Study On Genes Involved In Cuticular Wax Synthesis And Transport During Fruit Development Stage Of Bud Sports Of Navel Orange Using RNA-Seq

Transcriptome sequencing(RNA-Seq) was performed to examine differentially expressed genes of glossy navel orange and its female parent during fruit development using the Illumina RNA-seq system. quantitative real-time PCR were used to confirm their expression levels.Through gene cloning, the molecular differences between glossy navel orange and its female parent Newhall navel orange were explored. These studies will lay a theoretical foundation for improving the quality of navel orange. The main results are as following:1.Glossy navel orange and Newhall navel orange were sequenced by Illumina HiSeqTM 2000, results of RNA-Seq appear below:(1)Over 77 million high-quality reads were generated. Among them,38,734,326 and 38,605,154 reads of MT and WT respectively. The coverage of each gene were calculated. 17% genes’coverage is more than 80% in both MT and WT.(2)3,217 differentially expressed genes were identified. Compared with WT,1,542 genes were found to be up-regulated and 3,417genes were down regulated in MT.(3)Differentially expressed genes were categorized into three main categories by gene ontology (GO):biological process, cellular component, and molecular function. Organelle, catalytic activity and cellular process shared the largest portion of three main categories.(4)3,217 differentially expressed genes were assigned into 124 pathway by KEGG. Of them,23.22% were involved in metabolic pathway, and 14.05% were involved in the biosynthesis of secondary metabolites.(5)9,296 gene structures were redefined, supplement or extend their 3’ UTR or 5’ UTR.(6)216 and 222 novel transcript were obtained from MT and WT, with mean length between 732.76 and 637.63.(7)707 and 607 genes were undergoing intron retention in MT and WT, which is one form of alternative splicing.(8)44,389 and 39,701 SNP were identified in MT and WT respectively.All of the results revealed the overall transcription differences of glossy navel orange and its female parent during fruit growth and development.2.Combined RNA-Seq data with waxy different composition results got from previous studies, differentially expressed genes related to wax synthesis were selected from KEGG pathway. To understand the formation mechanism of glossy navel orange at gene level, RT-qPCR were used to examine the expression pattern during navel orange fruit development. Main results are as follows:(1)16 differentially expressed genes which blong to 7 gene familis in 6 pathway were choosen randomly to demonstrate the RNA-seq results.3 were up-regulated and 13 were down-regulated among them.13 genes expression were continual while the rest performed a discontinuity. The RT-qPCR data were basically consistent with the RNA-seq.(2)The RNA-Seq data were consistent with waxy difference composition results got from previous studies.3. cDNA sequence of Cs4g02580, Cslg02760 and Cs4g06430 was obtained from skins of both MT and WT using RT-PCR and gene cloning, named CER1-1, CER1-2 and KCS-1. Sequence analyses results are as follows:(1) cDNA of CER1-1 was 1,492bp bp in full length, containing an ORF of l,275bp bp encoding 360 amino acids.11 mutation site were found in MT alignment to WT amino acid sequence.(2) The full length cDNA of CER1-2-MT was 1,146bp, including a 1,128bp ORF encoding 375 amino acids while CER1-2-WT was 3 less in length of cDNA and ORF and 1 less in numbei of amino acids than CER1-2-MT.2 mutation site were found between MT and WT and one of them was lost in MT.(3) KCS-1 cDNA spaned 1,304bp and contained a 1,266bp ORF encoding 421 amino acids.2 mutation site were found between MT and WT.