Cloning And Stress-induced Expression Analysis Of Tobacco Cytoplasmic Small Heat Shock Protein HSP17.8 Gene

During the life of plant, most of them rarely survive in entirely appropriate environment. Abiotic stress have more effects on growth and development of plants and the range of cultivation, Small Heat Shock Proteins (sHSPs) is the response of the planst to external stress, which is one kind of important stress-induced proteins. Under stress, increasing expression can strengthen the ability of the plants to resist various stress factors which play a very important role in plants and the environment long-term co-evolution process as well as has an important significance in taking it as a study of Stress Physiology.In this paper, the full-length cDNA of sHSPs gene was coloned from the laminae of Nicotiana tabacum L. by RT-PCR and RACE. The open reading frame (ORF) of NtHSP17.8 gene was subcloned to construct recombinant plasmid. Northern blot showed that NtHSP17.8 gene expression was induced by heat shock and drought. The major experiment results of this research are as follow:1. Molecular cloning and characterization of NtHSP17.8 gene:The total RNA was extracted from the laminae of Nicotiana tabacum L. A pair of specific primers were designed according to the conserved nucleotide sequence of different plants.446 bp sequence was obtained by RT-PCR. By application of RACE, the full length cDNA was 876 bp, including an open reading frame(480 bp) which encoded 159 amino acids with a deduced molecular weight of 17.8 KDa. The gene was named as NtHSP17.8.2. Bioinformatics analysis:Compare the gene with other small heat shock protein homologous sequence alignment, results show that the gene and cytoplasmic calss I sHSPs family clustered together. From the amino acid sequencewe can draw the conclusion that the gene with other plant cytosolic class I sHSPs highly homologousso. So we speculate that the gene is cytosolic class I sHSP genes. We can also make a speculation of Nicotiana tabacum L. small heat shock protein secondary structure and three-dimensional structure that secondary structure are highly consistent and three-dimensional structure is very similar to the template crystal structure of the complex transition state which sequence consistency is about 70%.3. Northern blot:Tobacco leaves under normal growth conditions could not detect NtHSP17.8 gene transcripts, however, tobacco leaves at sing stress of different heat temperatures or drought stress detected the accumulation of NtHSP17.8 mRNA, indicating that NtHSP17.8 is a gene of specific expression of induction of heat shock and drought. The contrast the results of tobacco seedlings under a single stress (heat shock or drought) and the cross-stress (heat shock+drought) show that the NtHSP17.8 expression under the cross-stress (heat shock+drought) obviously increase, indicating that the overexpression of NtHSP17.8 can improve stress resistance of tobacco under temperature stress and drought stress.4. Stress induced expression:Induction results show that expression situation of the plant small heat shock protein expression under single stress has made great progress, however, the study of cross-stress which often encountered in the natural environment should be further study; If we contrast the three Plant, the induce expression of small heat shock protein of tabcoo is in need of more in-depth study.