Cloning And Expression Analysis Of Genes Related With β-carotenoid Biosynthesis In Cucumis Sativus L.var. Xishuangbannanesis

Xishuangbanna cucumber (Cucumis sativus L.var. xishuangbannanesis Qi et Yuan) is a specialvariation germplasm only found in China until now. Orange flesh and placenta of mature fruit wereobviously different from cultivated cucumbers.-carotene content of flesh in Xishuangbanna cucumberwas nearly one hundred times than that of common cucumbers, which was induced by the accumulationof-carotene.-carotene was an important kind of carotenoid and essential for our health. In this study,main genes in the-carotenoid pathway were focused on and key genes were cloned based on reportedresearches and related information of Cucumber Genome Database. Compared with the referencesequences of9930and GY14, specific sequences in Xishuangbanna cucumber were selected.Compared with the resequence database of113cucumber germplasm, the specific sequences relatedwith-carotene biosynthesis in Xishuangbanna cucumber were determined further and new molecularmarker was developed preliminarily. In order to evaluate the correlation of carotene accumulation withtranscriptional regulation of carotenoid biosynthesis related genes, using real-time PCR technique, theexpression of5genes during fruit development were studied in BN35(var. xishuangbannanesis) andJD7(var. sativus) respectively. Above results were benefit to understand the molecular mechanism ofcarotenoid accumulation, explicate elite genes of Xishuangbanna cucumber and improve qualitybreeding in cucumber. Main results were as followed:The main results were as followed:1. Four cDNA sequences encoding CsPsy1, CsZds, CsLcyB, CsChxB in-carotenoid biosynthesispathway and only three DNA sequences encoding CsPsy1, CsLcyB, CsChxB were cloned fromXishuangbanna cucumber. The sequence analyses showed that CsPSY1、CsZDS、CsCHXB containchloroplast leading peptide. There were three predicted transmembrane profiles for the deduced acidsequences of CsCHXB, while others’ weren’t detected. Conserved sequence analysis showed thatCsPSY1had two characteristic sequences, CsZDS and CsLCYB contained NADB-Rossmannsuperfamily sequences, and CsCHXB had a L-histidine receptor. The result of mutiple sequencealignment indicated that these genes were very conservative and shared high sequence identity withother orthologous genes. Above related enzymes had their own characteristic sequences. Phylogeneticanalysis showed predicted amino acids in cucumber were closest to cucurbitaceae.2. Compared with9930and GY14, CsPsy1gene showed five differences which were4SNPand a13bp InDel. CsLcyB gene showed a SNP site within coding region, which brought about thechange of amino acid. CsChxB gene showed a TTTTTT insert region in the sixth intron and a SNP sitein coding region, which brought about the change of amino acid. CsZds gene showed no variationdefinitely. Blast the genome resequence data of113germplasm in cucumber, the SNP of CsChxB was100%specific in Xishuangbanna cucumber.3. The expression level of CsPsy1, CsPds, CsZds, CsLcyB and CsChxB genes at different stagesduring fruit development were studied. The result of real-time PCR analysis showed that the expressionof-carotene biosynthesis relative genes of BN35were first increased and then decreased during fruit development, and the highest expression was at color turning period. The up regulation expression ofJD7was later than that of BN35. It was showed to some extent that CsZds, CsLcyB, CsChxB wererelated to-carotene accumulation.4. The method of PCR-SSCP was optimized. According to the specific SNP of CsChxB gene,primer SNPChxb1-178was screened effectively while Xishuangbanna cucumber and cultivatedcucumber could be distinguished well.