leghemoglobin. [Methods] By using the MatchmakerTM Library Construction & Screening Kit (Clontech), we constructed a yeast AD-cDNA library basing on the total RNA, which was isolated from the root and nodule tissues of A. sinicus at different developmental stages infected by Mesorhizobium huakuii 7653R. [Results] The quality examination of the AD-cDNA library showed that the transformation efficiency was 1.0×106 transformants/3μg pGADT7-Rec DNA, and the average length of cDNA inserts was around 1.0 kb. The library was then screened with the leghemoglobin AsB2510 as bait by yeast two-hybrid system, and 26 positive clones was obtained on SD/-Leu/-Trp/-His/-Ade containing X-gal. 10 of them were individually further confirmed by resuing the plasmid, amplifying the cDNA insert and retesting the protein-interacting phenotype. [Conclusions] The cDNA inserts of positive clones were sequenced and undertaken a blast analysis in NCBI database, it was found that clone LY-53 contained a tify domain and divergent CCT motif, which was an important transcription factor needs in-depth investigation.

Key words:Keywords: Astragalus sinicus; Mesorhizobium huakuii 7653R; AD-cDNA library; AsB2510; yeast two-hybird; interacting protein