The Effect Of Proline On The Physiological Indexes Of Cold Tolerance Of Chinese Honeybee And Its Transcriptome Analysis

As a unique bee species in China,Apis cerana cerana Fabricius can adapt to the complex topography of mountain forests,and its cold tolerance is stronger than that of western honeybees.Therefore,it has important ecologieal and eeonomic significance for the plants pollination of the mountain forests in the early spring and late autumn.Even so,the mortality of A.c.cerana during the overwintering period is still high,so overwintering management for A.c.cerana has been an important factor influencing the development of the bee industry.In the study of Apis mellifera ligustica,there is evidence that proline may be an important antifreeze.Based on the systematic detection of the effect of proline on the cold tolerance index of A.c.cerana,and combined with transcriptome analysis,this study discusses the possibility of proline as a winter feed additive.The results of this study wil]help to optimize the preparation of winter bees for bees,thereby improving the wintering success rate of A.c.cerana.The main results of this study are as follows:1.The main cold tolerance physiological indexes of A.c.cerana in different seasons were measured.The change rules are found as follows:(1)The supercooling point(SCP)and freezing point(FP)of the adults bees show obvious seasonal changes,both of which show an increase with the rising of temperature.And the supercooling point during the overwintering period is significantly lower than the growth season(P<0.05).(2)The free water content and bound water content of the bees show obvious change trend with the seasons.It shows that the free water content increases with the rising of temperature,while the bound water content shows the opposite trend.The free water content in bees during overwintering period is significantly lower than that in the growth season(P<0.05),but there is no significant differences in the growth season(in June and August)(P>0.05).(3)The total fat content of the bees increases with the decrease of temperature,and the total fat content during overwintering period is significantly higher than that of the growth season(P<0.05),but there is no significant differences in fat content during the growth season(P>0.05).(4)The glycogen content of the bees first increases and then decreases.In the early winter,the glycogen content is the highest.During the overwintering period,the glycogen content decreases significantly(P<0.05).(5)The protein content of the bees decreases with the rising of temperature.At the early winter,the protein content increases significantly(P<0.05).After the wintering period,the protein content decreases significantly with the rising of temperature(P<0.05).2.Feed the winter bee with the proline-free syrup and the sugar water containing 1%,3%,and 4.35%proline for 2 weeks,(S0,S1,S3,S4.35),and then measure the over-cooling point SCP.The results show that the SCPs of S1,S3 and S4.35 are significantly lower than S0(P<0.05),while the SCPs of S1,S3 and S4.35 have no significant differences(P>O.05).Feed the overwintering bees with the syrup containing 1%proline and the syrup without proline for 2 weeks(C0,C1),and then measure the cold tolerance physiological indexes.The results are as follows:(1)The supercooling point and freezing point of C1 are significantly lower than CO(P<0.05).(2)The free water content of Cl is significantly lower than CO(P<0.05),and the bound water content is significantly higher than CO(P<0.05).(3)There is no significant difference in fat content between the two groups(P>0.05).(4)The glycogen content of C1 is significantly higher than C0(P<0.05).(5)The protein content of C0 is significantly higher than that of C1(P<0.05).3.Through the conduct of RNA-seq for 12 samples of LT0,LT1,NT0,NT1(LT0:0%,0?;LT1:1%,0?;NT0:0%,25?;NT1:1%,25?),a total of 82.20 GB of clean bases are obtained,the samples data amount is above 6.2 GB,and the Q30 value is between 93.6%-94.6%.By comparing LT0-LT1,LT0-NT0,NT1-LT1 and NT1-NT0,820 differential expression genes(408 up-regulated genes,412 down-regulated genes),644(307 up-regulated genes and 337 down-regulated genes),229(129 up-regulated genes and 100 down-regulated genes)and 673(287 up-regulated genes and 396 down-regulated genes)are obtained.There are 28 differential expression genes in bees fed with 1%proline syrup and without proline at low temperature stress.There are 45 differential expression genes in bees fed with 1%prolme syrup and without proline at low temperature(LT0-LT1)and normal temperature(NT0-NT1).Analysis of transcriptome data reveals that bees can adapt to cold environments at low temperatures in the following ways:(1)Under low temperature stress,the A.c.cerana changed the expression of DLAT,PDHB,AKR1A1,GALM and NAGLU genes in the Glycolysis/Gluconeogenesis pathway,so that the TCA cycle is weakened,the glycogen utilization is reduced,and the energy is stored to protect the cold;Under the same conditions,the addition of proline to the diet also weakens the glycolytic pathway,and the Insulinreslstance and Glucagon signaling pathways are activated,which increases the glycogen content in the body and can make them adapt to colder environments..(2)Under low temperature stress,the bees can increase the content of free amino acids in the body by change the expression levels of DLD,PHGDH,CHDH,GRHPR and other genes in the metabolic pathways of glycine,serine and threonine,as well as tyrosine,alanine and tryptophan metabolism,to enhance the cold tolerance.After feeding the proline-containing syrup,the bees may have a reduced metabolic pathway of amino acids and increased protein content.Thus,the bees do not need to produce more amino acids to protect against cold,thereby improving cold tolerance.(3)Under low temperature stress,the bees can enhance the lipid metabolism and produce energy to protect against cold through changing the genes expression levels of CPT1A,ACSBG,ACADSB,GCDH and other genes in the lipid metabolism pathway.While adding proline to the diet has no obvious effect on lipid metabolism.4.The expression levels of 8 differentially expressed genes in the transcriptome were verified by qRT-PCR.The experimental results were consistent with the sequencing results,indicating that the RNA-seq data is reliable.