Differential Expression Gene Analysis Of Fruit Development In Nitraria Tangutorum And Clone And Function Analysis Of NtUFGT

Nitraria tangutorum Bobr., a shrub belonging to the Nitraria genus in the family Zygophyllaceae, is a typical desert plant with strong resistance to drought, salinity, alkalinity, and high temperature. Beside of its substantial ecological value, N. tangutorum has a high potential economic value as a source of edible berries and medicinal compounds. However, recent research on N. tangutorum has focused on its ecology, classification, chemical composition, and stress-resistance genes. There has been little research on the molecular basis of its fruit development and ripening. In this study, we performed de novo transcriptome sequencing of N. tangutorum fruit using the RNA-Seq technology. Using a digital gene expression system, we identified a large number of differentially expressed genes (DEGs) relate to fruit development and nutrition material synthesis. This work will provide valuable resources to understand the molecular mechanism of fruit development and ripening in N. tangutorum. The main research results are as follow:1. We performed de novo transcriptome sequencing of N. tangutorum fruit using the RNA-Seq technology. At last 69,306 unigenes (average length,587 bp) were assembled. All the assembled unigenes were compared with the NCBI Nr, Swiss-Prot, GO, COG, and KEGG database. As a result,42,929,26,809,33,363,15,537, and 24,592 of the 69,306 Unigenes were annotated based on these database, respectively.2. DEGs among different stages of N. tangutorum fruit development were identified by digital gene expression profiling analysis. A total of 8,240 DEGs were detected between the S1 and S2 libraries; 4,994 DEGs between the S2 and S3 libraries; 5,985 DEGs between the S1 and S3 libraries. According to the GO functional enrichment analysis, a total of 50,171,28,071, and 32,891 DEGs were significantly enriched in 59,58, and 58 GO term in S1 vs. S2, S2 vs. S3, and S1 vs. S3, respectively. According to the KEGG functional enrichment analysis, a total of 6, 8, and 34 significantly enriched pathways were found in in S1 vs. S2, S2 vs. S3, and S1 vs. S3, respectively.3.28 DEGs associated with plant hormone signal transduction were selected out, and then cluster analysis of gene expression patterns were performed. These genes were classified into 4 groups according to their expression patterns. The largest group (Group 2) included 21 DEGs, which showed down-regulated from S1 to S2 and then up-regulated from S2 to S3. The remaining three groups contains only 7 DEGs. The cluster analysis showed that most of the DEGs associated with plant hormone signal transduction were down-regulated from S1 to S2 and then up-regulated from S2 to S3.4.61 DEGs associated with transcription factors were selected out, and then cluster analysis of gene expression patterns were performed. These genes were classified into four groups according to their expression patterns. The largest group (Group 1) included 42 DEGs, which also showed down-regulated from S1 to S2 and then up-regulated from S2 to S3. In addition, Group 3 contained 9 DEGs, Group 4 had 6 DEGs, and Group 2 only included 4 DEGs. The clustering results of transcription factors are similar with that of plant hormones.5. Based on Nr annotations 14 DEGs encoding enzymes involved in flavonoid biosynthesis were selected. In addition,7 genes with RPKM values>100 which annotated as flavonoid biosynthesis were also selected for cluster analysis. The high expression levels of PAL, C4H, and 4CL at the early ripening stage may have resulted in high accumulation of 4-coumaryl-CoA, the substrate for down-stream pathways in the late stage of fruit ripening. Different expression patterns of CHS, CHI, and F3H may be involved in the biosynthesis of different types of flavonoids. Only one F3’H gene (CL7210.Contigl) with a high expression level was down-regulated from S1 to S2 and then upregulated from S2 to S3.This gene encodes the enzyme that catalyzes the formation of cyanidin, which gives the purple-red color to N. tangutorum fruit.5. One anthocyanin synthesis gene NtUFGT was cloned from N. tangutorum fruit which contain 1407 bp ORF. The protein secondary structure, subcellular localization, signal peptide, transmembrane topological structure, hydrophilicity and hydrophobicity were predicted by bioinformatics software. And then this gene was transferred into Arabidopsis thaliana. Transgenic T3 generation homozygous plants were obtained. The content of anthocyanin in transgenic A. thaliana was higher than that of wild type, and showed a trend of growth with the increase of UV-B stress. While, the content of the procyanidolic showed a decreasing trend with the increase of UV-B treatment, and the content in transgenic was lower than the wild type A. thaliana.6. We have measured the content of starch, sucrocse, glucose, and fructose during N. tangutorum fruit development and ripening. In addition, the activity of SS, SPS, AI, and NI were measured. The changes of sugar content and enzyme activities in different development stages of N. tangutorum fruit were analyzed.25 DEGs related to sucrose metabolism and 13 DEGs related to starch synthesis were selected for cluster analysis. The changes of expression levels of these DEGs and the activities of related enzyme were also discussed.