Expression Analysis Of Genes Related To β-carotene Biosynthesis And Fruit Development In Cucumis Sativus L.var. Xishuangbannanesis

Cucumis sativus L. var. xishuangbannanesis, which found only in Xishuangbanna county of Yunnan province in China, is a specific variety resource with orange flesh in cucumber. The β-carotene content of their flesh shows obvious difference among xishuangbanna population and is about one hundred times as germplasm of Cucumis sativus L. var. sativus. CsPsy, CsPds, CsZds, CsLcyb, Cschyb and CsZep are key enzymes in the carotenoid biosynthesis pathway, and their regulation in expression level might be one of the most important factors of carotenoid accumulation in plants. The fruit shape of xishubanna cucumber is nearly circular or cylindrical when fruit reaches physiological maturity, and the maximum diameter of fruit is significantly greater than that of common cucumber, which might be related to a large number of genes associated with fruit development. In this study, inbred lines of C. var. xishuangbannanesis and C. var. sativus resectively were selected as main materials. Firstly, based on previous transcriptome sequencing data, genes with stable expression during fruit development were screened out. Traditional reference genes as control, the expression of 9 genes were determined and their stablity was evaluated. Furthermore, using the screened reference gene, expression level of the genes related to β-carotene biosynthesis and fruit development was detected using real-time fluorescence quantitative PCR technology in order to understand their expression pattern, which might be related with high β-carotene accumulation and specific fruit shape of Xishuangbanna cucumber. Above studies will develop to excavate elite genes of Xishuangbanna cucumber. The main results are as follows:1. Five genes showing stable expression were selected as candidate reference genes based on transcriptome sequencing data. Four commonly used reference genes as control, the expression stable of 9 genes were analyzed using real-time fluorescence quantitative PCR technology and geNorm and NormFinder software. Generally, the genes screened based on the transcriptome sequencing data expressed stably than traditional reference genes. The gene of Ubiquitin-conjugating enzyme E2(Csa4G045000) showed relatively stable among the samples of different varieties and pollination methods during fruit development stage, which should be optional reference gene of real-time fluorescence quantitative PCR for the fruit of cucumber.2. The relative expressional quantity of 6 genes including CsPsy, CsPds, CsZds, CsLcyb, Cschyb and CsZep during fruit development stage was analyzed using real-time fluorescence quantitative PCR technology. It resulted that the quantity 6 genes showed the trend of firstly increased and then decreased, and reached a maximum value in color turning stage(about 50 d after pollination) in Xishuangbanna cucumber, which expressed the biggest difference with common cucumber far more than 4 times, 1.5 times, 3 times, 3 times, 6 times and 19 times respectively. In addition, the quantity of CsPsy1 of fruit endocarp was significantly higher than that of pericarp in Xishuangbanna cucumber while the difference of CsPsy1 in common cucumber and other 5 genes in two cucumber materials were not obvious. Results suggest that the highly accumulation of β-carotene might be influenced and co-regulated by CsPsy, CsZep, CsChyb combining with other genes.3. Based on sequencing data, 251 genes related to the fruit development of cucumber were selected including 51 cyclin genes, 40 expansion genes, 44 kinesin genes, and so on. Variance analysis showed that the different expression of 122 genes were significant at different fruit development stages, among which 59 genes found in both two materails group and 25 genes were only in Xishuangbanna cucumber which may be related to their specific fruit shape develpment. The number of cyclin-dependent protein kinase genes and fasciclin-like arabinogalactan genes showing increasing trend at the stage of 20d/30 d was much more in Xishuangbanna cucumber than in common cucumber. The qRT-PCR of 8 selected genes at 7 different fruit development stages expressed that their expression quantity showed the trend of increased firstly and then decreased or increased continuously, except for the gene of MAP 70-5(Csa6G511060). The results of qRT-PCR were similar to that of the transcription sequencing data except for Csa6G078670 and Csa6G423400.