杜梨胆碱单加氧酶基因克隆及胁迫表达
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家自然科学基金(31101529);江苏省农业科技自主创新资金[CX(11)4050]


Cloning and Expression Analysis of a Choline Monooxygenase Gene in Pyrus betulaefolia Bunge under Abiotic Stress
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    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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李 慧,丛 郁,常有宏,等.杜梨胆碱单加氧酶基因克隆及胁迫表达[J].西北植物学报,2012,32(6):1093-1098

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  • 在线发布日期: 2012-07-06
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