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

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.

By using the Matchmaker 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.

The quality examination of the AD-cDNA library showed that the transformation efficiency was 1.0 x 10(6) transformants/3 microg 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.

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.