Gene Clone And Expression Of Heat Shock Protein Genes From Pardosa Pseudoannulata

Heat shock protein(HSP)widely exist in prokaryotic and eukaryotic cells in nature.It is a group of ancient and highly conserved special protein that can be induced and expressed by a variety of extermal stress factors.It makes the organism quickly and temporarily resist damage from adverse stress.Pardosa pseudoannulata(B?senberg et Strand),belonging to Arthropoda,Arachnoidea,Araneae,Lycosidae,is an important predatory nature enemy of rice field pests and faced with frequent changes of field conditions such as temperature.In order to understand the characteristics and temporal expression dynamics of HSP in P.pseudoannulata when exposed to high temperature or other stress environments,this paper studied its three HSP genes and molecular characteristics of HSP,and the relative expression levels of the m RNA at different time periods under different high temperature stresses.The main results are as follows:1.Obtined three HSP genes hsp70,hsp83 and hsp90By using polymerase chain reaction(PCR)and rapid amplification of c DNA ends(RACE)technology,the full-length c DNA sequences of three HSP genes of P.pseudoannulata were cloned and named Pphsp70,Pphsp83 and Pphsp90,and their Gen Bank accession numbers are MN820834,MN820835 and MN820833,respectively.2.Interpreted the main bioinformatic characteristics of Pphsp70,Pphsp83 and Pphsp90The full-length c DNA sequence of Pphsp70 is 2331 bp and contains a 1917 bp open reading frame(ORF).The polypeptide deduced from the ORF comprises 638 amino acids with a predicted molecular weight of 70.4 k Da and a putative p I of 5.6.It has a putative polyadenylation signal(AATAAA)at nucleotide 2287-2292 and EEVD sequences at the C-terminus.The full-length c DNA sequence of Pphsp83 is 2466 bp and contains a 2175 bp ORF.The polypeptide deduced from the ORF comprises 724 amino acids with a predicted molecular weight of 83.7 k Da and a putative p I of 4.91.It has a putative polyadenylation signal(AATAAA)at nucleotide 2419-2424.The pentapeptide MEEVD is at the C-terminus.The complete c DNA sequence of Pphsp90 is 2663 bp,including a 2346 bp ORF encoding 781 amino acids with a predicted molecular weight of 90.0 k Da and a putative p I of 4.88.It has a putative polyadenylation signal(ATTAAA)at nucleotide 2615-2620 and the sequences of HEEL at the C-terminus.Amino acid multiple sequence alignment shows that the amino acid sequences of Pphsp70,Pphsp83 and Pphsp90 were highly similarity to those of other spider families.Phylogenetic tree analysis revealed that Lycosidae is closely related to Theridiidae,Araneidae and Eresidae.3.Revealed temporal expression dynamics of heat shock genes in male and female adults of Pardosa pseudoannulata under different high temperature stressesThe relative expressions of Pphsp70,Pphsp83 and Pphsp90 m RNA were detected by q RTPCR in female and male adults of P.pseudoannulata under stress of different temperatures(25,28,31,34,37,40,43℃)for 12 h.The relative expression of m RNA of Pphsp70,Pphsp83 and Pphsp90 in the female and male adult spiders are the highest at 43℃.The expression levels of Pphsp70,Pphsp83,and Pphsp90 in female spiders at 43℃ were 143.4 times,4.5 times,and 3.6 times than that at 25℃(control group),and had highly significant differences(P<0.01);the expression levels of Pphsp70,Pphsp83,and Pphsp90 in male spiders at 43℃ were 152.7 times,4.2 times,5.4 times than that at 25℃,respectively,which were also highly significantly different from the control group(P<0.01).When exposed to 43℃ for different durations(4,8,12,16,20 h),the relative m RNA expression levels Pphsp70,Pphsp83 and Pphsp90 genes of male and female adult spiders were the highest at 4 h,which was significantly different from the control group(P<0.05).In conclusion,Pphsp70,Pphsp83 and Pphsp90 in P.pseudoannulata can be induced to express under high temperature stress and played a protective role within a certain temperature range and time range.The results implied that transitorily fast and efficient expresion of the three HSP genes,espicially Pphsp70,may be a survival strategy by balancing biological activities for the organism.The results provided certain theoretical basis and basic information for further understanding of the physiological and biochemical mechanism of paddy field spiders’ adaptation to temperature stress and research on protection and utilization of the spider in pest biocontrol.