INTERACTION NETWORK OF TaRHA2b OF WHEAT (TRITICUM AESTIVUM L.) BASED ON HIGH-THROUGHPUT YEAST TWO-HYBRID SCREENING.

To explore the signal transduction pathway that TaRHA2b gene is involved in regulating, yeast two-hybrid system was used to screen and identify proteins interacting with TaRHA2b. The homogenized cDNA library was constructed from the embryo of mixed barley. The decoy protein vector pGBKT7- TaRHA2b was constructed and transformed into yeast AH109 cells for self-activation detection. Candidate positive clones were screened by one to one yeast co-transfection, SD/-Leu/-Trp and SD/-Leu/- Trp/-Ade/-His medium screening and β-galactosidase chromogenic reaction. The in vitro co-transfection and GST-pull down experiments were used for further verification. The homogenized cDNA library of the barley embryos with titer of 1.2 10⁶ cfu/ml and storage capacity of 1.1 10⁶ was successfully constructed. The inserted fragment size was between 1000-3000 bp. The plasmids of the cDNA library were extracted and sequenced on a large scale, and 5000 plasmids were obtained. The decoy vector without self-activation function was successfully constructed. The x-gal filter paper of eighty-four hybrid clones showed strong blue color and forty-nine pieces of effective sequence information were obtained. Eight of them were selected for co-transfection verification. YTH2450 was selected for GST-pull down verification. The results showed that they were positive hybrid clones. TaRHA2b interacted with YTH2450 and other proteins. The information of above genes could be used for genetic improvement of crops, further improving the adaptability of crops to the environment. [ABSTRACT FROM AUTHOR]