AUTHOR=Jin Jisu , Zhao Meiting , Wang Yao , Zhou Zhongshi , Wan FangHao , Guo Jianying 

TITLE=Induced Thermotolerance and Expression of Three Key Hsp Genes (Hsp70, Hsp21, and sHsp21) and Their Roles in the High Temperature Tolerance of Agasicles hygrophila

JOURNAL=Frontiers in Physiology

VOLUME=10

YEAR=2020

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2019.01593

DOI=10.3389/fphys.2019.01593

ISSN=1664-042X

ABSTRACT=<p>Thermal adaptation plays a fundamental role in the expansion and distribution of insects, and heat shock proteins (Hsps) play important roles in the temperature adaptation of various organisms. To determine the roles of Hsp genes (<italic>Hsp70</italic>, <italic>Hsp21</italic>, and <italic>sHsp21</italic>) on the high temperature tolerance of <italic>Agasicles hygrophila</italic>, we obtained complete cDNA (complementary DNA) sequences for <italic>Hsp70</italic>, <italic>Hsp21</italic>, and <italic>sHsp21</italic> by rapid amplification of cDNA ends (RACE), analyzed their expression profiles under different high temperature treatments by quantitative reverse transcription polymerase chain reaction (RT-qPCR), and performed functional verification by RNA interference (RNAi). The open reading frames of <italic>Hsp70</italic>, <italic>Hsp21</italic>, and <italic>sHsp21</italic> were 1940, 543, and 567 bp, encoding 650, 180, and 188 amino acids, respectively. Their molecular weights (MWs) were 71.757, 20.879, and 21.510 kDa, and the isoelectric points were 5.63, 6.45, and 6.24, respectively. Phylogenetic tree analysis showed that the <italic>Hsp70</italic>, <italic>Hsp21</italic>, and <italic>sHsp21</italic> genes of <italic>A. hygrophila</italic> were relatively conserved in evolution. The <italic>Hsp70</italic> and <italic>Hsp21</italic> genes in <italic>A. hygrophila</italic> were homologous to those in <italic>Leptinotarsa decemlineata</italic> (87 and 79% similarity, respectively), and the <italic>sHsp21</italic> gene in <italic>A. hygrophila</italic> was homologous to that in <italic>Lissorhoptrus oryzophilus</italic> (74% similarity). The amino acid polypeptide chain had highly conserved sequences of DLGGGTFD, VLVGGSTR, and GPTIEEVD. The sequence of EEVD was the characteristic motif of cytoplasmic <italic>Hsp70</italic>, and the highly conserved sequences of MALFR and MSLLP were characteristic sequences of <italic>Hsp2</italic> and <italic>sHsp21</italic>, respectively. Relative quantitative real time PCR showed that the three <italic>Hsps</italic> could be induced by 4-h treatment at high temperatures. Significant upregulation of these <italic>Hsps</italic> was observed when the temperature was further increased. The RNAi results showed that the injection of the three <italic>Hsps</italic>’ dsRNA could suppress the expression at the gene level significantly. Compared with the control group, high temperature heat shock reduced the fecundity of <italic>A. hygrophila</italic> significantly, and the fecundity decreased with the increase in temperature. Our results suggest that <italic>Hsp70</italic>, <italic>Hsp21</italic>, and <italic>sHsp21</italic> might play key roles in high temperature adaptation of <italic>A. hygrophila</italic> and help improve our understanding of their mechanism of thermotolerance.</p>