The Distribution And Metabolism Of Carotenoid And The Identification And Functional Analysis Of Laccases In Carrot

Carrot(Daucus carota L.)is an important Apiaceae family root vegetables,abundant carotenoids accumulating in its edible fleshy root are essential nutrients for the human body,which are also the precursor of vitamin A and a good antioxidant and anticancer agent.In plants,carotenoids play an important role in photosynthesis,light protection,etc.,and in the fleshy root of carrot,carotenoid is not only nutrients but also the main pigment for its various colour spectrum.Although the carotenoids biosynthesis genes in carrots have been identified,the mechanism of synthesis and degradation of carotenoids and lutein is not very intact.In this study,we investigated the carotenoid contents and carotenoid biosynthesis pathway genes in six different color carrot cultivars,3 key carotene hydroxylase genes were identified to be essential for carotene degration to xanthophyll;then we identified the transcripts of carotenoid biosynthesis genes in ’Kuroda’ carrot leaf and root by using high-throughput sequencing technology;in addition,iTRAQ technology was used to analyze proteome identification of ’Kuroda’ carrot,the expression levels of carotenoid cleveage dioxygenase(CCD)were determined,and CCD genes expression were also investigated by qRT-PCR,the mechanism of the degradation pathway of carotene and lutein was preliminarily studied.Then the 3 key carotene hydroxylase genes were constructed to pYN7736 vector for transforming into orange ’Kuroda’ carrots,the over-expressed lines are expected to degrade the a-and/or(3-carotene in orange carrots to different types of xanthophylls to get insights into the substrate preference and function cross overlapping of hydroxylase gene in carrot.In addition,the purification and enzyme activity parameters of two carrot laccase proteins were also studied,the basic properties of the laccase were verified.The main results are as follows:1.Six different carrot cultivars were used to analyze simultaneously carotenoid contents by HPLC.The expression levels of carotenoid biosynthesis genes in carrot were also detected by qRT-PCR.Yellow carrot cultivars showed high expression levels of lutein formation genes with high proportion of lutein contents in total carotenoid contents;while orange carrot cultivars exhibited low expression levels of lutein formation genes,and high proportion of a-and P-carotene content in total carotenoid contents.The results indicated that a-carotene accumulation and lutein formation might be related to the transcriptional levels of carotene hydroxylase genes in carrot.In addition,the expression levels of two carotenoid cleveage dioxygenase genes indicated that carotenoid may degrade into other derivant through carotenoid cleveage pathway.2.Next-generation sequencing has become an efficient tool for generating a great number of annotated sequences,expressed genes and quantitative expression levels.Here,we have obtained 46,119,008/48,414,508 raw reads and 45,394,846/47,887,648 clean reads from carrot,var.’Kuroda’,leaf and root which accumulated α-and β-carotene in both tissues investigated by HPLC.Then using Trinity assembled into contigs and mapped back to contigs,these reads were assembled 56,267 and 62,427 unigenes after removing Ns and paired-end extending.In addition,a total of 18,354 differentially expressed genes(DEGs)were detected between carrot leaf and root,and 99 DEGs were assigned to carotenoid biosynthesis using the integrated function annotation.In carotenoid pathway DEGs,DcPSY1,DcZ-ISO,DcCRTIS02,DcLBCY1,DcLECY,DcZEP1,DcZEP2,DcVDE1,DcVDE2,DcNXS1,DcNXS2,DcAA03a,DcA8H-CYP707A1.2,DcCCD4 and DcMAX1 expressed dramatically in carrot leaf than root,which is consistent with our qRT-PCR analysis of DEGs expression profiles.3.And apocarotenoid is the main provider of the aroma and odor of fruits and flowers in plant,which is synthesized by the CCD(carotenoid cleavage dioxygenase)catalyzing oxidative cleavage of carotenoids.Here,high accurate and high sensitive iTRAQ were utilized to analyze the proteome of carrot(D.Carota L.cv.’Kuroda’)leaf and root.The differentially expressed proteins(DEPs)were mainly distributed in the chlorophyll synthesis and photosynthesis proteins,and we found 3 carrot CCD DEPs:CCD la,CCD1d and CCD4.Moroever,the protein levels of CCDla and CCDld were higher in roots than those in leaves,while the expression of CCD4 was more active in leaf.This may be related to its function in different tissues.qRT-PCR experiments show that,DcCCD genes differing in relative expression may caused by their different functions for plant structure and development.4.Three key carotene hydroxylase genes were identified to be involved in carotene degradation into xanthophyll.Among them,a cytochrome P450 family of carotene β-ring hydroxylase gene in carrot orange have nucleotides insertion resulting in its protein functional deficiency,which led to the accumulation of a-carotene,another cytochrome P450 family of carotene ε-ring hydroxylase gene encoding by CHXE(CYP97C1)and a non heme type carotene(3-ring hydroxylase encoding by CHXB2 were identitied to be involved in accumulation of(3-carotene carrot by various varieties of carrot and transcriptome analysis of orange carrot leaf and root.Based on the phylogenetic tree and protein properties of two kinds of carotene hydroxylase amino acid sequence found that the properties of two types of carrot carotene hydroxylase are significantly different,but the same type are very similar.In addition,we transformed the CHXB2,CYP97A3 and CHXE(CYP97C1)gene from ’Bejo-1719’ into orange ’Kurada’,and obtained over-expressed transgenic seedlings with GUS blue stain for subsequent functional verification.5.Laccase is a kind of blue copper oxidase with multiple copper atoms.Two laccase-encoding genes DcLacl and DcLac2 were isolated from carrot,and their proteins were successfully expressed and purified using the Escherichia coli expression system BL21(DE3).DcLacl and DcLac2 had Km values of 3.9043 and 1.255 mM with ABTS as the substrate,respectively,and Vmax values of 54.0832 and 81.7996 μM mg-1·min-1,respectively.Moreover,DcLacl and DcLac2 had optimal pH values of 2.8 and 2.6,respectively,and optimal temperatures of 45 ℃ and 40 ℃,respectively.The activities of the two enzymes were promoted by Ca2+,Mg2+,Cu2+,and Na+ but inhibited by Fe2+,Zn2+,Mn2+,K+,SDS,and EDTA.Expression profiles showed that the two DcLac genes had almost identical responses to high and low temperature stresses but different responses to salt,drought,and metal stresses.This study provided insights into the characteristics and tolerance response mechanisms of laccase in carrot.