杜梨CPI基因的克隆、序列分析及表达  

Cloning,sequencing and expression of a cysteine proteinase inhibitor gene(PbCPI) from Pyrus betulaefolia Bunge

作　　者：李慧[1] 丛郁[2] 常有宏[1] 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,江苏南京210014 [2]中国科学院南京土壤研究所,江苏南京210008

出　　处：《江苏农业学报》2011年第5期1070-1077,共8页Jiangsu Journal of Agricultural Sciences

基　　金：江苏省农业科学院科研基金项目(6110816);国家"948"项目(2009-Z18);国家农业科技成果转化基金项目(2008GB2C100100)

摘　　要：植物半胱氨酸蛋白酶抑制剂(Cysteine proteinase inhibitor,CPI)在植物的抗逆基因工程中发挥着越来越重要的作用,分离和克隆植物CPI基因进而研究该基因的功能是植物抗逆基因工程研究的热点。为从分子水平上揭示CPI基因在杜梨防御机制中所起的作用,利用RACE和PCR方法,从杜梨种子中克隆CPI基因的cDNA和DNA序列,并采用跨内含子表达引物进行半定量RT-PCR来分析该基因在不同胁迫条件下的表达情况。结果表明:PbCPI基因cDNA长度为987 bp,开放阅读框包含738个核苷酸,编码1个由信号肽(26个氨基酸)和成熟肽(219个氨基酸)组成的多肽。该多肽预测的等电点为6.68,估计的相对分子质量为27 190。其对应基因组DNA序列由3个外显子(1～302 bp,401～772 bp,1 615～1 897 bp)和2个内含子(303～400 bp,773～1 614 bp)组成。通过PSORT进行亚细胞定位分析发现PbCPI蛋白位于内质网上。PbCPI基因编码的多肽具有植物CPI产生抑制活性所必需的一级结构:2个甘氨酸残基(Gly46-Gly47)、假定的反应域QXVXG(Q90-V91-V92-A93-G94)和A/PW基序(P120-W121);并包含植物CPI家族高度保守的特征序列模式LARFAVQEHN、QVVAG和YQAKVWVKPW。进化树分析表明PbCPI和蔷薇科植物CPI蛋白位于分子进化树的同一发育分支上,并且与苹果MdCPI(AAO19652)蛋白具有较高的一致性(95.92%)。杜梨叶片中PbCPI为诱导型表达,高温(30℃)、低温(4℃)、NaCl、机械损伤、MeJA或ABA处理4 h后其表达量明显上调,即其对温度胁迫、盐碱、机械损伤和外源激素处理均存在转录响应,这表明该基因参与了杜梨对生物或非生物胁迫的防御机制。Plant cysteine proteinase inhibitor（CPI） has played more and more important roles in the fields of plant genetic engineering for resistance to adverse environments.It is one of the hot issues to isolate and validate CPI gene functions in the stress-tolerance gene engineering at present.The objective of this study was to illuminate the role of CPI gene in the defense mechanism of birch-leaf pear（Pyrus betulaefolia Bunge）.The full-length cDNA and DNA sequences of a CPI gene were cloned from birch-leaf pear seeds by rapid amplification of cDNA ends（RACE） and PCR.Its expression patterns under different stresses were analyzed by semi-quantitative reverse transcription polymerase chain reaction using cross-introns primers.The results showed that PbCPI cDNA sequence length was 987 bp,which contained a 738-bp-length open reading frame（ORF） and encoded 245 amino acid residues.Sequence analysis indicated that PbCPI protein contained a signal peptide（26 amino acids） and a mature peptide（219 amino acids）.Predicted isoelectric point and relative molecular mass of PbCPI protein were 6.68 and 27 190,respectively.PbCPI genomic DNA sequence consisted of 3 exons（1-302 bp,401-772 bp,1 615-1 897 bp,respectively） and 2 introns（303-400 bp,773-1 614 bp,respectively）.Subcellular localization result indicated that PbCPI protein was localized in the endoplasmic reticulum through the PSORT software analysis.PbCPI deduced polypeptide had the primary structure which was absolutely necessary for inhibitory activity of plant cysteine proteinase inhibitors.This structure included two glycine residues（Gly46-Gly47）,assumed response areas QXVXG（Q90-V91-V92-A93-G94） and A/PW motif（P120-W121）.PbCPI protein contained three typical motif domains,LARFAVQEHN,QVVAG and YQAKVWVKPW,which usually exists in plant cysteine proteinase inhibitors superfamily.PbCPI and other CPI proteins from Rosaceae plants belonged to the same branch in the CPI phylogenetic tree.Homology analysis exhibited that it had the highest sim

关 键 词：杜梨 半胱氨酸蛋白酶抑制剂 基因克隆 序列分析 表达特性 

分 类 号：Q78[生物学—分子生物学]