| The carmine spider mite,Tetranychus cinnabarinus(Boisduval),is one of the most important pests on cotton,vegetables and other crops;it is widely distributed in China.Because of high fecundity and short generation time,the mite can rapidly developing a certain number of population and resistance to acaricides easily in a short period of time,result in difficult to prevent and control.Heat shock protein 70 kDa(Hsp70s) is the most important proteins among members of heat shock proteins,and functions mainly as molecular chaperones.Moreover,they play important roles in cellular protection,anti-apoptotic effects,and immune therapy of tumor.Therefore,studies on Hsp70s have been one of important and prospective focuses in fields of life sciences.The four full-length cDNA of Tetranychus cinnabarinus(Boisduval) heat shock protein 70s were cloned by using the technique of rapid amplification of cDNA ends(RACE).The mRNA expression levels of four Hsp70 genes in female T.cinnabarinus which were dealed with different temperature and abamectin were detected by Real-time RT-PCR technology.In this study,we primarily discussed the inherent relationship between the expression of the Hsp70 genes in T.cinnabarinus and its fitness to environment stress from molecular level.This work was supported by the National Nature Science Foundation of China(No.30600059).The main results are as the following:Four Hsp70 cDNAs were isolated from carmine spider mite,Tetranychus cinnabarinus.They were tentatively named as TCHsp70-1,TCHsp70-2,TCHsp70-3 and TCHsp70-4.The complete cDNA of the T.cinnabarinus Hsp70-1 gene was deposited in GenBank under the accession numbers EU679412.The full length of TCHsp70-1 cDNA was 2446 bp,with a single open reading frame(ORF) of 1965 bp that encoded a protein of 655 amino acids.The theoretical molecular weight of TCHsp 70-1 based on the deduced amino acid sequence was calculated to be 71.28 kDa,with an isoelectronic point(pI) of 5.37.TCHsp70-1 cDNA included a 5' untranslated region(UTR) located 274 bp upstream of the putative start codon(ATG) and a 3' UTR of 207 nucleotides that ends in a poly(A) tail.A possible consensus signal sequence for polyadenylation (AATAAA) was located 14 bp upstream of the poly(A) tail.The complete cDNA of the T.cinnabarinus Hsp70-2 gene was deposited in GenBank under the accession numbers EU679413.The full length of TCHsp70-2 cDNA was 2305 bp,with a single open reading frame(ORF) of 2016 bp that encoded a protein of 672 amino acids.The theoretical molecular weight of TCHsp70-2 based on the deduced amino acid sequence was calculated to be 73.78 kDa,with an isoelectronic point(pI) of 5.26.TCHsp70-2 cDNA included a 5' untranslated region(UTR) located 189 bp upstream of the putative start codon(ATG) and a 3' UTR of 97 nucleotides that ends in a poly(A) tail.A possible consensus signal sequence for polyadenylation (AACAAA) was located 27 bp upstream of the poly(A) tail.The complete cDNA of the T.cinnabarinus Hsp70-3 gene was deposited in GenBank under the accession numbers EU679414.The full length of TCHsp70-3 cDNA was 2284 bp,with a single open reading frame(ORF) of 1980 bp that encoded a protein of 660 amino acids.The theoretical molecular weight of TCHsp70-3 based on the deduced amino acid sequence was calculated to be 71.86 kDa with an isoelectronic point(pI) of 5.39.TCHsp70-3 cDNA included a 5' untranslated region(UTR) located 134 bp upstream of the putative start codon(ATG) and a 3' UTR of 165 nucleotides that ends in a poly(A) tail.A possible consensus signal sequence for polyadenylation (AATAAA) was located 13 bp upstream of the poly(A) tail.The complete cDNA of the T.cinnabarinus Hsp70-4 gene was deposited in GenBank under the accession numbers EU977182.The full length of TCHsp70-4 cDNA was 2182 bp,with a single open reading frame(ORF) of 1956 bp that encoded a protein of 652 amino acids.The theoretical molecular weight of TCHsp70-4 based on the deduced amino acid sequence was calculated to be 70.9 kDa with an isoelectronic point(pI) of 5.40.TCHsp70-3 cDNA included a 5' untranslated region(UTR) located 106 bp upstream of the putative start codon(ATG) and a 3' UTR of 117 nucleotides that ends in a poly(A) tail.A possible consensus signal sequence for polyadenylation (AATAAA) was located 14 bp upstream of the poly(A) tail.The BLAST program analysis showed that the deduced amino acid sequences of the four TCHsp70 genes shared high homology with other known Hsp70 genes,they all contained the highly conserve functional motifs of the Hsp70 family,and the conservation of the N-terminus was higher than that of the C-terminus.Comparison of deduced amino acid sequences,the lowest identity of four Hsp70 genes was 57.33%(TCHsp70-1 and TCHsp70-2) and the highest identity was 91.90%(TCHsp70-3 and TCHsp70-4).The phylogenetic tree suggested that TCHsp70-1,TCHsp70-3 and TCHsp70-4 were cytoplasm Hsp70,TCHsp70-2 was endoplasmic reticulum Hsp70.Real-time comparative quantitative PCR was used to compare and analyze the expression levels of four Hsp70 genes in female T.cinnabarinus after being treated with different temperatures.The results showed that after cold shock(7℃,10℃),the expression levels of TCHsp70-1 genes was higher than that after heat shock(34℃,37℃),indicated TCHsp70-1 was more sensitive to cold shock.After cold shock(7℃,10℃),the expression levels of TCHsp70-3 genes was lower than that after heat shock(34℃,37℃),indicated TCHsp70-3 was more sensitive to heat shock.The expression levels of TCHsp70-4 genes increased both after cold shock(7℃,10℃) and heat shock (34℃,37℃),suggested it was sensitive to cold and heat shock.The expression level of TCHsp70-2 was stable after being treated with different temperatures and significant lower than that of TCHsp70-1,TCHsp70-3 and TCHsp70-4.These results possibly indicated the expression patterns of TCHsp70s were affected by their location in different cellular compartments and the four TCHsp70s, especially TCHsp70-1,TCHsp70-3 and TCHsp70-4 may play an important role in mediating tolerance to cold,thermal stress for T.cinnabarinus.Real-time comparative quantitative PCR was also used to compare and analyze the expression levels of four Hsp70 genes in female T.cinnabarinus within the susceptible strain(SS), abamectin-resistant strain(AbR) and 37℃high temperature domestication strain(HR).The expression levels of four Hsp70 genes in AbR and HR were higher than that in SS,and the highest expression level was in HR.All of the results have significant differences except the expression of TCHsp70-1 in SS and AbR and that of TCHsp70-2 in AbR and HR.The above results indicated abamectin and high temperature stress both can induce the expression of TCHsp70s,but the latter induced more;and abamectin induced the expression of TCHsp70s may play an important role on AbR which showed fitness advantages in high temperature. |
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