Cloning The Full-length CDNA Of TaMBD2in Wheat And Screening Its Interactation Proteins

In order to disscuess more regulatory functions of methyl-binding domain protein during the growth and development in wheat, the full-length cDNA encoding a methyl-binding domain protein TaMBD2were cloned by using RACE technology. Yeast expression vector （Bait） of TaMBD2were constructed, and screening of TaMBD2-interacted proteins by the method of Yeast two-hybrid system.The main results showed as follows:1. The full-length cDNA of TaMBD2was1511bp, which including the164bp of5’UTR,405bp of3’UTR and942bp of ORF. The gene encodes a protein containing313amino acids. The deduced molecular weight was34.65kD and isoelectric point was5.21. The amino acid sequence analysis showed that a typical methyl-CpG-binding domain （143～221） and a CW-type Zinc finger domain （80～132） were included in TaMBD2. Homophylic comparison of MBD domain with ZMMBD111（EU965071）、 ZMMBD108（AY029560） and AtMBD2（NM₂03121） were96%、92%and68%, respectively. The3D structure prediction showed that the specific spatial structure of MBD domain of TaMBD2was constructed by two alpha helixes and three beta strands.2. Yeast expression vector （Bait） of TaMBD2was successful constructed, which no toxicity and autoactivation in Yeast cells. Positive clone were verifirf by using the Western blot.3. Using Yeast two-hybrid system method, we screened TaMBD2interacted proteins. The37clones of screened91were sequenced, the Blast analysis showed that21of them were assumed nucleoside diphosphate kinase （NDPK3）,9of them were epsin N-terminal homology domain-containing protein, and others were zinc finger C2H2type family protein,agenet domain-containing protein,hypothetical protein respectively.4. NDPK proteins widely present in plants and highly conservative. It is a type of enzymes catalytic the γ-phosphate of ntp to the ndp, which play an important role in the maintenance the content of intracellular ntp. It also activates G protein by influencing cytoskeleton movement and mediated signal transduction. Recent studies showed that NDPK proteins may play other important regulatory functions. ENTH domain-containing protein is ubiquitously expressed in various plant tissues, and was a kind of proteins involved in membrane transportance. The proposed functions were recruited ubiquitinated cargo into coated pits, and helped to change the curvature of the membrane during vesicle formation. The main proteins of interacted with TaMBD2were NDPK and ENTH in this study, which implied that the interactation would play an important regulation function during the growth and development in wheat,but the specific mechanism of interaction and biological function will be a new point of further study.