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作 者 ：李 慧1,2，丛 郁3，常有宏1,2*，蔺 经1，盛宝龙1

期 刊 ：西北植物学报 2012年 6期 页码：1093~1098

关键词：杜梨；胆碱单加氧酶；基因克隆；表达特点；

Keywords:birch leaf pear (Pyrus betulaefolia Bunge), choline monooxygenase, gene cloning, expression features,

摘 要 ：为了解梨砧木—杜梨对非生物胁迫的防御机制，采用RT PCR、RACE和长片段PCR技术从杜梨幼苗中获得1个甜菜碱合成相关的胆碱单加氧酶基因(PbCMO)，运用生物信息学方法分析序列特点，并通过跨内含子引物进行半定量RT PCR研究其在非生物胁迫下的表达情况。结果表明：(1)PbCMO基因cDNA序列编码区长1 227 bp，编码由408个氨基酸组成的多肽。其对应基因组DNA序列长2 928 bp，由10个外显子和9个内含子组成。其推导的多肽预测的等电点为6.19，相对分子质量为46.27 kD，具备Rieske型铁硫［2Fe 2S］簇结合区域和非血红素单核态铁配位点序列，与枸杞CMO蛋白相似性最高（71%）。(2)PbCMO在幼苗根和叶中均为诱导型表达，100 mmol/L氯化钠、10%(W/V)聚乙二醇、180 mmol/L甘露醇或20 μmol/L脱落酸处理后其表达量明显上调，表明PbCMO对盐碱、干旱、渗透胁迫和ABA均存在表达响应，可能参与杜梨应对非生物胁迫的转录调节。

Abstract:To provide an insight into stress defense mechanism of birch leaf pear,a choline monooxygenase gene (PbCMO) was cloned via reverse transcriptase PCR,the rapid amplification of cDNA ends (RACE) and long PCR methods.At the same time,PbCMO sequence characteristics were analyzed by the bioinformatics software.Furthermore,semi quantitative PCR with cross intron primers was adopted to study the expression patterns of PbCMO gene under abiotic stresses.The results showed that:(1)PbCMO cDNA sequence contained a 1 227 bp length coding region which encoded 408 amino acid residues.PbCMO genomic DNA sequence length was 2 928 bp which consisted of 10 exons and 9 introns.Predicted isoelectric point and relative molecular mass of PbCMO were 6.19 and 46.27 kD,respectively.PbCMO encoded polypeptide included Rieske type iron sulfur ［2Fe 2S］ cluster binding region and the conserved residues of the mononuclear Fe binding motif.Homology analysis showed that the deduced PbCMO protein was highly similar with medlar CMO protein (71%).Phylogenetic analysis also indicated that PbCMO was more related to medlar CMO.(2)PbCMO gene expression was inducible and its transcription abundances climbed up quickly after 100 mmol/L NaCl,10% (W/V) PEG 6000,180 mmol/L mannitol or 20 μmol/L ABA treatment.It is showed that PbCMO expression was response to salt,drought,osmotic and ABA stress.Conclusively,PbCMO was involved in the defense mechanism against abiotic stress in birch leaf pear.

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