杜梨胆碱单加氧酶基因克隆及胁迫表达  

Cloning and Expression Analysis of a Choline Monooxygenase Gene in Pyrus betulaefolia Bunge under Abiotic Stress

作　　者：李慧[1,2] 丛郁[3] 常有宏[1,2] 蔺经[1] 盛宝龙[1] 

机构地区：[1]江苏省农业科学院园艺研究所,南京210014 [2]国家农业科技华东(江苏)创新中心--高效园艺作物遗传改良实验室,南京210014 [3]中国科学院南京土壤研究所土壤与农业可持续发展国家重点实验室,南京210008

出　　处：《西北植物学报》2012年第6期1093-1098,共6页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(31101529);江苏省农业科技自主创新资金[CX(11)4050]

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 227bp,编码由408个氨基酸组成的多肽。其对应基因组DNA序列长2 928bp,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46.27kD,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,100mmol/L氯化钠、10%(W/V)聚乙二醇、180mmol/L甘露醇或20μmol/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。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 bioinfor- matics 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 se- quence 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 mono- nuclear Fe-binding motif. Homology analysis showed that the deduced PbCMO protein was highly similar with medlar CMO protein （71~//00）. Phylogenetic analysis also indicated that PbCMO was more related to medlar CMO. （2）PbCMO gene expression was inducible and its transcription abundances climbed up quick-ly after 100 mmol/L NaC1,10% （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.

关 键 词：杜梨 胆碱单加氧酶 基因克隆 表达特点 

分 类 号：Q789[生物学—分子生物学]