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西双版纳黄瓜是我国特有的果肉橙黄色的黄瓜变种资源,不同种质间的β-胡萝卜素含量差异明显。PSY是胡萝卜素生物合成途径中的第1个限速酶。本文以西双版纳黄瓜为试材,分别克隆西双版纳黄瓜八氢番茄红素合成酶(Cs-PSY1)的DNA和c DNA序列,结果显示,DNA长2797 bp,包含5个内含子和6个外显子,c DNA序列长1385 bp,编码421个氨基酸。Psy1推测的氨基酸序列包含该家族的2个特征序列,保守性很高。该蛋白为不稳定蛋白,无明显疏水区,未预测到跨膜结构;系统进化分析结果显示,西双版纳黄瓜的Cs-PSY1蛋白与甜瓜的同源性较高;与栽培黄瓜深度测序材料“9930”和“GY14”的序列进行比较分析,结合115份黄瓜重测序结果,共发现5个SNP,其中2个位于起始密码子上游27 bp处和971 bp处,3个位于内含子区域。其中SNP4在重测序的19份西双版纳黄瓜中的突变率为100%,在96份栽培黄瓜中的特异性为5.3%。转录因子结合位点预测结果显示,在普通栽培黄瓜该位点处存在一个CTAG motif,在西双版纳黄瓜中该位点突变后则不存在该motif。利用实时荧光定量PCR技术分析Cs-Psy1的表达量变化趋势,结果表明,在黄瓜不同果实发育时期,该基因的表达量均呈现先上升后下降的趋势,在西双版纳黄瓜中表达量变化的差异明显,在授粉后50 d达到最大值,是果实发育初期表达量的8倍多,是同时期普通黄瓜的4倍多,而普通黄瓜表达量的总体变化相对平缓。西双版纳黄瓜果实内果皮的表达水平明显高于中果皮,最高相差约5倍,普通黄瓜差异不明显。从上述研究结果推测Psy1基因可能影响西双版纳黄瓜的β-胡萝卜素积累。
Xishuangbanna cucumber is an endemic orange-yellow cucumber variety resource in China, with significant differences in β-carotene content among different germplasms. PSY is the first rate-limiting enzyme in the pathway of carotene biosynthesis. In this paper, the DNA and c DNA sequences of Cucumber phytoene synthase (Cs-PSY1) were cloned from Xishuangbanna cucumber. The results showed that the DNA was 2797 bp in length and contained 5 introns and 6 exons The c DNA sequence is 1385 bp in length and encodes 421 amino acids. The predicted amino acid sequence of Psy1 contains two characteristic sequences of this family and is highly conserved. The phylogenetic analysis showed that there was high homology between Cs-PSY1 protein and melon in cucumber of Xishuangbanna. Compared with the sequence of cultivated cucumber deep sequencing material “9930 Five ”SNPs were found in 115 cucumber re-sequencing results. Two of them were located at 27 bp upstream and 971 bp upstream of the start codon, and three were located in the internal Sub-area. Among them, the mutation rate of SNP4 in 19 re-sequenced Xishuangbanna cucumbers was 100% and that of 96 cultivated cucumbers was 5.3%. Prediction results of transcription factor binding sites showed that there was a CTAG motif at this locus in Cucumber cultivars and the motif was absent in Cucumber at Xishuangbanna. The results showed that the expression level of Cs-Psy1 at different developmental stages of cucumber increased firstly and then decreased, and the difference was significant in the expression of Cs-Psy1 in cucumber , Reached the maximum value at 50 d after pollination, which was more than 8 times of the early stage of fruit development, more than 4 times of that of common cucumber in the same period, while the general change of cucumber expression was relatively gentle. The expression level of endocarp in cucumber fruit in Xishuangbanna was significantly higher than that in mesocarp, the highest difference was about 5 times, but the difference was not obvious in common cucumber. It is speculated from the above results that the Psy1 gene may affect the β-carotene accumulation in the cucumber of Xishuangbanna.