| The total length of Hsp70 cDNA sequence was cloned by RT-PCR from the total RNA extracted from the heat stressed mice livers at 40°C for 1h with the method of TRIzol, afer a pair of primers was designed according to the Hsp70 cDNA sequence in Genbank, and the sequence and its molecular evolution were analysised. At the same time, embryos that cultured to blastocyst stage were divided into 7 groups with 50 blastocysts in each group. They were exposed to either 40°C or 38°C for 1h, 2h and 3h, and followed by 37°C for 3h, 2h and 1h, respectively. Another group of blastocysts was cultured at 37°C as a control, and all the groups were replicated for 3 times. After the total RNA of embryonic cells was extracted, a pair of primers was designed according to the sequence cloned by RT-PCR and published in Genbank. Then the expressive level of Hsp70 mRNA in heat stressed mice blastocysts was detected by semiquantitive RT-PCR and real-time RT-PCR with the interior standard. The results were as follows:1. The total length of Hsp70 cDNA sequence with 1926bp was cloned successfully by RT-PCR and was compared with other Hsp70 sequence in Genbank by BLAST. The homology with other sequence in Genbank was 99.3% by BLAST. Thirteen sites were mutated that induced replacement of 7 amino acids and the homology of protein was 98.8%. But the isoelectric point had no change and still was 5.516. The analysis of secondary structure of the protein showed that the percentage of α -Helix, β-Sheet,Turn and Coil were 39%, 10%, 6% and 45%, respectively.2. The molecular evolution of Hsp70 between mice and other species were analysised with DNA Star protean and the results showed that the homology of Hsp70 in mice and Hsp70-1A, Hsp70-1B and Hsp70 in rat, Hsp70-1A and Hsp70-1B in bovine, Hsp70-1A and Hsp70-1B in human and Hsp70 in dog are higher than in other species, and the molecular evolution of Hsp70 between mice and goat is 81.1% barely.3. The semiquantitive analysis of Hsp70 mRNA expression in heat stressed mice blastocysts was proceeded with 18S rRNA as interior standard and the result showed that each group of blastocysts, including the control group, had Hsp70 gene expression. The level of Hsp70 mRNA in cells of heat stressed mice blastocysts at 38°C 1h had no significant change, but peaked at 2h of heat stress and then declined; The level of Hsp70 mRNA in cells of heat stressed mice blastocysts peaked at 1h of 40°C heat stress, which was 5.893-fold higher than that in the control group and then declined with the duration of heat stress.4. The expressive level of Hsp70 mRNA in the mice blastocysts was detected by real-time RT-PCR with 18S rRNA as external standard and the result showed that the Hsp70 mRNA in the blastocysts increased slightly after heat stress at 38°C for 1h, which was 4.59-fold higher than that in the control group, and it reached a peak after 2h, which was 137.19-fold higher than that in the control group, then declined and reached 29.86-fold higher than that in the control group; the Hsp70 mRNA in the blastocysts reached a peak after heat stress at 40°C for 1h, which was 103.97-fold higher than that in the control group, then declined and it was only 51.98-fold and 27.86-fold, respectively, higher at 2h and 3h than that in the control group.The above results indicate that the mice blastocysts are sensitivity to heat stress and the inducible Hsp70 gene in mice blastocysts could be induced by a slight heat stress within a short time. The Hsp70 mRNA in embryonic cells reaches the peak at 2h and 1h, respectively, after 38°C and 40°C treatment.
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