Study Of The Structure And Function Of Conserved RegionⅡ Of ABI5Family

In arabidopsis, seven conserved regions are founded in ABI5famliy by Dot Matix Comparision. The conserved region II (CRII), with unknown function sharing the conserved sequence GKXXGSMNXDELLKXVLXXA EE, contained an a-helices in the acidic activation domains in Theoretical speculation. CRII, acted as a trans-activation domain, have no amino acid sequence homology compared with others reported. Therefore, study the CRII activate transcription of the molecular mechanism of the ABI5family on molecular level give us an insight into the mechanism of regulation of gene expression and late embryogenesis signaling processes, and, at the same time, is significance to cultivate drought-resistant transgenic crops in theoretical. Studies showed that the conserved regions II activates reporter genes when fused to the yeast GAL4DNA-binding domain. Whereby, this article selected the CRII of the AtDPBF4, the smallest member of ABI5family, as the research object. In order to understand the possible activating transcription mechanisms of the CRII, we constructed a series of substitution mutations and deletion mutants and analysised their transcriptional activation.Through analyzing the primary and secondary structure of the conserved region II by prediction software,12point mutation and deletion mutants were determined, and works of these mutants in vitro construction had been completed. Activity analysis showed that:(1) Two basic amino acid residues (Ks) were found presenting in the enrich acidic activation transcription domainin in the analysis amino acid Residues of CRII. Yeast one-hybrid experiments show that, when the No.51basic amino acid residue K in the AtDPBF4protein sequence were mutated to acidic amino E (p4IIK51E), activity from12.4U of CRII (p4II) decreased to7.3U; In the case of No.51residue mutating into E,mutated the No.39basic amino acid K into basic amino acid R (p4IIK39R/K51E) or acidic amino acid E (p4IIK39,51E), activity were10.4U and11.6U respectively, activity has been recover, and closed to the CRII. (2) Speculated that there is an a-helix structure (No.46-53aa, LDELLKTV)present in the conserved region II, and the activity of the structure alone is10.8U. When adding six amino acid residues to The C-terminal of the a-helix (p4II46-59aa, sequence LDELLKTVLPPAEE), the activity is11.6U. Compared to the wild-type CRII (p4II, sequence GKPLGSMNLDELLKTVLPPAEE), the two deletion mutants activity are not significantly decreased. This result suggests that, the a-helix of the CRII performed activity of transcription activation, and if its upstream and downstream sequences have transcriptional activation or enhanced activity needs further analytical work.(3) In the12substitution and deletion mutants,6mutants can not be measured reporter gene activity:1mutant (p4II38-53aa) resulting in the receptor yeast can not grow and the remaining5mutants (p4IIK39,51E, p4IIK39E/K51R, p4IIK51R, p4IIK39,51R, p4IIK50P) lead to the receptor yeast at a slow pace of growth.