Molecular Mechanism Of Heat Shock Transcription Factor AhHsf Regulating The Expression Of AhHsp70 In Agasicles Hygrophila

Alternanthera philoxeroides(Mart)Griesb(Amaranthaceae)is an aquatic weedy species native to South America.It has been recognized as a noxious weed all over the world.In China,the alligator weed was firstly introduced into China in the 1930 s initially as a forage crop and presently,it subsequently spread to the regions of southern China and has become one of the worst weeds in China.It is listed as one of the 16 most serious invasive species in China.At present,biological control is considered to be the most effective method for the control of A.philoxeroides.The Agasicles hygrophila Selman & Vogt was the first host-specific insect introduced into the countries of the world to control the A.philoxeroides and achieved great success.However,the biggest problem with the flea beetle in our country is that it is greatly affected by temperature.According to field surveys in Hunan Province,the A.hygrophila has two obvious peaks during June to July and October to November,but it drops sharply during August to September,leading to an unefficient control of the alligator weed.We all know that heat shock proteins(Hsps)play an important role in the temperature adaptation of various organisms,and the expression of heat shock proteins is closely related to the organism tolerance.Previous studies have confirmed that three heat shock protein genes(Hsp70,Hsp21 and s Hsp21)play a key role in the high temperature tolerance of A.hygrophila(Jin et al.,2020).However,the molecular regulatory mechanism of heat shock protein genes is unclear.In order to reveal whether the heat shock transcription factor Hsf regulates the expression of heat shock protein gene to resist the high temperature and ensure the survival of offspring,in this study,we explored the molecular mechanism of heat shock transcription factor Hsf regulates the expression of Hsp70.Our results improve our understanding of the molecular mechanisms of insect feel high tempreture activation the AhHsf-AhHsps signaling pathway to promote the expression of Hsp70 in A.hygrophila.Our study helps improve the understanding of the mechanisms of thermotolerance in A.hygrophila at a molecular level,understand the adaptability of A.hygrophila to temperature changes,and predict the efficacy of bio-control using A.hygrophila in the face of climate change.The main results are as follows:1.A heat shock transcription factor AhHsf was identified and demonstrated that AhHsf is involved in regulating the transcriptional expression of two potential heat shock protein genes AHhsp70 and AhsHsp21,and its role in heat shock response of A.hygrophilaIn this study,one heat shock factor gene(AhHsf)was isolated and its two potential target genes(AhHsp70 and AhsHsp21)were confirmed.Sequence analysis indicated that AhHsf belongs to the Hsfs gene family.q PCR results showed that the heat shock factor AhHsf and two heat shock protein genes could be induced by high temperatures.RNAi results showed that injection of double-stranded AhHsf could significantly inhibit the expression levels of two potential target genes AhHsp70 and AhsHsp21,shortened the adult lifetime,reduce the amount of eggs,and lead to the decrease of fecundity.In addition,injection of dsAhHsp70 or dsAhsHsp21 also significantly affected AhHsf expression levels.2.Cloning and bioinformatics analysis of the promoter of AhHsp70 gene in A.hygrophila.In this study,the upstream promoter sequence of AhHsp70 gene was cloned by I-PCR.Bioinformatics analysis showed that the promoter sequence of AhHsp70 gene contains many functional elements.BDGP gene promoter prediction showed that AhHsp70 promoter sequence had a common TATA box at about-30 bp upstream of the transcription start site and that the transcription site started at the purine base about upstream-137 bp of the coding region of ATG.JASPAR and Tfsitescan predicted that the promoter had transcription factor binding sites at about-850 bp.Methprimer analysis showed that the promoter did not contain Cp G islands.3.Detection of AhHsp70 gene promoter activity in A.hygrophilaFive dual fluorescent reporter vector of the AhHsp70 gene promoter successfully constructed and transfected sf 9 cells.The results showed that AhHsp70 gene promoter sequence may contain core functional areas between 744 bp and 944 bp.q PCR results showed that AhHsp70 gene could be expressed in transfected sf 9 cells,and the expression level of AhHsp70 gene increased with the extension of transfection time.The results showed that the AhHsp70 promoter expression vector was successfully transfected into sf 9 cells,and the AhHsp70 gene promoter may contain core functional areas and have promoter activity.4.Interaction analysis of AhHsf and AhHsp70 promoter of A.hygrophilaThe transcription factor(AhHsf)overexpression vector PIZ/V5-HIS-Hsf was successfully constructed and co-transfected with the AhHsp70 promoter expression vector into sf 9 cells.The double luciferase reporting system was used to detect the influence of overexpression of AhHsf on the activity of target gene AhHsp70 promoter and the RT-q PCR was used to determine the influence of overexpression of AhHsf on the expression level of target gene AhHsp70 at the transcriptional level.DLR system results showed that overexpressed AhHsf could enhance the activity of AhHsp70 gene promoter.q PCR results showed that overexpression of AhHsf could promote the expression level of AhHsp70 in transfected cells.In summary,this study found that an important transcription factor AhHsf can regulate the expression of AhHsp70,and the upstream regulatory mechanism of AhHsp70 was preliminarily revealed.The results showed that the combination of AhHsf and AhHsp70 upstream core promoter region(-744bp--944bp)activated the Hsf-Hsp pathway at the transcriptional level,and promoted the transcriptional expression of AhHsp70 to protect the A.hygrophila from high temperature damage..