苹果类泛素蛋白MdRAD23C2基因的分离及功能鉴定

Separation and functional characterization of a ubiquitin-like protein MdRAD23C2 in Malus

【目的】研究苹果类泛素蛋白RAD23基因(MdRAD23C2)的功能,为抗旱苹果新品种的培育奠定基础。【方法】以秦冠苹果(Malusdomesticacv.‘Qinguan’)为材料,克隆MdRAD23C2基因,对其进行生物信息学和定位分析,通过实时定量PCR分析其在不同逆境胁迫下(干旱、盐、碱和ABA)的表达模式。构建过表达载体转化农杆菌,采用叶盘法转化烟草,选取相应的转基因烟草株系,分析干旱胁迫下转基因烟草的相对电导率和丙二醛、叶绿素、过氧化氢(H2O2)、超氧阴离子(O-·2)含量以及二氨基联苯胺(DAB)、氮蓝四唑(NBT)染色情况,探讨MdRAD23C2过表达对转基因烟草抗旱能力的影响。【结果】苹果MdRAD23C2基因编码序列长1146bp,编码381个氨基酸,其定位于细胞核和细胞膜中,可响应干旱、盐和碱等逆境胁迫。干旱处理2周,野生型烟草株系萎蔫程度明显,且DAB和NBT染色加深,相对电导率、丙二醛、H2O2和O-·2含量均极显著高于转基因型烟草,而叶绿素含量极显著低于转基因型烟草,说明过量表达MdRAD23C2的烟草株系对干旱胁迫的耐受性明显增强。【结论】分离克隆了苹果MdRAD23C2基因,该基因可提高转基因植物对干旱胁迫的耐受性。

【Objective】This research studied the functions of a ubiquitin-like protein of MdRAD23C2 in Malus to provide basis for breeding new drought-resistant varieties.【Method】The full-length cDNA of MdRAD23C2 was cloned from‘Qinguan’apple(Malus domestica),and analysis was performance for bioinformatics and location.The qRT-PCR was carried out to analyze the expression pattern of MdRAD23C2 under different stresses(drought,salt,alkaline and ABA).Expression vector carrying MdRAD23C2 was constructed and transformed into wild tobacco via leaf disk method.Independent lines of transgenic tobacco were selected for further analysis based on their relative conductivity,MDA content,chlorophyll content,H 2O 2 content,O-·2 content,DAB staining and NBT staining under drought stress.【Result】The cDNA of MdRAD23C was 1 146 bp in length,encoding 381 amino acid residues.MdRAD23C2 was located in nucleus and cell membrane,and responded to drought,alkaline and salt stresses.After withholding water for two weeks,the leaves of wide-type tobacco plants exhibited obvious wilt,and DAB and NBT staining deepened.The relative electrical conductivity and contents of MDA,H 2O 2 and O-·2 were significantly higher than those of transgenic tobacco,but the chlorophyll content was significantly lower,suggesting that over-expression of MdRAD23C2 in tobacco(Nicotiana nudicaulis)plants led to enhanced drought stress tolerance.【Conclusion】MdRAD23C2 was isolated from‘Qinguan’apple(Malus domestica),and it would improve the tolerance of transgenic plants against drought stress.