Cloning And Functional Analysis Of 9-cis-epoxycarotenoid Dioxygenase Gene Of Carrot

Carrot(Daucus carota L.)is a biennial root vegetable crop of Apiaceae,which has a wide cultivation area all over the world.In the process of growth and development,carrot is always subjected to drought stress,resulting in the decline of yield and quality.Carotenoids accumulated in the carrot taproots are important nutrients,which can act as antioxidants to participate in the drought resistance process of plants.9-cis-epoxycarotenoid dioxygenase(NCED)is the most important rate-limiting enzyme in the pathway of carotenoid degradation and ABA synthesis,which can directly affect the drought resistance of plants.It is of great scientific significance to study the metabolic mechanism of carotenoids and the molecular mechanism of DcNCED gene under drought stress.In this study,the carotenoid contents and the expression of genes related to carotenoid metabolism in three different-colored carrots under simulated drought stress were analyzed.Two key genes DcNCEDs were identified as genes essential for drought stress.Bioinformatics analysis and expression specificity analysis of DcNCED1 and DcNCED2 genes were performed to preliminarily understand their regulatory roles in the growth and development of carrot.The selected DcNCED2 gene was constructed into plant overexpression vectors and transferred into Arabidopsis and carrot.Drought-resistant function of DcNCED2 gene was preliminarily verified,and the molecular regulation mechanism of DcNCED2 gene in carotenoid metabolism was discussed.The main research contents and results are as follows:1.In the present work,the carotenoid contents in three different-colored carrot cultivars,‘Kurodagosun’(orange),‘Benhongjinshi’(red),and ‘Qitouhuang’(yellow),were determined by UPLC under 15% polyethylene glycol(PEG)6000 treatment.Real-time fluorescence quantitative PCR(RT-qPCR)was then used to determine the expression levels of carotenoid synthesis-and degradation-related genes.Increases in lutein and decreases inα-carotene content in ‘Qitouhuang’ and ‘Kurodagosun’ under drought treatment were found and may be related to the expression level changes of DcCYP97A3,DcCHXE,and DcCHXB1.The expression levels of DcNCED1 and DcNCED2 in three cultivars significantly increased,thus suggesting that NCED genes could respond to drought stress and affect the drought resistance of carrot.Analysis of the growth status and carotenoid contents of carrots under PEG6000 treatment indicated that the orange cultivar‘Kurodagosun’ has better adaptability to drought stress,β-carotene and lutein may be the main carotenoids involved in the drought resistance response of carrot.2.DcNCED1 and DcNCED2 genes were cloned from ‘Kurodagosun’ by RT-PCR.Bioinformatic analysis of these two genes and their coding amino acid sequences were carried out.RT-qPCR was used to compare the expression levels of DcNCED1 and DcNCED2 in different growth stages and different tissues.The results showed that DcNCED1 and DCNCED2 contained an open reading frame(ORF)of 1767 and 1722 bp,encoded 588 and 573 amino acids,and contained a conserved PLNO2258 domain at sites22～588 and 2～573,respectively.Both of DcNCED1 and DcNCED2 belonged to the RPE65 superfamily.DcNCED1 and DcNCED2 proteins contained 48 and 52 phosphorylation sites,respectively.DcNCED1 and DcNCED2 were hydrophilic proteins with no signal peptide and transmembrane structure.Their secondary structures were composed of α-helix,β-fold,extended chain and random coil.The sequence alignment and phylogenetic tree analysis results showed that NCED was highly conserved in plant evolutionary process.RT-qPCR results showed that DcNCED1 and DcNCED2 genes were highly expressed in carrot leaves.The expression levels of the two genes increased first and then decreased during the growth and development process of carrot leaves and roots,and reached the top at 60～75 days after sowing.The expression level of DcNCED2 was higher than that of DcNCED1 in carrot leaves and taproots at 60～90 days after sowing,suggesting that DcNCED2 gene played a greater regulatory role in the growth and development of carrot.3.DcNCED2 gene was transferred into ‘Col-0’ Arabidopsis and ‘Kurodagosun’ carrot by constructing plant overexpression vector.The transgenic seedlings were verified by GUS staining and PCR identification.The root length,activities of SOD and POD,contents of MDA,ABA,and carotenoid,expression levels of related genes were determined in transgenic Arabidopsis under drought stress.The results demonstrated that the transgenic Arabidopsis showed the phenotype of drought tolerance,with longer root length,higher SOD and POD activities,lower MDA content,higher ABA content and related gene expression,and lower lutein and β-carotene content.The results indicated that DcNCED2 gene could improve the drought tolerance of the seedling.The ABA content in leaves of overexpressed DcNCED2 carrot was significantly increased,while the contents of lutein,α-carotene and β-carotene were decreased compared with the wild carrot.The changes of expression levels of most related genes were consistent with the above results.These results indicated that DcNCED2 gene could degrade carotenoids in carrot leaves and convert them into ABA.