Abstract: It is one of important strategies for many insects to produce antifreeze proteins (AFPs) to resist low temperature. In order to study the mechanism of cold resistance, an antifreeze protein gene Tm-afp was cloned from the larvae of yellow- and black-color varieties of Tenebrio molitor, the nucleotide sequence and the encoded amino acid sequence characteristics of Tm-afp were analyzed, and the mRNA levels were examined in the two varieties by reverse transcriptional PCR and rapid amplification of 5′ and 3′ cDNA ends. The results showed that the full cDNA sequences of Tm-afp cloned from the yellow- and black-color varieties of T. molitor were 579 bp and 588 bp in length, respectively. Both contain a 20 bp 5′ non-coding region, a 402 bp open reading frame and a variant 3′ non-coding region, and their nucleotide sequence identity is 95%. Because of two amino acid variation (D35→E35 and T130→S130) existing between the two mature peptides encoded by the two cDNA sequences, they were considered two isoforms of Tm-afp , i.e.,Tm-afp -1 and Tm-afp -2, respectively. Three amino acid variation (G2→A2, S9→T9 and Y27→N27) exists between signal peptides of the antifreeze protein isoforms (Tm-afp -1 and Tm-afp -2) encoded by Tm-afp-1 and Tm-afp -2. The 1st amino acid of the mature peptides of the two antifreeze protein isoforms is glutamine. Both the mature peptide sequences contain eight short-tandem repeats, each composed of twelve highly conservative amino acid residues. Furthermore, both the 2nd and 8th amino acids of each repeat are cysteines. In addition, Tm-afp -1 and Tm-afp -2 both belong to threonine- and cysteine-rich insect antifreeze proteins. Their secondary structure consists of beta sheets and random coils, and their tertiary structure has eight special right-handed beta helixes, composed of twelve amino acid residues per helix. Beta sheet, composed of conservative XCT, arrays on the lateral surface of the beta helixes. Meanwhile, low temperature up-regulated the expression of Tm-afp in yellow- and black-color varieties of T. molitor. But under long time of cold induction, the relative mRNA expression level of Tm-afp in the black-color variety of T. molitor was significantly higher than that in the yellow-color variety. The results suggest that Tm-afp among different color varieties of T. molitor may have several isoforms and Tm-afp in the black-color variety of T. molitor is more sensitive to cold than that in the yellow-color variety. This study may provide a useful reference to the further research on the molecular mechanisms of cold resistance in T. molitor.

Key words: Tenebrio molitor, color variety, antifreeze protein (AFP), gene isoform, sequence analysis, mRNA expression