Cloning And Functional Analyses Of Four Genes Related To Betalain Biosynthesis In Hylocereus

Pitaya(Hylocereus)has been commercially produced as a new type fruit crops for its high nutritional value and bright market prospect.Pitaya's peel and pulp are abundant in betalain.Red pulp pitaya(H.monacanthus)is the only fruit containing abundant betalains for consumer.Betalain is one of the four natural plant pigments which plays key roles in colouration,osmotic adjustment and stress adaptation in plant.Betalain can serve as food and drug colorants and has medicinal benefits in term of excellent ability of anti-oxidization.However,there were few reports of genes involved in selection of reliable references and betalain biosynthesis in pitaya.In this study,two pitaya varieties i.e.Guanhuahong(H.monacanthus)and Guanhuabai(H.undatus)were used as plant materials.The stability of the selected reference genes for normalizing the RT-qPCR data were identified from pitaya.Four candidate gene associated with CYP450 and GT were identified.The expression levels of CYP450(31591,36993)and GT(36238,32889)were determined in different development stages of pulp and different tissue or organs of the two pitaya.The main results are as follows:1.Twenty-four reference genes were selected from the pitaya transcriptome dataset and six reference genes were selected from the Arabidopsis Information Resource(TAIR).Five candidate reference genes i.e.ACT1(1),GAPDH,ACT1(2),TUA and EF1-? were selected from 30 typical candidate reference genes Based on melting curves and Ct values,five candidate reference genes i.e.ACT1(1),EF1-?,GAPDH,ACT1(2),TUA were selected to determine the most stable internal genes for RT-q PCR normalization in different tissues and fruit developmental stages of pitaya.Three algorithms,i.e.,geNorm,NormFinder and BestKeeper were used to evaluate expression stability of GAPDH,ACT1(2),ACT1(1),TUA,and ef1-? reference genes.GAPDH was the most stable gene according to calculations of three statistical method while TUA and EF1-? showed the lowest expression stability.2.Four regulatory gene i.e.31591,36993,36238 and 32889 inloved in betalain biosynthesis were obtained from pulp of Guanhuahong.The four genes showed high similarity with sequences from other plant species related to betalain biosynthesis.The gene of 31591 shared 71% and 78% homology with Beta vulgaris and Mirabilis jalapa's CYP450 at amino acid level.The gene of 36993 shared 66% and 62% homology with Beta vulgaris and Mirabilis jalapa's CYP450 at amino acid level.The gene of 36238 shared 66% and 61% homology with Beta vulgaris and Mirabilis jalapa's GT at amino acid level.The gene of 32889 shared 63% and 62% homology with Beta vulgaris and Mirabilis jalapa's CYP450 at amino acid level.This results show that 35191 and 36993 may be CYP450 genes related to betalain biosynthesis,36238 and 32889 are likely to be GT genes related to betalain biosynthesis.3.Results from the expression levels of 31591,36993,36238,32889 in different tissues and fruit developmental stages in pitaya indicated that 31591,36993,36238,32889 were related with the betalain accumulation.Therefore,31591,36993,36238,and 32889 were possible betalain-related genes in pitaya.4.Four genes of CYP450(31591,36993)and GT(36238,32889)were transformed into Arabidopsis thaliana and tobacco via Agrobacterium-meditated method,respectively.38,42,53 and 49 transformed Arabidopsis thaliana plantlets were obtained for 35191,36993,36238 and 32889 genes,respectively,based on PCR analyses.13,10,13 and 12 transformed tobacco plantlets were obtained for 35191,36993,36238 and 32889 genes,respectively,based on PCR analyses.