Study On The Difference Expression Patterns Of AFP Gene In Honeybees And The Preliminary Study On The Mechanism Of Cold Resistance In Individuals

Antifreeze proteins are a kind of candidate genes in the studies about cold resistance mechanism of insects,which could improve the adaptability and survival rate in response to the low temperature environment by reducing the freezing temperature non-colligatively and have no significant impact to melting point.Currently,the researches about AFP expression patterns and mechanism of cold resistant molecular in hymenopteran have not been reported.In this study,the candidate genes about cold resistance were investigated in different temperature treatments by transcriptome analysis of worker bees in A.c.cerana.To explore the physiological indexes of cold resistance in honeybees,the contents of glucose,glycerol and amino acids of A.c.cerana and A.m.ligustica with different temperature treatments were determined in this study.We cloned the full-length cDNA of AcerAFP and AmelAFP and determined the expression patterns of AFP in different development stages,tissues and genders.The expression levels of AcerAFP and AmelAFP of honeybee after cold treatments were measured by qRT-PCR and Western blot,and the expression levels of AFP were correlated with the contents of various cryoprotectants in A.c.cerana and A.m.ligustica.RNA interference was performed on the AFP gene in A.c.cerana and A.m.ligustica by feeding dsRNA,respectively.The expression levels of other cold resistance related protein genes were detected before and after RNAi.The thermal hysteresis activity of hemolymph in A.c.cerana before and after RNAi were measured by differential thermal scanners.Combined it with the THA test result of AcerAFP from heterologous expression,physiological functions of AcerAFP were presumed.The main results in our study were as follows:(1)501 DEGs were identified in A.c.cerana between different temperature treatments.A Gene Ontology analysis showed that DEGs were enriched in pathways related to sugar and amino acid biosynthesis and metabolism,as well as calcium ion channel activity and microtubule cytoskeleton.Additionally,heat shock proteins,zinc finger proteins and serine/threonine-protein kinases were differentially expressed between the two treatments.(2)The contents of glucose,glycerin and amino acids were significantly changed in different temperature treatments of A.c.cerana and A.m.ligustica.In order to against cold,A.c.cerana relied on glucose metabolism more than A.m.ligustica but A.m.ligustica relied on glucose accumulation more than A.c.cerana.There were 17 kinds of amino acids involved in the cold hardness of A.c.cerana,which were more than 15 kinds in A.m.ligustica.There were differences for change trendence of contents of amino acids between different temperature treatments.(3)A nucleotide alignment analysis showed that the entire coding sequence of AcerAFP and AmelAFP were 1095 bp and encoded a polypeptide of 365 amino acids.AcerAFP and AmelAFP both contained signal peptides but no transmembrane domain,and the physicochemical properties of the two sequences were neutral and hydrophilic proteins with stable structures.Amino acid sequence of AFP shared 94%homology with homologous proteins from other hymenopterans.AFP mRNA was higher expressed in the earlier development of A.c.cerana and later development of A.m.ligustica.AFP was highest expressed in Motor related organs and showed different expression patterns in different genders.(4)The expression levels of AFP were reduced by low temperature and process times and there were significant difference in expression patterns between different species.The expression levels of AFP were significantly correlated with the contents of glucose,glycerol and amino acids in the two species.The AFP mRNA expression levels were correlated with the expression levels of most antifreeze-related proteins genes in different species.By comparing THA results of hemolymph before and after the RNAi in A.c.cerana,and then combined it with the THA test result of AcerAFP from heterologous expression,these results showed AcerAFP could improve the cold resistance of the individuals by lowering the total freezing temperature.In conclusion,honeybees could improve cold resistance by regulating biosynthesis and metabolism of sugar and free amino acids,as well as with calcium channel activity and microtubule cytoskeleton.The cold tolerance mechanism of physiological level in honeybees was substance system including"glucose-glycerin-amino acids".There were significant differences in AFP expression patterns between different species,which may be closely related to the difference of biological characteristics and cold resistance in different species.Our study provided a new perspective for further study on the mechanism of cold resistance and gene function of AFP in honeybees and offered a scientific foundation for breeding of new cold-resistant strains.