| Heat shock proteins HSPS are a kind of stress proteins of a class,they spontaneously produce a defence mechanism while they are experiencing adverse biological environments such as heat stress and stimulation and so on.They can enhance the cell tolerance and recovery and act as molecular chaperone.Among this family the heat shock protein is the most conservative,including nulceic acid and amino acid sequences,both of them are high conserved.Monitoring the expression level of heat shock protein HSP70 in vivo can be used as a biological indicator of environmental pollution as a kind of evaluation.Now the chemical pesticide dosage of the agricultural field is very great,while we control the pest we do harm to natural enemies of insectd,thus HSP70 has become a kind of resistance indicator to the field stress stimulus.In the respect of natural enemies feeding and releasing to control the pest,we can make the expression levels of heat shock proteins increase by transgenic technology or other ways,thereby the defence capability to external adverse environment can be enhanced so that the natural enemies can withstand the intense stress conditions to preserve the natural enemies and at the same time control insect pest successfully,and this is a kind of protection of natural enemies from a genetic point of view,Now at home and abroad many articles have been reported about some of the gene sequence of heat shock protein of manu kinds of natural enemies,but the HSP 70 gene sequences of major natural enemies Chrysopa phyllochroma of legumes have not been reportes yet.In this study,we get the gene HSP 70 through cloning,and by heat shock and handling of peaticides which were commonly used in soybean fields,measured the changes of the expression levels of HSP 70,analyzed the action of protection of HSP 70 of Chrysopa phyllochroma under the stress environments.Chrysopa phyllochroma Wesmael belongs to Neuroptera,Chrysopidae.Feeding on Aphis glycines,Acyrthosiphon solani,bean thrips and other important pests of fields,but the study about the Chrysopa phyllochroma molecular biology is not reported.(1)Gene cloning:In this article,we have cloned the full-length sequence of Hsp70 cDNA of Chrysopa phyllochroma:this sequence contains a 2178 base including a length of 1944 bp open reading frame,encoding a protein with 648 amino acid residues of protein,its molecular weight is about 70.92kDa,the isoelectric point of pI was 5.27,and we had been submitted to GenBank and obtain login number KF311064.Cloning of the full-length sequence of Hsc70 cDNA of Chrysopa phyllochroma:this sequence contains a 2466 base,including a 1956 bp open reading frame,encoding a protein with 652 amino acid residues,and the molecular weight is about 71.5kDa,isoelectric point pI is 5.54,and we have been registered to the GenBank and got the login number KJ599671.(2)Prokaryotic expression:Western blot analysis showed that,the prokaryotic expression results of recombinant plasmid pET/Hsp70 and pET/Hsc70 got in line with the expected results,confirmed that the expression of gene belongs to Chrysopa phyllochroma Hsp70/Hsc70 indeed.(3)The expression under different temperatures:using Real Time PCR technology,the relative quantitative analysis of expression levels of Hsp70 and Hsc70 of Chrysopa phyllochroma under different and the different temperatures stress showed that:The expression level of HSP 70 of Chrysopa phyllochroma larvae at 1,2 instar and adults is increasing with the raise of temperature,and can reach the most at 33?,under the temperature of 33-43?,its expression level of Hsp 70 delined.Consider 24? as control,the expression level of Hsp 70 of 1st instar at 33? is times of it is under 24?,2.4 times 2nd instar larvaes,3.0 times adults.The expression levels of 3rd instar larvae and pupae of Hsp 70 in a range of 4-37? are increasing with the raise of the raise of the temperature significantly,at 37? it can reach the most,the expression levels of Hsp 70 declined in a range of 37-43 ?.Take 24? as control,the expression levels of 3rd instar larvae at 37? is 10.3 times of it is at 24?,1.4 times pupae.The expression levels of Hsp 70 of each instar did not change significantly,were about 2 times around.The analysis of the expression levels of Hsp 70 of Chrysopa phyllochroma changing with different temperature show that,the expression levels of Hsp 70 in each instar of Chrysopa phyllochroma,increasing in a range of 33-37?,thus we can tell that it is sensitive of Chrysopa phyllochroma to this temperature range,where the 3rd instar larvae is the most sensitive,followed by adults.The expression levels of Hsp 70 did not change significantly.It can be seen that Hsp 70 played an important role under the heat shock stress that insects suffered.(4)The expression of different chemical treatments:during the chemical treatments,use two kinds of commonly used herbicide mixture of Quizalofop and Imazethapyr and Fomesafen,and also use two kinds of pesticide Omethoate,Chlorine Fluorine · Profenofos,with water as ck.Firstly detect the effects of different agents on different instars:under the mixture of Quizalofop and imazethapyr treatment,the expression levels of Hsp 70 of Chrysopa phyllochroma Ist,2nd,3rd larvae and pupae are higher than others.The expression levels of Hsp 70 of spraying the mixture of Quizalofop and imazethapyr of 1st larvae is 1.3 times of ck,4.4 times 2nd larvae,8.4 times 3rd larvae,3.2 times pupaes.Adults are more sensitive to Chlorofluorocarbons-Profenofos,the expression levels of Hsp 70 is 7.6 times of ck,Among the four pharmaceutical treatment,the expression levels of Hsp 70 of each instar did not change significantly.Secondly,detect the impact of same drug treatment on different instar larvaes:the order of more sensitive instar to Quizalofop and Imazethapyr are as follows,3rd larvae,2nd larvae,pupae,1st larvae and adults;the order of more sensitive instar to Fomesafen are as follows,3rd larvae,2nd larvae,adults,1st larvae and pupae;the order of more sensitive instar to Omethoate are as follows,adults and pupae;the order of more sensitive instar to Chlorine Fluorine · Profenofos are as follows,3rd larvae,2nd larvae,1st larvae and pupae.The orders all above are according to the analysis of change of expression levels of Hsp 70,the change of Hsc 70 is not obvious,By analysis of the impact of 4 kinds of pesticides on different instar of larvaes,we can tell that the herbicides had greater impact on 2nd and 3rd larvae of Chrysopa phyllochroma,insecticides had greater impact on adults and 3rd larvaes.Therefore when applying these agents in the fields,we should have avoided the sensitive period of Chrysopa phyllochroma,in order to avoid the destruction of Chrysopa phyllochroma.Research of temperature and chemical treatments on the expression of HSP70 in Chrysopa phyllochroma,and furtherly indicates Hsp70 is a stress protein that is present in vivo,under outer stress conditions can be highly induced;Hsc70 is a kind of constitutive protein,the external environment stimulates did not change the expression levels very much.Thus,HSP70 played an important role on heat shock resistance on Chrysopa phyllochroma and stimulation of pesticides,we can not only choose when to release Chrysopa phyllochroma according to the temperature of the field conditions,but also can avoid the sensitive period of leaf herbicides and pesticides of Chrysopa phyllochroma to spray pesticides. |
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