苦瓜McAPX2基因及其启动子的克隆与表达分析

Cloning and Expression Analysis of the McAPX2 Gene and Its Promotor in Momordica charantia L.

基于苦瓜叶片均一化文库克隆得到McAPX2基因的cDNA全长序列(Gen Bank登录号:KJ722767.1),采用染色体步移法获得该基因的启动子序列,实时荧光定量PCR技术检测该基因在不同器官中的表达量及低温胁迫对McAPX2表达量的影响。结果表明:McAPX2基因全长1 086 bp,开放阅读框747 bp,编码249个氨基酸;McAPX2属于过氧化物酶家族ClassⅠ的成员,其脂肪族氨基酸指数为83.90,是一个亲水性蛋白,可推测定位于细胞质中;与甜瓜(NP_001284378.1)、黄瓜(ACO90193.1)、南瓜(AHF27424.1)、苦瓜(AGJ72851.1)同源性较高,分别为95%,94%,94%和93%;q RT-PCR结果显示,在苦瓜的根、茎、叶、雌花和幼果等组织器官中McAPX2的表达量存在显著差异,其中根的表达量最大,而在叶片中的表达量最低;在低温胁迫下,随着胁迫时间的延长,McAPX2表达量逐渐上调,处理12 h时达到最大,随后下降,说明该基因的表达与苦瓜耐低温胁迫相关。分离得到McAPX2基因上游调控序列1 267 bp,其启动子含有与GA、ABA、CTK、乙烯等激素和病原菌等生物胁迫以及热激、干旱、低温、光等非生物胁迫的相关元件。

The full length ascorbate peroxidase gene sequence of cDNA was gained from the normalized full-length cDNA library of bitter gourd leaves, which was named McAPX2（Gen Bank accession No. KJ722767.1）. The gene promotor sequence was isolated by chromosome walking. The gene expression pattern in different tissues and under low temperature stress was analyzed by RT-PCR. The results of sequence analysis showed that the McAPX2 cDNA sequence was 1 086 bp in length and had an open reading frame（ORF） of 747 bp coding for 249 amino acids. The protein was a hydrophilic with the aliphatic index of 83.90. The McAPX protein and shared the characters of ClassⅠof peroxidase family and might locate in cytoplast. The McAPX2 protein showed 95%, 94%,94 % and 93% similarities to other APX proteins homologs from Cucumis melo, Cucumis sativus, Cucurbia moschata and Momordica charantia, respectively. The 1267 bp promotor sequences were obtained, which possessed some elements induced by physiological and environmental factors. These results indicated that McAPX2 might play an important role in response to hormone factors such as GA, CTK, ethylene, ABA, and pathogens et el.,and abiotic stresses such as hot stress, low temperature, drought, and light. q RT-PCR revealed that there were significant differences of McAPX2 gene expression in roots, stems, leaves and young fruits, which was the highest in roots and lowest in leaves. The m RNA level of McAPX2 was remarkably up-regulated significantly until 12 h in a cold stress, implying that McAPX2 might play an important role in bitter gourd tolerance to cold stress.