Cloning And Molecular Characterization Of Unc45b Gene And Its Temporal Expression Of Grass Carp Fed Bean

Exp.1 C loning, molecular characterization, and expression analysis of the unc45 b gene from grass carpunc45 myosin chaperone b(unc45b) gene is a molecular chaperone that mediates the folding, assembly and accumulation of thick-filament myosin during sarcomere formation, which plays an important role in maintaining sarcomere stab ility and striated muscle development. In this study, the unc45 b gene c DN A sequence of grass carp was obtained by RAC E(rapidamplification of c DNA ends) method, and the characteristics of the unc45 b gene and its encoded protein sequence were predicted and analyzed by bioinformatics methods. The expression pattern in different tissues was also detected by Quantitative real-time PCR. The results showed that the full- length of unc45 b gene of grass carp was 3163-bp, which contained a 60-bp 5’UTR, a 298-bp 3’UTR, and a 2865-bp open reading frame(ORF) encoding a 934-amino acid peptide. The deduced Unc45 b protein exhibited 92%, 86%, 86%, 82%, 81% homology to the protein of zebrafish(Danio rerio), C hannel catfish(Ietalurus punctatus), Mexico Tetra(Astyanax mexicanus), Tilapia mossambica(Oreochromis niloticus), Salmon Atlantic(Salmo salar) respectively. The Unc45 b protein of grass carp was a hydrophilic and non-secretory protein with a molecular mass of 103,699.8 Da, and isoeletronic point of 7.39. The structural elements included a-helixes, and loops. The unc45 b gene expressed in all grass carp tissues, including blood, liver, spleen, kidney, heart, brain, intestines, gill, skin and muscle. It expressed in heart and muscle significantly higher than other tissues. The highest expression tissue was heart, which is significantly higher than other tissues(P<0.05), whereas a significantly lower expression tissue was muscle(P<0.05), and the lowest expression tissues were blood, liver, spleen, kidney, brain, intestine and gill. Those results matched with the function of the gene.Exp.2 The temporal expression of unc45 b of grass carp fed beanTwo groups of grass carp were collected in this experiment. Control group was fed with compound feed. The other one was fed with dry broad beans soaked in water for 24 hours.This experiment lasted for 12 weeks. and the tissues of liver, skin, skeletal muscle and heart were collected at 0 weeks, 4 weeks, 8 weeks, 12 weeks differently. And the m RNA expression pattern of unc45 b of those tissues were also detected by Q uantitative real-time PCR. The results showed that the unc45 b gene expression of skeletal muscle and heart decreased significantly with the increasing cultivation time(P<00.05), and increased in liver and skin significantly(P<00.05), which were so lower than in skeletal muscle and heart absolutely. At the same time, the two groups were compared with each other at different period. The unc45 b gene of skeletal muscle and heart of crispted grass carp with broad bean expressed significantly higher than common grass carp at each period(P<0.05), but there was no significant difference in liver and skin between two groups(P>0.05). All those results showed that the nutrition such as broad beans and the growth period all have a certain impact on the expression of m RNA of the unc45 b gene, the expression pattern were different between skeletal muscle and heart with liver and skin with the growth time. And the broad bean improved the expression of unc45 b gene in skeletal muscle and heart actually, which consistent with the fuction in sarcomere assembly.