Cloning And Bioinformatics Analysis Of Anthocyanins Biosynthesis Key Genes In Bok Choy

Bok choy(Brassica rapa ssp.Chinensis)is a type of Brassicaceae cruciferous vegetable.It was commonly known as leafy vegetables.It is deeply loved by the general public for its various species.In addition,it is easy to grow and is rich in vitamin C,mineral elements and so on.Purple bok choy contents much anthocyanins for its leaves are light purple or deep purple.In this study,deep purple material and its green mutant and the light purple lines were used to clone the key genes,which related to anthocyanins metabolism,such as BrcF3H、BrcLBD39 and BrcLBD38 gene,and related bioinformatics analysis were also performed.The results are as follows:1.In this study,we found that the sequences of the three cDNA clones from NJZX1-3,NJZX1-1 and NJZX1-0 were completely identical,with a full-length of 1077 bp(designated as BrcF3H),containing an opening reading frame(ORF)of 1074 bp and encoding 358 predicted amino acids.Structural analysis revealed that BrcF3H protein belong to the,20G-Fe(II)oxygenase superfamily.Multiple alignments reveled that BrcF3H had 100%identities and 98%identities to F3H genes from Brassica napus and Brassica rapa pekinensis,respectively.Phylogenetic tree analysis demonstrated that purple bok choy shared a close relationship with B.napus and B.rapa pekinensis.Shading treatment and RT-PCR analysis showed that low light would inhibited the synthesis of anthocyanin and down-regulated the expression of BrcF3H gene in leaves significantly.In a way,total anthocyanins content decreased along with the reduction of F3H mRNA transcript levels,while enzyme assay reveled that darkness would induce the BrcF3H activity with the increment of 39.43%,21.48%and 36.09%in NJZX1-3,NJZX1-1 and NJZX1-0,respectively.It is speculated that BrcF3H is not a light-independent enzyme.All the results in our paper provide a theoretical basis of molecular regulatory mechanism for anthocyanin synthesis.2.The study indicated that the sequences of the two cDNA clones form NJZX1-3 and NJZX1-0 were coincident completely,with a full-length of 699 bp.The ORF of LBD39 is 696 bp in length and encodes 232 predicted amino acids,which was designated as BrcLBD39.Subcellular localization predicted that the protein was distributed in cell nucleus.The molecular weight of BrcLBD39 is 25.30×103 and pI value is 8.85.The BrcLBD39 protein have 100%identities to BraLBD39,and shares the most close relationship with Brassica rapa,then Brassica napus and Brassica oleracea,with the identities of 99%and 93%,respectively.With the treatment of 6-BA,total anthocyanins content in the purple self-line NJZX1-3 increased and were higher than control after 24 h,however,the content of anthocyanins in leaves ofNJZXl-0 was lower than 0.03 mg·g-1,and almost no anthocyanins.At the same time,BrcLBD39 expressed in the two different self-lines with similar expression patterns,which declined first and then rised significantly and then fell again.The expression of BrcLBD39 increased to the maximum at 48 h,with the increment of 74.84%and 49.87%in the purple and green materials,respectively.Furthermore,the expression level of BrcLBD39 in NJZX1-0 was higher than that in NJZX1-3.That is the negative regulatory gene BrcLBD39 is responsive to exogenous 6-BA and had close relation with the accumulation of anthocyanins in bok choy,it suggests that the gene might play a key regulatory role in anthocyanins biosynthesis.3.The sequences of the LBD38 genes cloned from purple material NJZX1-3 and green material NJZX1-0 are not identical,but the fragment lengths are both 726 bp,of which containing an ORF of 723 bp long and encodes 241 amino acids,respectively.The cloned fragments were named as BrcLBD38-3 and BrcLBD38-0,respectively.Nucleotide sequence alignment showed that there were nine differences in the nucleotide sequences of BrcLBD38-3 and BrcLBD38-0 gene,and there were two differences in the sequences of BrcLBD38-3 and BrcLBD38-0 protein.The nucleotide sequence of BrcLBD38-0 gene was identical to BraLBD38 gene in Chinese cabbage.Protein sequence analysis revealed that the isoelectric values of the BrcLBD38-3 and BrcLBD38-0 proteins were 7.22 and 7.29,respectively,and their molecular weights were 26.56 KDa and 26.61 KDa,respectively.Conserved domain analysis revealed that BraLBD38 protein are identical to the BraLBD39 protein,which belongs to the DUF260 superfamily.In addition,BraLBD38 also contains a transmembrane region.What’s more,the genes BrcLBD37,BrcLBD38-0,and BrcLBD39 which inhibit the biosynthesis of anthocyanins are highly expressed at the 15-leaf stage in green mutant material NJZX1-0 of bok choy.In addition,the expression level of BrcLBD37 was higher in the 7-leaf stage of the two materials and the expression level of BrcLBD39 gene in NJZX1-3 was always lower than 0.2.The above results provided useful foundation for further clarifying the mechanisms of leaf color mutation in purple bok choy.4.A total of 55 Chinese cabbage LBD family genes were identified in this paper.Among them,the most genes were found on chromosomes 4 and 9 of Chinese cabbage,with 9 LBD members of each,followed by chromosome 5,which contains 8 LBD members,while chromosome 8 and 10 contain the least number of LBD members,with 1 LBD member of each.The gene structure analysis revealed that most of the LBD family genes contained 1 to 2 introns,and 4 genes contained no introns.In addition,the conserved domain analysis showed that the LBD family proteins of cabbage belongs to the DUF260 superfamily.According to the phylogenetic tree analysis,they were divided into two classes:class I(45 genes)and class II(10 genes).Type II genes can be further subdivided into four subfamilies:Ia,Ib,Ic,and Id.Both of classe I and II contain a CX2CX6CX3C motif,but class II does not contain the Gly-Ala-Ser block and the LX6LX3LX6L structure.In addition,the anthocyanins synthesis negative regulator LBD37,LBD38 and LBD39 gene belong to LBD class II.These results are helpful for the functional analysis of LBD genes in plants.