| Heat Shock Proteins (HSPs) are named Heat Stress Proteins, which are conservative and ubiquitous in prokaryote and eukaryote. Under the condition of stresses, such as heat shock, cold shock, organics, heavy metals, oxidant injury and ischemia, gene damage, tissue wound, microorganism infection and so on, synthesis of most of normal proteins and mRNA transcription are inhibited in vivo, simultaneously some stress proteins are synthesized immediately which are able to resist bad environment.Ratites norvegicus heat shock protein 4 (Hspa4), which are also named ischemia responsive 94 kDa protein (irp94), can keep organism avoiding damage by participating to heat shock response and responding unfolding protein. In normal physiological condition, it participates to ATP dependent protein trafficking, folding and assembling process. Hspa4(HSP70)is an important molecular chaperone in mammalian cell with strong mobility, and has the function of preventing protein denaturation, under the press of heat shock and other stresses, Hspa4 might participate to remove denatured proteins in vivo, to prevent denatured proteins coacervation and cell damage.Hspa4 gene was amplified by RT-PCR from Ratites norvegicus in this study, and was applied to construct overexpression vectors and derivation expression vectors, which were transformed into rice callus. The homomycin resistance gene in the vectors was used to select transgenic plants. The plant reconstructed by gene engineering could have better antireversion force. And the crop yield would also be increased.The main results were as follow:â‘ Cloning and sequence analysis of Hspa4 gene from Ratites norvegicus The full cDNA of Hspa4 was cloned by RT-PCR. The results showed that the cDNA was 2530 bps in length and contained an open reading frame (ORF) of 2523 bps, which encoded 840 amino acids. Compared to Hspa4 in Genbank, the sequence homology of nucleotide sequence were 99.96%. Only one base mutated, which located 1682bp, with T mutated into C in this base that induced Met changed into Thr. The sequence homology of amino acids sequence showed that either Met or Thr was in this situs. Furthermore the result of stereo chemical structure analysis certificated that the cloning sequence was the same stereo chemical structure as primitive sequence.â‘¡Cloning and transient activity analysis of higher plant promoters Three plant promoters were cloned by PCR in this study. There had two constitutive promoters: one was maize Ubi-1 promoter, and the other was rice Actin promoter (GenBank accession no: EU155408). Both two promoters had transient activity by GUS Histochemical Assay, and their transformation efficiency was much higher than CaMV 35S promoter, which made overexpression of genes. We also cloned Arabidopsis thaliana inducible promoter (Rd29A), which was induced by some factors including high salt, low temperature and drought. The promoter could drive the overexpression of genes when it was induced by those factors, which could raise crops resistance.â‘¢Construction of plant expression vectorsWe constructed six plant expression vectors. Three of them were pBI121-Ubi-GUS, pCAMBIA1301-Actin-GUS and pCAMBIA1301-Rd29A-GUS, and GUS gene were driven by Actin, Ubi-1 and Rd29A promoters respectively. The other three vectors were pCAMBIA1301-Ubi-Hspa4, pBI121-Hyg-Ubi-Hspa4 and pCAMBIA1301-Rd29A-Hspa4, and Hspa4 gene were driven by promoters Ubi-1 and Rd29A.â‘£Gaining and detection of transgenic plantsSix plant expression vectors were transformed into rice through Agrobacterium-mediated. Now we got about one hundred transgenic plants. 66 plants had been detected by PCR, and 27 plants were positive transgenic plants.
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