西双版纳黄瓜Cs-Psy1基因的序列特征与表达分析

Sequence Characteristics and Expression Analysis of Cs-Psy1 in Cucumis sativus L. var. xishuangbannanesis Qi & Yuan

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（Cucumis sativus L. var. xishuangbannanesis Qi Yuan） is a kind of specific cucumber germplasm resources with orange flesh which were found only in Yunnan province in China. The content of beta carotene showed obvious difference among different germplasm. Phytoene synthase（PSY） is the first speed limit enzyme in the carotene biosynthetic pathway. In this study,DNA sequence（2797 bp） containing 5 introns and 6 extrons and c DNA（1385 bp） sequence encoding 421 amino acids of Psy1 were cloned respectively in Xishuangbanna cucumber. In addition,amino acid sequence was deduced and contained two highly conservative sequences which were characteristic in PSY family. Its PSY protein was unstable,having no obvious hydrophobic domain,and the membrane structure was not predicted. Phylogenetic analysis showed that the Cs-PSY1 protein of Xishuangbanna cucumber expressed high homology with that of melon. Furthermore,nucleotide blast with references＂9930＂and＂GY14＂were performed and five specific SNPs and one 13 bp-In Del were found in Xishuangbanna cucumber. Based on the resequencing data of 115 accessions in cucumber,SNP4,which occurred in 27 bp of the up-stream of the initiation codon,expressed high specific percentage,whose mutation rate was 100% in 19 accessions of C. sativus L. var. xishuangbannanesis Qi Yuan population and 5. 3% in 96 accessions of cultivated cucumber population. The result of prediction for transcription factor binding sites showed that there was a CTAG motif in 27 bp of cultivated cucumber but it did not exist in Xishuangbanna cucumber because of SNP4. Finally,the relative expression of Cs-Psy1 at 7 different developmental stages of fruit was studied by real-time fluorescence quantitative PCR. The results showed that the expression level of Cs-Psy1 increased and then decreased during the fruit development,and the amount was significantly different in C. sativus L. var. xishuangbannanesis Qi Yuan. At about the stage of flesh color conversion（50 d after pollination）,it reached the maximum value,which was more than 8 times as 0 d in Xishuangbanna cucucmber and more than 4 times as the same stage in common cucumber. The expression amount of common cucumber changed mildly in general. Also,the expression level of the inner peel of Xishuangbanna cucumber was 5 times higher than that in the middle peel,meanwhile the difference between inner and middle peel of common cucumber was small relatively. From above analysis,it preliminarily concluded that Cs-Psy1 may affect the accumulation of beta carotene in Cucumis sativus L. var. xishuangbannanesis Qi Yuan.