Preliminary Study Of Carotenoids Between Tetranychus Cinnabarinus And Tetranychus Urticae With Underlying Its Mechanism

For a long time,the taxonomy between Tetranychus cinnabarinus and Tetranychus urticae is controversial.Based on body color difference,some foreign experts regared T.cinnabarinus as red form of T.urticae.However,the reason for body color difference between T.cinnabarinus and T.urticae remains unknown till now.The current study firstly conducted qualitative and quantitative analyses of carotenoids in T.cinnabarinus and T.urticae by non-targeted metabolomics and HPLC technique,which identify the difference in carotenoid composition and content between T.cinnabarinus and T.urticae.Then their differentially expressed genes related to carotenoid anabolism were analyzed by high-throughput sequencing.Finally,the mechanism of body color difference between T.cinnabarinus and T.urticae was explored by RNA interference(RNAi).The main results are as follows:1.Identifying composition and assaying content of carotenoids.Using non-targeted metabolomics and HPLC technique to identify and verify carotenoids in T.cinnabarinus and T.urticae,the result showed that both of them contained 13 kinds of carotenoids,but the total amount of carotenoids in T.cinnabarinus was higher than T.urticae,and the content of 5 kinds of differential carotenoids(neoxanthin,astaxanthin,α-carotene,β-carotene and γ-carotene)in T.cinnabarinus were higher than T.urticae.In addition,the content of β-carotene was highest of all.In order to further confirm the result,3 kinds of red spider mites(T.cinnabarinus kept in the laboratory,Panonychus citri Mc Gregor,Tetranychus truncatus Ehara)were verified by HPLC.It was also found that the total amount of carotenoids in these red spider mites was higher than T.urticae,and the content of β-carotene was highest in all.The result revealed that red body color of T.cinnabarinus might be derived from more accumulated carotenoids than T.urticae.2.Screening and analyzing differentially expressed genes related to carotenoid anabolism.There were 4079 differentially expressed genes between T.cinnabarinus and T.urticae by transcriptome sequencing,and 12 differentially expressed genes associated with carotenoid anabolism were identified by function annotation.Using real time quantitative PCR(RT-q PCR)to verify expression levels of these genes,the result confirmed that 2 genes(SDR,PSLC)were highly expressed in adult mites of T.cinnabarinus,and 8 genes(β-UGT,UGT,PPs,CYP385C4,Caa X PPR,PLAT10,PLAT11,PDs)were highly expressed in adult mites of T.urticae.The m RNA expression levels of 2 genes(KST,RDH2)had no significant difference between T.cinnabarinus and T.urticae.Comparing full-length sequences of 10 differentially expressed genes between T.cinnabarinus and T.urticae,it was found that the similarity of their nucleotides was over 88%,and there was little difference on protein(number of amino acids,molecular weight,isoelectric point).The result implied that there might be no differences on physiological function of these genes between T.cinnabarinus and T.urticae.Then their m RNA expression levels at different development stages detected,the result revealed that m RNA expression levels of 2 overexpressed genes(SDR,PSLC)of T.cinnabarinus at different development stages were irregular.The m RNA expression level of SDR at the first stage of nymph in T.cinnabarinus was lower than T.urticae,and m RNA expression level of PSLC at the second stage of nymph was higher in T.cinnabarinus than T.urticae.For 8 overexpressed genes of T.urticae,their m RNA expression levels at each stages were higher than T.cinnabarinus,and fold change at different development stages varied by several to tens of thousands.The result implied that differential expression of these genes was probably correlated with body color difference between T.cinnabarinus and T.urticae.3.Functional verification of differentially expressed genes related to carotenoid anabolism.According to different lengths and characteristics of these genes,RNAi was studied by si RNA and ds RNA to verify function on carotenoid anabolism and body color difference.The silence efficiency detected were distributed between 30% and 95%by RT-q PCR.After target genes silenced successfully,HPLC technique was used to assay the conten of 6 kinds of differential carotenoids,respectively.It was found that 7genes(PSLC,PDs,PLAT10,PLAT11,CYP385C4,β-UGT,UGT)slienced could change carotenoid content.PSLC decreased the content of γ-carotene after slienced.PDs,CYP385C4,PLAT10 and PLAT11 increased the content of β-carotene and γ-carotene after slienced.UGT and β-UGT increased the content of α-carotene,β-carotene andγ-carotene after slienced.It demonstrated that these genes were related to carotenoid anabolism.UGT and β-UGT influenced the content of α-carotene,β-carotene andγ-carotene at one time,therefore they were verified for phenotypic function.With micro-injection of UGT and β-UGT,the body color of T.urticae turned yellow obiviously after 2 days.The result indicated that β-UGT and UGT down-regulated could make body color of T.urticae deepen.According to above research,the reason of body color difference was probably correlated with differential carotenoid content between T.cinnabarinus and T.urticae.The mechanism of body color difference is their differential expressed genes related to carotenoid anabolism,which results in different accumulation capacity of carotenoids.