| Apolygus lucorum is the primary pest in the cotton area of North China and even the whole country.Peristenus spretus is a dominant doparasitoid which can effectively control the mirid bug A.lucorum.However,the relevant reports have focused on the morphological characteristics,host species survey and reproductive biology related contents of P.spretus so far,,the reproductive-related genes and their regulation mechanisms are still unclear.Previous studies had found that Heat Shock Protein 83(HSP83)was higher expressed in the ovary of P.spretus,and there is a hypothesis that HSP83 may play an important role in the reproductive regulation of P.spretus.HSP83 is a molecular chaperone required for the growth and development of eukaryotes which belongs to the HSP90 protein family.In present study,hsp83 of P.spretus was cloned firstly,and the effect of heat shock on oogenesis and ovarian development was studied by immunofluorescence staining.At the same time,hsp83 effect on reproduction was analyzed by RNAi and heat shock.The results of this study are helpful to clarify the regulation mechanism of hsp83 on the reproduction of P.spretus,and lay a theoretical foundation for A.lucorum management,especially the field release system construction.The main results as follows:1.The full-length sequence of the HSP83 gene of P.spretus was obtained and analyzed.Degenerate primers were designed in the conserved region according to the amino acid sequence which was obtained by mass spectrometry sequencing,the full-length HSP83 cDNA(Pshsp83,accession number:MK165459)of P.spretus was obtained by RACE technique and cloned fragment.Pshsp83 complete cDNA is 2175 bp in length and encodes a protein of 724 amino acids with a calculated molecular mass of 83.4 kDa and a theoretical isoelectric point of 4.87.The full-length acquisition of this gene laid the foundation for antibody synthesis and its functional exploration.2.The expression of Pshsp83 in the female of P.spretus at different days after emergence and with different temperatures treatment was detected.The results showed that the expression level of Pshsp83 was different at different ages,and the gene was detected from 1-13 days post emergence and peaked at 13th day.At the same time,the effect of high temperature on the expression level of Pshsp83 was determined.It was found that with heat stress at 35? for 1h,the expression of Pshsp83 presented highest point,which was significantly higher than that at other temperatures.With the other temperature treatment(26?,29?,32? and 38?),there was no significant difference in the amount of Pshsp83 expression compared to the control(23?).3.Synthetic antibody and HSP83 protein subcellular localization ELFDEMAEDKENYK was selected as the antigenic peptide and used for the polyclonal antibody of HSP83.Firstly,antigenic peptide was injected subcutaneously with New Zealand white rabbit for 4 times.Polyclonal antibody was obtained by the whole blood.The ELISA determined the titer found that antibody titer,rabbit A>256 K,rabbit B>512 K,rabbit C>512 K,rabbit D>512 K.Immunofluorescence staining was used to detect the specificity of the antibody.The results showed that the antibody obtained from rabbit B had higher specificity and could bind well to HSP83 protein.And with the antibody screened,the HSP83 protein localization experiment showed that HSP83 protein expressed in the whole ovarian tube of P.spretus,among which the growing follicles were more expressed and germarium was less expressed,the expression of HSP83 protein at 35?was significantly higher than that at 23?.The results of expression level were consistented with the qPCR results.4.Preliminary study on the function of HSP83 in the ovary of P.spretus.The reproduction regulation of hsp83 in the P.spretus was studied with RNAi and heat shock.Firstly,Pshsp83 dsRNA(404 bp)was synthesized in vitro by RT-PCR.Pshsp83 dsRNA was injected into the female of P.spretus by microinjection.The expression level of the target gene Pshsp83 was detected by qPCR,and its effect on reproductive index was observed in P.spretus.The results showed that the expression level of Pshsp83 in female decreased by 21.8%after injection of target dsRNA comparing with the control group(injected gfp dsRNA),while the other parameters including longevity of female,sex-ratio and hatchability of the offspring were not significantly different.Furthermore,the effect of high temperature on the reproduction of P.spretus was detected with heat shock(35?).The results showed that the expression of Pshsp83 in the female of P.spretus was significantly increased after heat shock compared with the control group(23?),and longevity of female was extended by 51%,indicating the high temperature had an significant influence on the expression of Pshsp83,and heat shock(35?)exerted an affect on reproduction of P.spretus. |
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