Screening Of The Reference Gene And Expression Analysis Of The Heat Shock Protein Genes Of Dendroctonus Valens

Dendroctonus valens is an alien invasive pest that occur heavily in pine in China.It mainly invades the cadres and roots of pine such as pinus tabulaeformis and pinus camphoris by means of larvae,resulting in pine growth impaired,even the whole plant dead.Dendroctonus valens mainly wintered in the roots of pine trees as adult and as mature larvae,since it was introduced into shanxi from the United States in 1998,it has been spreading northward to Inner Mongolia,liaoning and other places,and its cold tolerance has been gradually enhanced.Because of the heat shock proteins(HSPs)can induce the peptide chain to refold when it has been misfolded by external stimul,in order to define the role of hsps in the resistance to low temperature stress,this research through the transcriptome data of Dendroctonus valen to obtain the candidate internal reference genes,and the real-time quantitative PCR was used to analyze the expression of genes in each sample,and the stability of genes was evaluated by three biological software,ge Norm,Norm Finder and Best Keeper.Meanwhile,real-time quantitative PCR was used to analyze the expression patterns of hsps in adult and mature larvae under different low temperature treatment.The results are as follows:1.The stability of candidate reference genes in adult and mature larva under different low temperature stress conditions was determined.By mining and analyzing transcriptome data,gene sequences of six candidate reference genes(ACT,TUB,SHDA,PRS18,18 S r RNA and CYP4G55)were obtained in this study.Under different low temperature stress,the stability sequencing of the six candidate reference genes in adult was 18 S r RNA> PRS18> TUB> SDHA> ACT> CYP4G55,and that of mature larvae was TUB>18S r RNA> SDHA> PRS18> CYP4G55> ACT.Among them,18 S r RNA and TUB was the most stable internal reference gene expressed in the adult and mature larva of Dendroctonus valens under different low temperature stress,respectively.2.The expression patterns of heat shock protein in the mature larva and adult under different low temperature stress were revealed.The gene sequences of four heat shock proteins(hsp20-1,hsp20-2,hsp70-1 and hsp70-2)were obtained from transcriptome data.In both mature larva and adult,four heat shock proteins all responded to low temperature stress,but the degree of response to temperature was different.Under the present experimental conditions,hsp20-2 and hsp70-2 can be induced by low temperature in both mature larva and adult,and when the expression level is highest in mature larva,the temperature is-5? and-10? respectively,and in adult is 5? and 10? respectively;Hsp20-1 and hsp70-1 can be induced by low temperature in mature larvae,and the expression levels reach the highest at-10? and 5?,respectively.The expression levels of hsp20-1 and hsp70-1 in adult all decrease compared with the control at each temperature,and were not induced by low temperature.To sum up,the stability sequencing of each internal reference gene in different developmental stages under the same low temperature treatment is not the same,at the same time,the types and expression patterns of the heat shock proteins that can be induced by low temperature are different,and there may be a synergistic effect among the heat shock proteins to resist the influence of low temperature stress,which provides an important reference for further revealing the cold-resistance mechanism of Dendroctonus valens.