The Identification Of Lily Small Heat Shock Protein Protein Gene And Its Preliminary Research

When exposed to heat, cold, drought, water or other stresses, heat shock proteins are induced in plants, among which low molecular weight of 12-40 kD small heat shock protein are the most abundant and they are highly conservative in its C-terminal which contains conserved domain of ACD. HSP synthesis is carried out quickly, even though plants are keeping in the state of heat shock, generally the synthesis of HSP last only a few hours.There are at least five kinds of sHSP in higher plants, which are localized in the cytoplasm, chloroplast, endoplasmic reticulum, mitochondria and peroxisome. The HSP mainly involve in peptide folding in vivo, protein assembly and transport, protection of irreversible protein denaturation, maintain the protein in normal folding state, promote degradation of misfolded proteins under variety stresses and have chaperone function, In recent years, particularly the sHSP research is the most important field about plant resistance to variety environmental stresses. We use Lily as research materials, the main results are as follows:1. According to EST sequence (GenBank Acc:ES370516) we design nested PCR primers, use RACE technology to obtain the full length cDNA sequence of sHSP, which has high expression in lily anther meiosis prophase I late zygotene to pachytene and sequencing, define it as LimHSP 16.45.2. Bioinformatics analysis shows:LimHSP 16.45 has homology with other plant sHSP genes. Amino acid sequence alignment and phylogenetic analysis show that the amino acid sequence of LimHSP 16.45 has high homology with other plant cytoplasmicⅡsHSP, the amino acid sequence of these genes have two highly conserved regionsⅠandⅡin ACD domain and have lower conservative in the N-terminal. Arabidopsis mesophyll protoplast transformation for transient expression of LimHSP 16.45 gene shows it located in the cytoplasm, which is identical with homology analysis and phylogenetic tree classification.3. Expression analysis shows the expression of LimHSP 16.45 is universality in lily, that is it expresses in the root, stem, leaf and anther (meiosis prophasel late zygotene to pachytene period), but significantly higher in the anther than in other tissues. When exposed the anther which in meiosis prophase I late zygotene to pachytene at 42℃high temperature and 4℃low temperature, treated 0h,2h,4h, 6h,8h, 10h respectively, then carried out RealTime-PCR detection, the result showed the expression of sHSP mRNA significantly increased in pace with the treated, both show a expression peak at the fourth hour, the expression level gradually decreased subsequently.4. Cloned the LimHSP 16.45 gene into the pET28b prokaryotic expression vector, transformed prokaryotes E. coli to study its function, the result showed the growth curve of transgenic lines was identical with the control when culture at 37℃. IPTG induction of protein expression, found the growth curve of transgenic lines into the exponential phase and the plateau was earlier than the empty vector control, implied the cell viability of transgenic lines higher than empty vector cells; IPTG induced of protein expression and 45℃high temperature heat shock 2h, found transgenic lines into the exponential growth and the plateau significantly earlier than the empty vector control, implied the cell viability of transgenic lines higher than the empty vector cells, showed LimHSP 16.45 had an important influence on cell viability of E. coli.