Gene Cloning And Expression Pattern Of Hsp70 From Aleuroglyphus Ovatus (Troupeau) Under Heat Stress

Aleuroglyphus ovatus(Troupeau) is affiliated to the Acaridae, as a stored grain pest it has a wide distribution in our country. It has serious harm to human’s economy and health. Temperature has a great influence on the population dynamics of A.ovatus. Biont has a range of suitable temperature for survival. When temperature exceeds this range will cause heat stress, this situation will induce the body to produce heat shock protein to deal with adversity. For these reasons we choose the heat shock protein 70(Hsp70) from A.ovatus as research material. Firstly, we cloned two full-length c DNA of AoHsp70 gene using reverse transcription polymerase chain reaction(RT-PCR) and rapid amplification of cDNA ends(RACE), then analyzes its bioinformatics. Secondly, we used realtime fluorescence quantitative PCR(RT-qPCR) to analysis the expression level of AoHsp70 during different developmental stages and thermal stress. The main conclusions are as follows:1. We cloned two full-length cDNA of AoHsp70 gene and were named AoHsp70-1 and AoHsp70-2 respectively. The AoHsp70-1 gene was deposited in GenBnak with the accession number KX237517. The overall length cDNA of AoHsp70-1 is 2307 bp. Its open reading frame(ORF) is 1974 bp which can encode 657 amino acid residues. Its protein molecular mass was 71.92 KDa and the isoelectric point is 5.27. Its amino acid sequence contains three signature sequences of Hsp70 family. There is a GGXP 4 peptide repeat sequence in C-terminal. It also has EEVD motif in C-terminal end which is the mark of cytoplasmic Hsp70. The AoHsp70-2 gene was deposited in Gen Bnak with the accession number KX237518. The overall length cDNA of AoHsp70-2 gene is 2623 bp. Its ORF is 1923 bp which can encode 640 amino acid residues. Its protein molecular mass was 70.64 KDa and the isoelectric point is 5.56. Its amino acid sequence contains three signature sequences of Hsp70 family. It also has EEVD motif in C-terminal end which is the mark of cytoplasmic Hsp70. There are some similarities between the amino acid sequences of AoHsp70-1 and AoHsp70-2. Such as both of them are missing signal peptide, they have six N-glycosylation sites and there are several hydrophobic regions in the amino acid sequence. Both of protein tertiary structure has Nucleotide binding domain(NBD) and Substrate binding domain(SBD). Multiple sequence alignment showed that Hsp70 had high degree of homology, the similarity of amino acid among different species was more than 60%. Used NJ method to build the system tree, the results showd that: first AoHsp70-1 forming a branch with AoHsp70-2, then they are formed a bigger branch with others Hsp70 from mites while all the other Hsp70 from insects are clustered into a large branch.2. The HSP70 expression levels at different decelopment(eggs, larvas, nymphs and female mites) are not the same. The expression level of AoHsp70-1 in female was slightly higher than other decelopment, but there was no significant difference. While the expression level of AoHsp70-2 was 5.01 folds of the control group. There was no significant difference in the expression level of AoHsp70-1 under low temperature stress. But the expression level of AoHsp70-2 was significantly increased under low temperature stress and achieve the maximum level at 5℃ which was 9.62 folds of the control group, then with the temperature further decreased the expression level of AoHsp70-2 began to decline. There was no significant difference in the expression level of AoHsp70-1 under high temperature stress. But the expression level of AoHsp70-2 was significantly increased under hige temperature stress and achieve the maximum level at 39℃ which was 23.97 folds of the control group, then with the temperature further increased the expression level of AoHsp70-2 began to decline. The different reponse between AoHsp70-1 and AoHsp70-2 to heat stress indicate: AoHsp70-1 and Ao Hsp70-2 are constitutive type and inducible type, respectively. The inducible type AoHsp70-2 has played a key role in combat stress in A.ovatus. It had a higher expression level at high temperature stress than at low temperature stress. This phenomenon indicated that A.ovatus is more sensitive to high temperature.