西双版纳黄瓜Cs-Psy1基因的序列特征与表达分析

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。

Sequence characteristics and expression analysis of phytoene synthase gene in Cucumis sativus L. var. xishuangbannanesis

Cucumis sativus L.var. xishuangbannanesis is characteristic varieties resource with orange flesh in our country. The beta carotene content has obvious difference between different germplasm. Phytoene synthase (PSY) is the first speed limit enzyme in the pathway of carotene biosynthetic. Full length cDNA and DNA sequences of Psy1 were cloned respectively according to Cucumis sativus L.var.xishuangbannanesis. Corresponding DNA sequence was 2797bp, containing 5 introns and 6 extrons. cDNA sequence was 1385bp, encoding 421 amino acids. The deduced amino acid sequence contains two sequences with high conservation which were characteristic sequences in PSY family. The protein was unstable, having no obvious hydrophobic domain, and the membrane structure was not predicted. The deduced protein PSY1 showed the highest homology with melon, watermelon and bitter gourd based on phylogenetic tree. Four SNPs and one InDel of 13bp were found in Cucumis sativus L.var.xishuangbannanesis by sequence alignment with deep sequencing material "9930" and "GY14". The SNP1 occurred in 27bp of the upstream of the initiation codon, other mutations were located in the introns. SNP1 had the highest specificity based on the sequence analysis of 115 resequencing material. Its mutation rate was 100% in 19 Cucumis sativus L. var. xishuangbannanesis, and 5.3% in 96 cultivated cucumbers. The relative expression of Psy1 in 7 different developmental stages of fruit was studied by real-time fluorescence quantitative PCR. The results showed that the expression level of Psy1 gene increased and then decreased in different cucumber fruit development period, and the amount was significantly different in Cucumis sativus L.var.xishuangbannanesis. In the conversion period (50d after pollination), it reached the maximum value which was more than 8 times of the initial stage of fruit development and more than 4 times of the same stage in common cucumber. The expression amount of common cucumber changed mildly in general. In addition, the amount of expression of the inner peel of Cucumis sativus L.var.xishuangbannanesis was significantly higher than that in the middle peel, and the maximum difference was 5 times. The difference between the inner and middle peel of common cucumber was small relatively. Psy1 may affect the accumulation of beta carotene in Cucumis sativus L.var.xishuangbannanesis.