Clone And Characterization Of The Key Genes Involving In The Watermelon Flesh Coloration

Watermelon（Citrullus lanatus L.）is one of the most important vegetable worldwide.Due to its fleshy,juicy,sweet and convenience,watermelon are appreciated by comsumers with significant economic value.The fruit color of watermelon depends on the carotenoid composition and content,which are the result of genetic fluctuations and highly regulated processes.Research on the function of the genes and proteins involving in carotenoid metabolism pathway will be not only useful in understanding the mechanism of the color formation in watermelon fruit,but also provide the way to improve the flesh color of modern watermelon.Previously,genetic linkage and quantitative trait locus（QTL）analysis of the two watermelon inbred lines LSW-177（red flesh color）and Cream of Saskatchewan（short for‘COS’,pale yellow flesh color）showed that lycopeneβ-cyclase（lcyb）may act as the dominant gene to distinguish the red colour fruits with others in watermelon.In carotenoid metabolism pathway,lycopeneβ-cyclase is the key enzyme to catalyze lycopene intoβ-carotene,whose activity is closely related to the color formation of watermelon fruit.So far,only a single copy of LCYB（Cllcyb）is present in the watermelon genome.The study was conducted to characterize the two polymorphic genes encoding lcyb enzymes,from two watermelon inbred lines（LSW-177 and COS）.Three nucleotide differences had been identified and two amino acid substitutions（226（Phe/Val）and 435（Lys/Asn））were present in the two proteins.Multiple sequence alignment indicated that all the lcyb proteins contained the strictly conversed amino acid Phe226 in higher plants.Color complentation assay with carotenoid engineering bacteria and high-performance liquid chromatography（HPLC）analysis showed that the two proteins could fully convert lycopene intoβ-carotene with no significant difference in prokaryotic expression system.Meanwhile,some amino acids had been site-directed mutated to explore the acitve site of lcyb enzyme.HPLC results showed that mutagenesis of Ala168 into Glu168 will inactivate the Cllcyb protein.Changing Phe190-Ile208 of Cllcyb into Leu190-Phe208will also make the lcyb loss of the cyclase activity.When Leu190-Phe208 found in the mutated Cllcyb protein,Phe226 will retain 80%of the lycopene cyclase ablity.Another site-directed mutagenesis of Pro349-Thr376 into Ser349-Aln376 in Cllyb makes the protein keep 30%cyclase ability.These results provide some strategies to manipulate the activity of Cllcyb to increase or decreaseβ-carotene contents in watermelon fruit.The co-action of LCYB and lycopeneε-cyclase（LCYE）generatesα-carotene.Color complentation assay with carotenoid engineering bacteria and high-performance liquid chromatography（HPLC）was also used to identify the function of LCYE,another member of lycopene cyclase family in LSW-177 and COS.Results showed that there was no color change when carotenoid engineering bacteria was transformed with with p ET28a-Cllcye.A small HPLC peak ofδ-carotene in the tranformed bacteria indicated the weak enzyme ability of Cllcye,which can only catalyze trace lycopene.In this study,we cloned and compared the promoter sequences in two watermelon inbred lines:the red fleshed LSW-177 and pale-yellow fleshed COS,which were 1,775 bp and 1,644 bp in length at the upstream of the start codon of Cllcyb,respectively.The two promoters shared 895 bp（-895～+1）consensus sequences.Analysis by Plant CARE program revealed that both promoter sequences contained some basic cis-elements such as TATA-box and CAAT-box,circadian control,photo-responsive elements such as Box 4,G-box,GT1-motif,as well as hormone related elements like the GARE and CGTC Amotifs.An AT-rich element and a TCA-element of salicylic acid were present only in the promoters of LSW-177 and COS Cllcyb,respectively.The full promoter length of Cllcyb in LSW-177 and COS,the 895 bp consensus sequence of the two promoters,and two5’-deleted promoter fragments in COS were obtained to drive GUS gene and the plant overexpression vectors were named P177,Pcos,P3,C1 and C2,respectively.Transient transformation of tobacco leaves showed that Pcos,C1 and C2 had promoter activity and the activity of Pcos was stronger than that of C1 and C2,whereas the GUS activities driven by P177and P3 were nearly undetectable.Combined with the results of promoter activity and Cllcyb gene expression pattern in the fruit ripening of LSW-177 and COS,it was speculated that Cllcyb may be involved in different regulatory networks during fruit coloration in LSW-177 and COS.Many genes have been reported to participate in plant carotenoid metabolism pathway.Based on the sequences provided by Cucurbit Genomics Database（Cu Gen DB）,some genes including three phytoene synthase gene（Cl PSY1,Cl PSY2 and Cl PSY3）,ζ-carotene desaturase（Cl ZDS）,carotene isomerase（Cl CRTISO-1 and Cl CRTISO-2）,9-cis-epoxycarotenoid dioxygenase（Cl NCED1,Cl NCED2 and Cl NCED3）,and a Dna J cysteine-rich domain containing protein OR（Cl OR）,had been cloned.Sequences anaysis show that a 23 nt deletion in Cl PSY1 from COS lead into a frame shift mutation compared with the one in LSW-177.In LSW-177 and COS,there is only one nucleotide variation leading to one amino acid change of Cl PSY2,Cl PSY3 contain the consensus sequence in the both materials.In LSW-177 and COS,a new transcript Cl ZDS（one more exon than reference sequence）had been obtained.Six nucleotide variations making three amino acid substitutions had been identified in the Cl ZDS coding region.Two new transcripts named Cl CRTISO-1 and Cl CRTISO-2 had also been ampilfied from the two watermelon inbred lines.The transcript of Cl CRTISO-1 can be translated into continuous amino acids,but lack N-terminal;the transcripts of Cl CRTISO-2 can not be translated into a function protein.Cl NECD gene family has been cloned in LSW-177 and COS.Sequence analysis showed that three nucleotide variations in Cl NCED1,eight nucleotide variations in Cl NCED2 and one in Cl NCED3were identified in both materials,which caused two,five and one amino substitution in the deduced proteins,respectivly.In LSW-177 and COS,a new Cl OR gene had been amplified.Compared with the reference sequence in Cu Gen DB,the new one lacks the 8^th exon and displays only one nucleotide change in the both materials with no amino acid substitution.Conversed domain analysis showed that all the deduced proteins were highly conseved in high plants and shared the similar structures.