Identification Of Carotenoids Composition And Carotenoids-related Genes In Antheraea Pernyi Larvae

Antheraea pernyi is one of the economically valuable model insects.After long-term natural selection and artificial breeding,the four body colors of cyan-yellow,yellow,blue and white have developed.In practice,larval body color has always been an important marker for breeding of A.pernyi.Carotenoids are one of the main component of insect pigments.In this study,representative body color varieties of A.pernyi.Was used as experiments material,including QingLiuhao(cyan yellow),Jiaolan(blue),LuHong(red)(red individual;red),LuHong(yellow)(yellow individual;yellow),and Xiaobaican(white).The optimal method for extracting carotenoids in A.pernyi larvae was determined by surface method.The composition and content of carotenoids in A.pernyi larvae were identified by using liquid chromatography-tandem mass spectrometry(LC-MS/MS)method.Carotenoids transport/binding-related genes of A.pernyi were cloned and the expression patterns of these genes were analyzed by using real-time quantitative PCR(qRT-PCR)technology.This study will lay the foundation on the role of carotenoids in body color differentiation of A.pernyi larvae.The main results were as follows:1.The optimal process conditions for response surface optimization of ultrasonic-assisted extraction of carotenoids in A.pernyi larvae was determined with the solvent of acetone: n-hexane(v: v = 3: 5),the particle size of 40 mesh of the lyophilized powder,the liquid-solid ratio of 20 mL/g,the temperature of60?,and the extraction duration of 40 min.The total carotenoids content in five body colors of A.pernyi larvae was different.QingLiuhao had the highest content of total carotenoids(16.20±0.27 ?g/g),followed by LuHong(red individual;red)(15.66±0.11 ?g/g),JiaoLan(15.33±0.18 ?g/g),LuHong(yellow individual;yellow)(12.93±0.22 ?g/g)and XiaoBaican(11.73±0.20 ?g/g).Significant difference of the total carotenoids content was found between XiaoBaican and other varieties except for LuHong(yellow individual;yellow).2.Using solvent [(n-hexane: acetone: ethanol(v: v: v = 2: 1: 1)): methanol(containing 0.1% BHT,v:v = 9: 1)] to extract carotenoids of A.pernyi larvae,based LC-MS/MS method,8 carotenoids were detected from 18 candidate carotenoids standards,including lutein,zeaxanthin,?-carotene,?-carotene,?-cryptoxanthin,antherxanthin,violaxanthin and neoxanthin.All the 8 types of carotenoids were detected in five body colors of A.pernyi larvae,but the ratio of carotenoids was different.The total carotenoids content in five body colors of A.pernyi larvae was different.QingLiuhao had the highest content of carotenoids(16.445 ?g/g),followed by LuHong(yellow individual;yellow)(11.208 ?g/g),XiaoBaican(6.947 ?g/g),LuHong(red individual;red)(5.834 ?g/g)and JiaoLan(3.802 ?g/g).The main carotenoids in A.pernyi larvae were lutein,?-carotene and zeaxanthin.3.Based on the transcriptome database,5 carotenoids transport/binding-related genes were screened and cloned,including 4 SR-B family(Scavenger receptor class B)genes and 1 carotenoid binding protein(CBP)gene.Those genes were the ApCameo1,ApCameo2,ApSCRB1,ApSCRB12,and ApCBP genes.4.qRT-PCR analysis results showed that ApCameo1 and ApCBP were expressed in all tested tissues and organs except the hemolymph of the XiaoBaican.ApCameo2 were expressed in all tested tissues and organs of five body colors of A.pernyi larvae.ApSCRB1 and ApSCRB12 were expressed in all tissues and organs except hemolymph.The relative expression of ApCameo1 and ApSCRB12 was 10 times lower than ApCameo2,ApSCRB1 and ApCBP.It was speculated that ApCameo1 and ApSCRB12 were not related to body color differentiation of A.pernyi.The expression of ApCameo2 and ApSCRB1 in integument was significantly higher than that in other tissues and organs.The expression level of ApCameo2 and ApSCRB1 in the integument of JiaoLan and LuHong(yellow individual;yellow)was significantly higher than that inother varieties,respectively.The expression of ApCBP in hemolymph was significantly higher than that in other tissues and organs except XiaBaican.The expression of ApCBP in hemolymph of JiaoLan was significantly higher than that in other varieties.Therefore,it is speculated that ApCameo2 and ApCBP may be related to the blue body color of A.pernyi,and ApSCRB1 may be related to the formation of yellow individuals in LuHong population.