Abstract: 【Aim】 Monochamus alternatus, an important stem borer in pine forests in southern China, is a major vector of pine wilt disease. It has a wide range of distribution and strong resistance to temperature stress. The study aims to better understand the molecular mechanism of resistance to temperature stress in M. alternatus. 【Methods】 The full-length cDNA encoding small heat shock protein (sHSP) was cloned
from M. alternatus by RT-PCR and RACE techniques. The sequence characteristics of this sHSP were analyzed by bioinformatics methods. The expression patterns of this gene in different developmental stages, various tissues of the 4th instar larvae and the 4th instar larvae stressed under different low and high temperatures were detected by qPCR, and the stability of the reference genes at different temperatures was analyzed by geNorm, NormFinder and BestKeeper tools. 【Results】 The complete cDNA of the gene encoding sHSP was obtained from M. alternatus, and named MaltHSP21.20 (GenBank accession no.: MH091811). The complete cDNA (871 bp) encodes a protein of 187 amino acids, with a signal peptide of 18 amino acids in N-terminus, the molecular weight of 21.20 kD and the theoretical pI of 8.65. The domain prediction conforms to the characteristics of the sHSP family, containing 10 β-sheets and 7 β-sheets in the α-domain. Phylogenetic tree analysis showed that the sHSP sequence shares high homology with sHSP of Anoplophora glabripennis. The most stable reference gene was different identified by different methods, and assessed by three methods the most stable reference gene was RPL10. MaltHSP21.20 was expressed in all developmental stages of M. alternatus, with the highest expression level in diapause larvae, and moderate in egg and pupal stages. MaltHSP21.20 was expressed in different tissues of the 4th instar larvae, with the highest expression level in fat body. The expression of MaltHSP21.20 in the 4th instar larvae showed no response to low temperature, while was up-regulated significantly at 35℃, reached the maximum after exposure to 45℃ for 2 and 3 h, and down-regulated at 50℃. 【Conclusion】 RPL10 is a more stable reference gene under different temperature stress. Compared to other heat shock protein genes, MaltHSP21.20 is less sensitive to low temperature and presumably plays an important role in larval diapause during overwintering and the resistance to high temperature.

Key words: Monochamus alternatus, small heat shock protein, temperature stress, reference gene, expression pattern