Cloning and Expression of Phytoene Desaturase Gene, PpPDS, from Peaches
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摘要: 利用基因克隆技术获得桃果实八氢番茄红素脱氢酶基因（PDS）的全长核苷酸序列，命名为PpPDS。PpPDS全长2 056 bp，编码区长度1 722 bp，共编码573个氨基酸。进化树分析发现PpPDS与杏果实PaPDS、草莓FaPDS亲缘性较高。蛋白质序列分析表明PpPDS含有二核苷酸[NAD（H）/NADP（H）或FAD（H）]结合基元，其处于NAD（P）-binding Rossmann-like保守结构域内。实时荧光定量PCR结果显示，PpPDS基因在黄肉品种‘金丽’桃果实发育过程中均有表达，且在果肉中的表达普遍高于果皮。果实处于硬核期的PpPDS基因在果实中的表达显著高于软核果时期，随后其表达处于较高水平并呈平缓趋势。本研究对桃果实PpPDS基因的克隆及表达分析为研究桃果实类胡萝卜素生物合成分子机理奠定了良好的基础。Abstract: The full-length cDNAs of phytoene desaturase (PDS) from peaches was isolatedby gene cloning, and named as PpPDS. It was 2 056 bp in length and encoded a predicted protein of 573 amino acids (ORF length at 1 722 bp). A phylogenetic analysis indicated that PpPDS closely resembled the PDSs from Prunus armeniaca and Fragaria×ananassa. The deduced amino acid sequence suggested that PpPDS protein contained dinucleotide binding motifs, NAD (H), NADP (H) or FAD (H), which belonged to the NAD(P)-binding rossmann-like conserved domain. Quantitative real-time PCR showed that PpPDS gene was generally expressed higher in mesocarp than exocarp in fruits of Jinli yellow peach at different maturity stages. After the peach stone hardened, the gene expression increased significantly, then leveled off at the peak. The information would be useful for further studies on the carotenoid biosynthesis pathway in peaches.
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Key words:
- peach /
- PDS /
- gene cloning /
- expression analysis
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图 1 桃果实PDS基因cDNA PCR扩增
注：M为1 500 bp DNA Marker；1为RT-PCR产物；2为3'RACE产物；3为5'RACE产物。
Figure 1. PCR ampliation of cDNA from peaches for cloning PDS gene
图 2 PpPDS蛋白功能结构域预测及氨基酸多重序列比对
注：a为蛋白功能结构域预测；b为基酸序列同源性比较。
Figure 2. Prediction of protein functional domains and multiple alignment of putative amino acid sequence on PpPDS gene
图 5 PpPDS基因在桃果实成熟期间的相对表达量
注：图中不同小写字母表示在P＜0.05水平上差异显著。
Figure 5. Relative expression of PpPDS gene during fruit development and maturation of peaches
表 1 本研究所用引物
Table 1. Primers applied
基因名称 引物序列*[5'-3'] 用途 PpPDS Forward: CACTGAAGGTTGTTTGCGTNGAYTAYCC
Reverse: TCCTTAACAGTCAGTCCATCYTGNGCYTC中间片段扩增 3'-GSP1: CCTCGTCCAGCTAAGCCGTTGAAGGTCGTGAT
3'-GSP2: GGCCATATTGAAGAACAATGAGATGCTGACTT3'-RACE PCR扩增 5'-GSP1: ACAGTCAGTCCATCCTGGGCCTCAACATAAGC
5'-GSP2: TATCTTTCCATGCTGCCACCTTTCCGCCCAGA5'-RACE PCR扩增 Forward: CCGTTGAAGGTCGTGATTG
Reverse: CTTTCCGCCCAGAACATC实时荧光定量PCR PpTEF2 Forward: GGTGTGACGATGAAGAGTGATG；
Reverse: TGAAGGAGAGGGAAGGTGAAAG内参基因 注：* Y=C/T；N=A/T/C/G。 
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