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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (28): 128-133.

所属专题： 生物技术； 园艺

• 林学 园艺 园林 • 上一篇    下一篇

甘蓝多聚半乳糖醛酸酶抑制蛋白BoPGIP基因的克隆及序列分析

张弢   

  • 收稿日期:2012-05-03 修回日期:2012-06-26 出版日期:2012-10-05 发布日期:2012-10-05
  • 基金资助:

    青花菜多聚半乳糖醛酸酶抑制蛋白BoPGIP1基因的功能验证及其启动子序列研究;芸薹属作物PGIP基因的克隆及进化研究

Cloning and Sequence Analysis of a Polygalacturonase Inhibiting Protein BoPGIP Gene in Brassica oleracea L.

  • Received:2012-05-03 Revised:2012-06-26 Online:2012-10-05 Published:2012-10-05

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摘要：

PGIP是一种特异性结合和抑制真菌内切PG活性的细胞壁结合蛋白，在植物分子抗病育种中占有十分重要的地位。甘蓝PGIP基因的克隆和序列分析，将为进一步通过转基因技术培育抗病甘蓝新品种奠定基础。本研究通过同源序列克隆法，根据GenBank已登录的青花菜BoPGIP1(Accession: JF325875)基因全长序列设计基因特异性引物，利用PCR扩增技术从甘蓝中克隆基因，利用生物信息学的方法对其编码蛋白的结构和功能进行了预测。将获得的PGIP基因命名为BoPGIP。该基因DNA全长为1065 bp，包含1个内含子和2个外显子。外显子全长975 bp，编码342个氨基酸，分子量为36.2 kD，等电点是6.26。该基因属于PGIP基因家族，具有典型的LRR结构域；功能位点分析显示编码蛋白序列中包括4个N-糖基化位点(65~68，106~109，130~133，254~257)，1个蛋白激酶C磷酸化位点(189~191)，5个酪蛋白激酶II磷酸化位点(73~76，78~81，151~154，182~185，304~307)和4个N-豆蔻酰化位点(157~162，188~193，236~241，273~278)；N末端具有一明显跨膜区域和一个典型信号肽序列；二级结构中包含31.79%helix，13.89sheet和54.32%loop，二级结构以无规则卷曲为主。通过对所克隆的甘蓝BoPGIP基因分析可知，该基因属于PGIP基因家族的新成员，基因的克隆及序列分析为进一步验证甘蓝BoPGIP的功能奠定了基础。

关键词: 水溶性壳聚糖, 水溶性壳聚糖

Abstract:

PGIP is a cell wall binding protein specific binding and inhibiting activity of fungal endo-PG activity and occupies a very important position in plant molecular resistance breeding. Cloning and sequence analysis of PGIP gene in Brassica oleracea L. will lay the foundation for breeding resistant disease varieties through the transgenic technology. Therefore, in the paper, through the homologous sequence cloning method, a new PGIP gene, designed as BoPGIP, were cloned from Brassica oleracea L. by PCR amplification, with two pairs of gene specific primers designed according to the full-length sequences of known BoPGIP1 gene (Accession: JF325875) from Brassica oleracea var. italica. The DNA full-length of BoPGIP was 1065 bp and consisted of one intron and two exons. The full length of the exons was 975 bp and encoded a protein of 342 amino acids with a predicted molecular mass of 43.8 kD and a 6.256 Isolectric Point. Bioinformatics analysis revealed that the BoPGIP gene belonged to the PGIP gene family and have typical LRR domains; moreover, it had four potential N-glycosylation sites (65-68, 106-109, 130-133, 254-257), one protein kinase C phosphorylation site (189-191), five casein kinase II phosphorylation sites (73-76, 78-81, 151-154, 182-185, 304-307) and four N-myristoylation site (157-162, 188-193, 236-241, 273-278); signal peptide analysis showed BoPGIP might encode a membrane protein with a signal peptide at the N-terminus. The predicted secondary structure composition for the protein had 31.79% helix, 13.89% sheet, and 54.32% loop. These results will lay the groundwork for further studying the function of the BoPGIP gene. Cloning and sequence analysis of BoPGIP gene revealed that it belonged to PGIP gene family and will provide a basis for further verify its function.