油菜Bnpgip2-1基因的克隆表达与生物信息学分析  

Cloning,prokaryotic expression and bioinformatics analysis of polygalacturonase-inhibiting protein gene(Bnpgip2-1) from rape

作　　者：陈夕军[1] 张磊[1] 陈羽[1] 张家豪[1] 童蕴慧[1] 徐敬友[1] 

机构地区：[1]扬州大学园艺与植物保护学院,江苏扬州225009

出　　处：《中国油料作物学报》2014年第6期701-706,共6页Chinese Journal of Oil Crop Sciences

基　　金：江苏省自然科学基金(BK2010305)

摘　　要：据GenBank及相关文献提供的序列设计引物,从油菜基因组DNA中扩增出Bnpgip2-1基因完整开放阅读框。该基因全长1 068bp,与已发表的Bnpgip2基因序列(登录号:AF529694)有98%的相似性。RT-PCR分析表明,该基因(已申请序列号为KJ820998)具一个72bp的内含子(544-615),编码区为996bp,含4个限制性内切酶酶切位点(AvaⅠ,185;HindⅢ,536;EcoRⅠ,929;Apa LⅠ,972)。原核表达该基因,表达产物能显著抑制油菜菌核病菌多聚半乳糖醛酸酶(PG)活性,抑制率达52.96%。生物信息学分析表明,表达产物Bn PGIP2-1有331个氨基酸,理论分子量为36.99k Da,p I为8.35,具强疏水性,主要存在于细胞壁。信号肽切点位于第22和23位氨基酸之间(SFS-KNL),N端和C端各存在5个和3个半胱氨酸残基,可形成3个二硫键;二级结构以α-螺旋(31.12%)、β折叠(16.01%)和无规则线圈(52.87%)为结构元件,具典型的LRR结构;三级结构为10个LRR按右手螺旋规则形成的一个开放裂隙区,可能负责其与病菌PG的互作。该基因的表达受病原菌侵染的强烈诱导,但对水杨酸(SA)处理不敏感,茉莉酸(JA)处理反而使其下调表达。According to the sequence of GenBank and related references, the open reading frame of gene Bnp- gip2 - 1 （GenBank accession number KJ820998） was amplified, which was 1 068 bp, 98% similarity to Bnpgip2, containing a single intron of 72 bp （position 544 -615 ） and a coding region of 996 bp. 4 restriction enzymes cut- ting sites ofAva I （185）, Hindm （536）, EcoR I （929） and ApaL I （972） were in this gene. Prokaryotic ex- pression product of the gene could inhibit the polygalacturonases （PGs） activities of Sclerotinia sclerotiorum, the pathogen of rape Sclerotinia stem rot. The inhibition ratio was 52.96%. Bioinformatics result showed that BnPGIP2 - 1 was a 331 amino acids hydrophobic protein with theoretical molecular weight 36.99 kDa and pI 8.35. The pro- tein was mainly located on cell wall. Its signal peptidesplice site was between 22th and 23th amino acid residue. 5 and 3 cysteines were in the N - and C - terminal, forming 3 disulfide bonds. The main structural elements of the deduced protein, which showed the typical leucine - rich repeat （LRR） modular organization, were ct - helix, 13 - sheet and random coil. Tertiary structure was a right - handed helix, consisted of ten repeated LRR modular organi- zations, and formed an opening activity cleft which might be responsible for the interaction of PGIP and PGs from the pathogen. Bnpgip2 - 1 expression was strongly induced by pathogen infection, not affected by SA （ salicylic acid）, and down- regulated by JA （jasmonic acid）.

关 键 词：多聚半乳糖醛酸酶抑制蛋白 原核表达 生物信息学分析 核盘菌 

分 类 号：S565.403[农业科学—作物学]