Study On Regulation Of Prominent Collar At Stalk Of Huangguogan Based On RNA-Seq

The research material Huangguogan that has researched previously has flat collar type(FT) and prominent collar type(PT).To study the inner mechanism of the regulating its collar's characteristic, first step, we analysis and compare of the two types from huangguogan to check whether have difference in fruit quality by conventional fruit quality analysis method, and then from ISSR molecular marker method to check whether have changed in nucleotides,combined with real-time PCR to study the regulation of cell wall metabolism related key genes XET, analyses the biology information and respectively in FT and type PT from the expression level, analyze its relationship with the collar characters.Finally combining with RNA-Seq transcriptome method analyzes genes expression in FT and PT, the results show that:1. By comparing the citrus fruit quality between FT and PT located in xianfeng and fengle, found that from the total sugar content and sugar acid ratio, FT is better than PT in the total sugar content and sugar acid ratio.It can not distinguish the two type by using designed ISSR primers,and there may be no difference may in nucleotides between FT and PT.2. Useing the improved CTAB method to extract total RNA citrus fruit,reverse transcribed into cDNA, and design XET gene amplification primers, cloned XET gene (CitXET) from huangguogan and study CitXET of expression patterns in FT and PT. The results showed that the length of CitXET was 960 bp, highly homologous to other XET genes from different species. CitXET deduced a protein of 319 amino acid residuals with a molecular mass of 37.4547 kD, a pI of 9.05 and a molecular formula of C1724H2548N448O466S14, and it was a hydrophilic protein with transmembrane structure. Compared with other species, amino acid sequence encoded by XET was almost conserved in evolution. Experimental results showed that protein encoded by CitXET had GH16 domain, which meant that this gene should belong to glycoside hydrolase family, contained a motif of DEIDFEFLG.qPCR display that expression patterns of CitXET is different between FT and PT, but it did not reach a significant level, indicating CitXET did not play a key role in controling growth of stalk part of huangguogan, and there may be co-regulation by multiple genes.3. By RNA-Seq transcriptome in huangguogan two growth periods, respectively 483, 284 significant genes, and these genes are enriched respectively to 42.31 GO functional classification. KEGG pathways showed, the differences between FT and NT may caused by different hormone levels and cell wall metabolic.