Genome-wide Identification Of Lycopene Cyclase Gene Family And Function Analysis Of Lycopene Epsilon Cyclase Gene In Celery

Celery(Apium graveolens L.)is a leafy green vegetable,belonging to Apiaceae family.It is widely cultivated worldwide,which is popular among urban and rural residents due to its rich nutrition.In addition,it also plays an important role in anti-oxidation,anti-inflammatory,anti-cancers and so on.Celery also has a wide range of applications in biopharmaceuticals and even cosmetics industries.AsA and lignin are both important indicators to measure the quality of celery.Lutein and β-carotene are two important carotenoids in celery,which play an important role during the growth of celery.Lycopene epsilon cyclase(LCY-ε)is an important branching enzyme in the carotenoid metabolic pathway,which restricts the accumulation of various carotenoids in downstream branches.This study locating on lutein and β-carotene synthesis and related genes in celery will provide certain reference to improve the quality of celery.In this paper,different color celery cultivars were used to analyze the relationship between the accumulation and m RNA abundance of AsA and lignin.Majoring in lutein andβ-carotene,the relationship between the expression levels of related genes in metabolic pathway were analyzed.The response of lycopene cyclase genes(LCY genes)under different abiotic stresses were also been determined.In our study,the lycopene cyclase family(LCY family)in celery were identified,and the Ag LCY-ε gene was screened.In order to further study the effect of Ag LCY-ε during the synthesis process of lutein andβ-carotene in celery,the gene was transformed into Arabidopsis and celery to verify its function.The main findings of this article are as follows:1.The results displayed that AsA content in leaf blades of ‘Xuebaiqincai’ was the highest,which was the highest in petiole of ‘Baiganshiqin’.In addition,the lignin content was the highest in leaf blades and petioles of ‘Baiganshiqin’ among several white celery cultivars.Yellow celery ‘Best’,white celery ‘saixue’ and green celery ‘Jinnan Shiqin’ were chosen as materials,and the content of lutein and β-carotene in leaf blades and petioles were measured by UPLC.The results showed that the content of lutein and β-carotene were highest in ‘saixue’,followed by ‘Jinnan Shiqin’,and the lowest in ‘Best’ among the three celery cultivars.Regarding to β-carotene,there was little difference in the leaf blades of‘Best’ and ‘Jinnan Shiqin’.In the petioles,the lutein and β-carotene level were lowest in‘saixue’,which was opposite to the situation of lutein content.There was little difference between lutein and β-carotene levels in ‘Best’ and ‘Jinnan Shiqin’.Except for AgLCY-β-2,lutein and β-carotene-related genes were mostly expressed at higher levels in celery leaf blades than that in petioles.In addition,the expression trend of Ag LCY-ε in celery was consistent with the content of lutein and β-carotene in three celery cultivars.2.Based on the celery transcriptome and genome database,3 LCY family members were been identified.They were lycopene ε-cyclase(LCY-ε)and lycopene β-cyclase(LCY-β-1,LCY-β-2).Blast comparison results illustrated that the homology was highest between Ag6G02176.1(Ag LCY-β-2)in celery and the AT3G10230.1(At LCY-β-1)in Arabidopsis,with a similarity of 59%.And the highest homology appearred in Ag8G00262.1(Ag LCY-ε)in celery and AT5G57030.1(At LCY-ε)in Arabidopsis,with a similarity of 73%.The homology was highest between Ag10G02300.1(Ag LCY-β-1)in celery and the AT3G10230.1(At LCY-β-1)in Arabidopsis,with a similarity of 77%.The phylogenetic tree displayed that the LCY genes of celery clustered together with the LCY genes in other species.Analysis of the promoter region demonstrated that the light response element was identified in all LCY genes in celery,and the auxin response element was identified only in the promoter region of AgLCY-ε.Chromosome mapping results illustrated that Ag LCY-ε,Ag LCY-β-1 and Ag LCY-β-2 were distributed on chromosomes 8,10 and 6,respectively.The LCY genes performed different responses under high temperature,low temperature,drought and salt stress.The results showed that Ag LCY-ε responded obviously under salt stress.3.Dipping method through agrobacterium-mediated was used to transform Ag LCY-εinto Arabidopsis and celery.The positive plants were identified by GUS staining and PCR verification methods.The subcellular localization results showed that Ag LCY-ε was located at the chloroplast.UPLC measurement results displayed that the lutein andβ-carotene content in transgenic Arabidopsis were significantly up-regulated comparing with that in wild-type plants.The transgenic plants grew more vigorously under salt stress,with higher SOD and POD activities,and lower MDA content.The root length of transgenic Arabidopsis seedlings was longer than that of the wild type plants,revealing stronger salt tolerance.The analysis of transcription levels illustrated that the transcript abundance of key genes(At PSY and At CRTISO)increased with the overexpression of Ag LCY-ε in transgenic Arabidopsis lines.Celery genetic transformation efficiency was time-consuming,and further verification will be implemented.