甜瓜抗旱性相关基因MeP5CS的克隆、序列分析及表达

Cloning,Analysis and Expression of a Drought-related Gene MeP5CS from Melon

用GenBank登录的抗旱相关基因序列进行比对,在保守区域设计一对引物,利用RT-PCR获得了一个甜瓜抗旱相关基因,命名为MeP5CS。生物信息学分析表明,该基因全长1000bp,开放阅读框(ORF)753bp,编码250个氨基酸;MeP5CS蛋白大小约82.18kD,理论pI值为4.90;MeP5CS编码蛋白与桐花树、猕猴桃和葡萄同源性较高,分别为94%、81%和73%。该蛋白含约40.2%的α-螺旋,25.2%的β-转角,34.6%的不规则卷曲,在第19～20氨基酸之间存在信号肽的剪切位点;MeP5CS编码蛋白是疏水性蛋白,有2个跨膜螺旋结构和13个磷酸化位点。半定量RT-PCR表明,MeP5CS在甜瓜根系中表达最高,茎中次之,叶片中表达较低。MeP5CS基因在甜瓜组织中的表达受丛枝菌根真菌(AMF)和水分胁迫双重诱导,与甜瓜的组织特异性和水分胁迫处理时间有关。水分胁迫条件下,接种AMF可以显著增加甜瓜幼苗脯氨酸的积累量,菌根甜瓜幼苗的脯氨酸积累与MeP5CS基因的表达呈正相关。

Using designed primers based on the conserved amino acid sequences of known drought-related genes to amplify cDNA fragments from melon(Cucumis melo L.)by RT-PCR,a drought-related gene named MeP5CS was obtained.Bioinformatics analysis indicated that the full-length of cDNA sequence was 1 000 bp,which contained an open reading frame of 753 bp and encoded a protein of 250 amino acid residues with a calculated molecular weight of 82.18 kD and isoelectric point of 4.90.The MeP5CS protein showed 94%,81% and 73% similarity to the P5CS from Aegiceras corniculatum,Actinidia deliciosa and Vitis vinifera.The protein include α-helix(40.2%),β-turn(25.2%),random coil(34.6%)and a cleavage site between nineteen and twenty amino acid residues.The protein is a hydrophobic protein and there is two transmembrane helix and thirteen phosphorylation sites.The result ofRT-PCR analysis indicated that MeP5CS expression levels were different in roots,stems and leaves,and it was the highest in roots and middle in stems,lower in leaves.The results showed that the AMF can induce expression of MeP5CS gene in melon under water stress,and enhance the drought resistance of melon;Expression differences in tissues were related to different tissues and the duration of water stress.Under water deficit,AMF could increase Pro accumulation in melon leaves,gene expression and proline accumulation were positively correlated.