Transformation And Expression Analysis Of Gene On Histone H2B Interacting Proteins In Citrus

Biochemical studies of histone proteins have shown that they can be extensively modified through the addition of acetyl, methyl, phosphoryl, and so on. And these modifications can have major effects on the structure of chromatin and therefore may regulate gene expression. Our previous work showed that histone H2B some kind of modification occurred and mediated kumquat's freezing resistant during cold acclimation. However, the molecular mechanism of modification is unclear. In this study, we use cold acclimation citrus leaves for material. Firstly, we screen and analyze interacting proteins or complex with H2B by Co-immunoprecipitation and LC-MS/MS. Secondly, through bioinformatics method, to analyze, screen and isolate genes related to histone H2B interacting proteins, and identify these genes via gene-clone, expression validation and functional analysis in order to unclose the molecular foundation of histone H2B modification.The results are as follows:(1) We screen and analyze the interacting proteins with H2B by Co-immunoprecipitation and LC-MS/MS, which was identified seven proteins, including carbonic anhydrase, calcium-dependent protein kinase, and five kinds of unknown function proteins:miraculin-like protein 2 [Citrus jambhiri] (named MLP2-1); miraculin-like protein [Citrus x paradisi] (named MLP-1); miraculin-like protein 2 [Citrus jambhiri] (named MLP2-2); putative miraculin-like protein 2 [Citrus hybrid cultivar] (named PMLP2-1; predicted protein [Populus trichocarpa] (named PP-1)?Through bioinformatics method, to analyze and predict the structural and functional characteristics of these five kinds of protein. These five proteins have a signal peptide, and the secondary structures are made from a spiral, ? folding and random coil components. MLP2-1, MLP2-2 and MLP-1 protein contains only reactive site loop, then PP-1 protein containing a plurality of the binding sites.(2) The gene expression levels in the cold acclimation process were determined by fluorescence quantitative PCR. The expression levels of genes have different degrees of increase by over time at 4? low temperature processing time five stages. The expression level of MLP2-1 gene increased within 0?6h, then declined slightly in 12 h, and gradually increased steadily in the 16?24 h. The expression level of MLP2-2 gene greatly enhanced within 6 h, and the fold induction about 5.00; It expression is halved to 12 h, but significantly increased to nearly 8.00 times in 18 h, then declined. The expression level of MLP-1 gene increased within 0?6h, then decreased slightly in 12 h, increased steadily at 16 h?24 h, and similar with the expression of MLP2-1 gene. The expression level of PMLP2-1 gene was stable increase in five stages of hypothermia treatment.(3) The MLP2-1 gene cloning to obtain 712bp fragments, and constructed the plant expression vector with p1300M. The MLP2-1 gene was successfully transferred into Arabidopsis, obtained four transgenic seedlings for further research.