Screening And Analysis Of Key Genes For Malate Metabolism In Loquat Fruit

Loquat(Eriobotrya japonica Lindl)is a member of the Rosaceae family,being highly appreciated for its refreshing taste,high medicinal value and fruit ripening is earlier than other fruits(late spring and early summer).However,high fruit acidity has been a major factor affecting fruit quality and commodity value.In this study,low acid ‘Bai Li’ and high acid ‘Jiefangzhong’,which are two leading cultivars of loquat growing in Fujian province,were used as the experimental material.three stages(S65,early development stage;S95,commercial harvest stage;S125,delayed harvest stage)of loquat fruits were sequenced on Illumina Hi SeqTM 2000 platform.Some key genes for malate metabolism,such as cy NADP-ME2 and VHP1 were cloned and analyzed on bioinformatics.Maic acids content in pulp were determinated by high performance liquid chromatogaphic during fruit development.Key differential gene expression were analyzed by q RT-PCR.At the same time,the relationship between gene expression and malic acid content was studied.an important basis for understanding the molecular mechanism that leads to malic acid accumulation was provided during fruit development.The main results were as following:1.Transcriptome of the two cultivars was analyzed using RNA-seq at three different stages during the development of fruit.Approximately 270 million high quality reads in eighteen libraries were de novo assembled by Trinity,yielding 450,44 unigenes with more than 79.9% functionally annotated.6531 unigenes participates in the 127 metabolic pathways,of which 14 unigenes are involved in the malate metabolic pathway.transcriptome profiles of the two cultivars were compared in the pulp,identifing 248,5,271 and 2,391 differentially expressed genes in three developmental stages.Gene ontology enrichment analysis of these genes indicated that there were 41 GO categories,in which the cellular processes and metabolic pathways were significantly in the biological process.KEGG analysis of differentially expressed genes indicated that 121 metabolic pathways were significantly enriched,mainly enriched in metabolic pathways such as glycolysis,flavonoids,amino acids,TCA and so on.In addition,the genes related to malic acid accumulation in fruits were analyzed,and 14 candidate key genes were screened.2.Based on the expression levels of 14 malate metabolism genes(rpkm),the full-length CDS of cy NADP-ME2(Unigene0019373)and VHP1(Unigene0025326)with high expression and differences between varieties were screened for cloning,and biological sequence analysis.The full-length c DNA sequence length of cy NADP-ME2 is 1836 bp,encoded 621 amino acids;The full-length CDS sequence length of VHP1 is 2073 bp,encoded 691 amino acids.The similarity of two genes to apples,pears,cherries was above 95%.3.Malic acid content in the pulp of the two cultivars accumulated slowly from a low level,then increased.There was no significant difference in the malic acid content between the two cultivars at the early stage of fruit development(S65).The malic acid content of the two cultivars reached the maximum at the ripening period(S95).The degradation rate of malic acid in low-acid atcultivar was significantly higher than high acid at the late stage of fruit development(S125),which was the main cause of the malic acid content difference between the two cultivars.the expression of 14 candidate genes associated with malic acid accumulation were determinated during the development of fruit.At the same time,the relationship between malic acid content and candidate genes was analyzed.The difference in malic acid content between cultivars may be regulated by cy NADP-ME2 and VHP1 expression.The quantitative PCR results of 12 genes were consistent with the transcriptome data analysis results,Indicating the accuracy of the transcriptome results.