Banana Leaves Of Low Temperature Stress Ssh Library Construction And Differential Protein Study

Banana (musa nana Lour.) Is a large-scale Musaceae banana is a perennial evergreen herb, found in tropical and subtropical more than 100 countries, but in most areas, bananas, cold winter and spring all year round are subject to the invasion, ranging from banana leaves, Fruit injury, cut, re the whole plant died. Therefore, studying the mechanism of cold damage to banana produce to improve cold hardiness of the banana, in theory, research and production practice, is of great significance.To resolve the molecular mechanism of cold damage to banana, respectively, using suppression subtractive hybridization (SSH) and protein two-dimensional electrophoresis technology, through bioinformatics, the process of low temperature stress on some of the specific expression of genes, proteins carry out systematic research.Application of suppression subtractive hybridization (suppression subtractivehybridization, SSH) was successfully constructed under low temperature stress and the natural growth conditions, the banana leaf blade differentially expressed cDNA subtractive library. The blue, white spot screening, a total of 142 positive clones were obtained. Colony PCR analysis showed that 95% of the positive clones containing the size of 200～700 bp of the inserts. Randomly selected 20 clones were sequenced and used tools for genetic search (Genbank) on access to the EST was Blastx analysis results show that the eight EST sequences are no clues to the unknown sequence, four belong to an unknown function of speculation protein gene 2 are unknown functions of the protein genes,3 with the known functional genes with high homology. Semi-quantitative PCR technology, No.28 fragments of the expression analysis results show that the fragment for the chilling stress-specific gene, in the low temperature stress within 10-24 h after treatment with the processing time increases the expression of enhanced under natural conditions the growth of the banana leaf does not detect the gene expression.Application of protein two-way technology system (including the banana leaf protein extraction methods and optimization of two-dimensional electrophoresis conditions for optimization), carried out comparative proteomics banana cold stress experiments, pairs of differential proteins were identified by mass spectrometry and functional analysis. Respectively TCA/acetone precipitation, phenol extraction of total protein banana leaves and to compare the two methods of extraction results show that the banana leaf phenol extraction method is the best method of total protein, extracted the most complete protein. Analysis of low temperature stress banana seedling leaves and banana seedling leaves normal growth pattern of protein and found that low temperature stress-specific expression of the protein after 11,2.5-fold increase protein spots 4, 0.5-fold lower protein 13. Pairs of 28 different proteins were identified by mass spectrometry,2 of acetyl coenzyme A carboxylase,3 and 6 samples of ribulose 1,5 bisphosphate carboxylase/oxygenase, as well as 8 and functional proteins are known to have a high homology, seven belong to an unknown function of speculation proteins,5 are unknown functions of proteins, while five samples were not retrieved in the database and matching higher homologous proteins.This result shows that, by building the suppression subtractive cDNA library and a comprehensive analysis of differences in protein, there may be a considerable number of unknown functional genes involved in the process of cold banana. In-depth understanding of its function will provide a comprehensive analysis of the molecular mechanisms of chilling injury of bananas to clarify the process of cold damage in the signal transduction process basis.