豆梨半胱氨酸蛋白酶抑制剂基因的克隆及胁迫表达  

Molecular Cloning of a Cystatins Gene PcCPI and Its Expression under Stress in Pyrus calleryana Dcne

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,南京210014 [2]中国科学院南京土壤研究所,南京210008

出　　处：《西北植物学报》2011年第5期861-867,共7页Acta Botanica Boreali-Occidentalia Sinica

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

摘　　要：采用RT-PCR结合RACE技术,从中国梨砧木——豆梨成熟种子中克隆获得豆梨半胱氨酸蛋白酶抑制剂基因（PcCPI）的cDNA全长序列,并通过半定量RT-PCR分析不同胁迫处理对该基因表达水平的影响.结果表明：（1）PcCPIcDNA序列长度为980 bp,开放阅读框78~812 bp,编码的多肽由信号肽（29个氨基酸）和成熟肽（216个氨基酸）组成,具有3个植物半胱氨酸蛋白酶抑制剂基因家族的高保守特征序列模式——LARFAVQEHN、QX-VXG和YQAKVWVKPW.（2）该蛋白与蔷薇科植物的CPI蛋白处于系统发育树的同一分支,且与苹果CPI蛋白的一致性最高（95.9%）.（3）半定量RT-PCR结果显示：PcCPI基因在豆梨叶片中为诱导型表达,在人工模拟逆境条件下（包括喷施50μmol/L MeJA或100μmol/L ABA、刀片2次机械损伤2、00 mmol/L NaCl胁迫、4℃低温或30℃高温胁迫）处理4 h后,豆梨叶片中PcCPI基因表达量明显上升,表明该基因参与了豆梨对生物或非生物胁迫的防御机制.Cystatins gene,plant cysteine proteinase inhibitor（CPI） gene plays an important role in the field of plant defense mechanism.In this study,reverse transcription polymerase chain reaction（RT-PCR） and rapid amplification of cDNA ends（RACE） methods were used for obtaining full-length cDNA sequence of PcCPI from bean pear（Pyrus calleryana Dcne.） mature seed.Then the effects of different stress treatments on the expression levels of PcCPI were investigated via semi-quantitative RT-PCR.The result showed that the cDNA sequence length of PcCPI was 980 bp,which contained an open reading frame from 78 bp to 812 bp that would encode a signal peptide（29 amino acids） and a mature peptide（216 amino acids）.PcCPI deduced polypeptide includes three highly conserved characteristic sequence patterns（LARFAVQEHN,QXVXG and YQAKVWVKPW） of plant cystatins gene family.PcCPI and other CPI proteins from Rosaceae plants belonged to the same branch in the CPI phylogenetic tree.Moreover,PcCPI had the highest identity（95.9%） with Malus×domestica MdCPI.Semi-quantitative RT-PCR results showed that PcCPI gene was inducible expression and its expression was enhanced under artificial stress conditions.Furthermore,PcCPI transcription level climbed up quickly after 50 μmol/L MeJA,100 μmol/L ABA,twice mechanical damage by blade,200 mmol/L NaCl,4℃ or 30℃ treatments for 4 h.It was indicated that PcCPI involved in bean pear against biotic or abiotic stress defense mechanisms.

关 键 词：豆梨 半胱氨酸蛋白酶抑制剂 基因 克隆 表达 

分 类 号：Q789[生物学—分子生物学]