Genome-wide Analysis, Expression And Molecular Function To Cold Response Of RNA Binding Glycine-rich Protein (RB-GRP) Family In Maize

Maize is the most important cereal, energy, forage and industrial material crop in the world. However, chilling stress has been becoming an environmental factor that limit the increase of corn yield. Therefore study on the mechanism of maize chilling tolerance and improve chilling tolerance of maize seedlings is a key target for maize breeders. RB-GRP belong to the RNA-binding glycine-rich protein gene family existing in plants extensively, which were regulated by different biotic and abiotic stresses, such as low temperature, flooding, high temperature, high salinity, UV radiation, heavy metal and so on. In this study, Zm RB-GRP were identified by bioinformatic analysis, then gene expression under varied abiotic stresses and the function on chilling tolerance of some of these genes were studied. Main results of this study are as follows:1. By screening the whole genome database, 23 Zm RB-GRP were identified. They distributed on 10 chromosomes of maize, and 15 of the gene products targeted to nucleus; Phylogenetic analysis showed that the Zm RB-GRP can bedivided into four subgroups. A total of 5 pairs of paralogous genes distribution on different chromosomes were detected, suggesting that segmental duplication is predominant in the expansion of the Zm RB-GRP in maize; In comparison with the RB-GRP in rice, Arabidopsis and wheat there are 2 subgroups were exclusively derived from maize. This imply that these Zm RB-GRP turned up after maize appeared, or may due to that homologs of these genes have not been identified in other plants; Promoter and phosphorylation analysis showed that the Zm RB-GRP have varieties of biotic and abiotic factors related cis-elements.2. We chose 2 Zm RB-GRPs from each subgroups of Zm RB-GRPs. q RT-PCR assay of 8 of these genes showed that the Zm RB-GRP were responded to salt, low temperature, temperature recovery and ABA.Besides, candidate gene approach association mapping analysis showed that Zm RB-GRP4, Zm RB-GRP5, Zm RB-GRP7 and Zm RB-GRP14 were significantly associated with at least one of the four chilling tolerance related traits,soluble sugar content, water content, leaf roll degree and ratio of root-to-shoot, suggesting that the four genes shouldbe associated with the chilling tolerance of maize.3. Complementation of a low temperature sensitivity of an E. coli csp A, csp B, csp E, csp G quadruple mutant(BX04) with four Zm RB-GRP indicated that they can enhance cold tolerance in certain degree.4. In vitro nucleic acid binding assays determined that recombinant Zm RB-GRP4, Zm RB-GRP7 and Zm RB-GRP14 were capable of binding to ss DNA, suggesting that these Zm RB-GRP may function as RNA chaperons.5. 35S::Zm RB-GRP4, 35S::Zm RB-GRP5 and 35S:: Zm RB-GRP14 were transformed into Arabidopsis thaliana T-DNA mutant atgrp7 and atgrp2, suggesting that these Zm RB-GRP can strengthen the cold resistance to the Arabidopsis mutants atgrp2 and atgrp7.6. Yeast two hybrid analysis shown Zm RB-GRP4 and Zm RB-GRP5 interacted with Dna J, suggesting that Zm RB-GRP4 and Zm RB-GRP5 might be response to chilling as chaperones. Respectivelly, Zm RB-GRP4 was also associated with RH, indicating that Zm RB-GRP4/RH protein complex may involved in r RNA processing.In conclusion, on the base of genome-wide identification of RB-GRP in maize, four members of them, Zm RB-GRP4, Zm RB-GRP5, Zm RB-GRP7 and Zm RB-GRP14 were found to play important roles in chilling response. Results in this study would provide important information for genetic improvement of chilling tolerance in maize.