Characterization of cDNAs Encoding Two Distinct Miraculin-like Proteins and Stress-related Modulation of the Corresponding mRNAs in Citrus jambhiri Lush
Characterization of cDNAs Encoding Two Distinct Miraculin-like Proteins and Stress-related Modulation of the Corresponding mRNAs in Citrus jambhiri Lush
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DOI：
10.1007/s11103-005-2941-4
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发表时间：
2005
影响因子：
5.1
通讯作者：
S. Tsukuda;K. Gomi;Hiroyuki Yamamoto;K. Akimitsu
中科院分区：
文献类型：
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作者：
S. Tsukuda;K. Gomi;Hiroyuki Yamamoto;K. Akimitsu
根据消减PCR获得的序列信息，通过随机扩增cDNA末端（RACE）方法从粗柠檬中分离到两个编码奇迹素样蛋白的全长cDNA，命名为RlemMLP1和RlemMLP2。RlemMLP1和RlemMLP2的转录本在叶片和茎中均未检测到，但在果实中积累。受伤后，接种孢子或茉莉酸甲酯蒸汽处理后，转录本在叶片中积累到较高水平。用水杨酸甲酯处理拮抗了创伤的信号通路。水杨酸甲酯处理2h后，可显著降低创伤诱导的RlemMLP1和RlemMLP2基因表达。利用原核表达系统获得了这两个基因的蛋白产物，并具有蛋白酶抑制剂活性。RlemMLP2含有奇异果甜蛋白基序，而RlemMLP1没有，只有RlemMLP2对柑橘链格孢菌具有抗真菌活性。利用粒子轰击法在洋葱细胞中进行瞬时翻译后，将RlemMLP1或RlemMLP2融合到绿色荧光蛋白基因上进行细胞定位分析，结果表明RlemMLP1和RlemMLP2均定位于细胞质中。这些证据表明，粗柠檬RlemMLP可能具有防御功能，至少当宿主细胞被病原菌感染破坏时。
Two distinct full-length cDNAs from rough lemon that encoded miraculin-like proteins were isolated by random amplification of cDNA ends (RACEs), based on sequence information from subtractive PCR previously described, and designated asRlemMLP1andRlemMLP2. The transcripts of bothRlemMLP1andRlemMLP2were not detected in leaves, or stems but accumulated in fruits. Transcripts accumulated to higher levels in leaves after wounding, inoculation with conidia ofAlternaria alternata, or treatment with methyl jasmonate vapors. Treatment with methyl salicylate antagonized the signaling pathway of wounding. Treatment with methyl salicylate at 2 h after wounding significantly reduced wounding-induced gene expression of bothRlemMLP1andRlemMLP2.Protein products of these genes were obtained by using a prokaryotic expression system, and had protease inhibitor activity. RlemMLP2, but not RlemMLP1, contained a thaumatin motif, and only RlemMLP2 showed anti-fungal activity againstAlternaria citri. Cellular localization analysis withRlemMLP1orRlemMLP2fused to a green fluorescence protein gene following transient translation using a particle bombardment in onion cells indicated that both RlemMLP1 and RlemMLP2 were localized to the cytosol. These evidences revealed that rough lemon RlemMLPs are likely to have defensive function against pathogens at least when host cells are broken by their infections.