新疆红肉苹果PGIP基因的克隆及原核表达  

Cloning and expression in E.coli of polygalacturonase-inhibiting protein gene in Malus sieversii f.neidzwetzkyana

作　　者：孙华[1] 王春燕[1] 宋杨[1] 吴树敬[1] 张芮[1] 冯守千[1] 陈晓流[1] 陈学森[1] 

机构地区：[1]山东农业大学作物生物学国家重点实验室,山东泰安271018

出　　处：《果树学报》2013年第1期1-7,共7页Journal of Fruit Science

基　　金：国家重点基础研究发展计划课题(2011CB100606);国家自然科学基金项目(31171932)

摘　　要：【目的】研究克隆新疆红肉苹果[Malus sieversii f.neidzwetzkyana(Dieck)Langenf]PGIP基因并进行原核表达,探讨其抗病机制。【方法】根据Genbank中已经发表的‘金冠’苹果PGIP保守区域设计1对特异引物,以新疆红肉苹果叶片总RNA为模板,T/A克隆后进行序列测定,并对该序列进行分析。随后将该蛋白成熟肽cDNA片段连接到原核表达载体pET30a(+)中,构建融合表达质粒,转化到E.coli BL21(DE3)中进行表达。【结果】序列分析表明,新疆红肉苹果PGIP基因cDNA编码区全长993 bp,编码330个氨基酸残基,命名为MsPgip,GenBank登录号为JQ001783。MsPgip分子质量为36.6 kD,等电点为7.05,有6个潜在的N-糖基化位点,信号肽为N端24个氨基酸残基。该蛋白质还具有2个连续的24个氨基酸残基大小的LRR基序(LSQLKNLTFLDLSFNNLTGAIPSSLSQ LPNLNALHLDRN-KLTGHIPIS)。与已克隆的‘澳洲青苹’、‘金冠’、‘富士’苹果PGIP氨基酸序列同源性均高达99%。原核表达产物经SDS-PAGE分析表明,表达蛋白的分子质量与预期一致。【结论】克隆了新疆红肉苹果PGIP基因,并可在大肠杆菌中表达。【Objective】 PGIPs（polygalacturonase-inhibiting protein） can specially bind Polygalacturonases（PGs） secreted by Plant Pathogenic fungi and inhibit or reduce its hydrolytic activity,and limit the growth of Plant Pathogens.Disease resistance mechanism was studied by cloning and expression of the pgip gene of Malus sieversii f.neidzwetzkyana.【Method】A fragment which was about 1 kb in size was amplified from the total RNA by reverse transcription PCR（RT-PCR）with a pair of specific primers based on the conserved sequences of pgip gene in ＇Golden Delicious＇apple.The fused expression plasmid was constructed by inserting the cDNA fragment encoding the mature peptide of PGIP into the prokaryotic expression vector pET 30a（＋）,and then transformed into E.coli BL21（DE3）.【Result】Sequence analysis showed that the fragment contains a full coding region of 993 bp encoding 330 amino acid residues with a molecular mass of 36.6 kD.Its GenBank accession number is JQ001783.The deduced protein has a pI of 7.05,six potential N-glycosylation sites,two LRRs,and a hydrophobic region of 24 amino acid residues in the N-terminal which was considered to be a signal peptide.The protein exhibits a homology of 99% with PGIP in ＇Granny Smith＇,＇Fuji＇and ＇Golden Delicious＇apple.The SDS-PAGE indicated that the size of expressed protein was consistent with that of expected protein.【Conclusion】A pgip gene of Malus sieversii f.neidzwetzkyana was cloned and expressed in E.coli.

关 键 词：新疆红肉苹果 多聚半乳糖醛酸酶抑制蛋白 基因克隆 表达 

分 类 号：S661.1[农业科学—果树学]