Characterization and functional analysis of a novel PGIP gene from Pyrus pyrifolia Nakai cv Huobali
Characterization and functional analysis of a novel PGIP gene from Pyrus pyrifolia Nakai cv Huobali
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DOI：
10.1007/s11738-012-1164-y
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发表时间：
2013-04
影响因子：
2.6
通讯作者：
Diqiu Liu;Wenxian Li;Xin He;Yuanming Ding;Chaoyin Chen;F. Ge
中科院分区：
文献类型：
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作者：
Diqiu Liu;Wenxian Li;Xin He;Yuanming Ding;Chaoyin Chen;F. Ge
多聚半乳糖醛酸酶抑制蛋白(PGIPs)是与植物自身免疫相关的多功能蛋白，属于植物细胞外富含亮氨酸重复序列(ELRR)蛋白超家族。在许多植物中，PGIP在寄主防御中发挥作用。本研究从火八梨品种Nakai中分离到一个新的PGIP基因PpPGIP。PpPGIP的核苷酸序列与其他植物的PGIP高度同源，其编码的蛋白质具有多个保守的LRR结构域。在系统发育树上，PpPGIP与园艺植物中的几个PGIP紧密聚在一起。构建的PpPGIP的同源模型表明，PpPGIP的主链构象和折叠模式与菜豆PvPGIP2的结构特征高度相似。用RT-PCR方法分析了‘火八梨’健康组织器官中PpPGIP的表达水平，发现PpPGIP在幼叶中有少量积累，但在‘火八里’果皮中大量表达。此外，为了验证PpPGIP的功能，构建了PpPGIP的植物表达载体，并将其导入烟草。Cv Xanthi)。Southern杂交和实时荧光定量PCR检测表明，PpPGIP基因已整合到烟草转化体的基因组中，并在转基因株系中高效表达。在体外平板上检测了PpPGIP的抗真菌活性，转基因烟草的粗蛋白提取物对拟南星、链格孢霉、青霉和黑曲霉的菌丝生长有不同程度的抑制作用。
Polygalacturonase-inhibiting proteins (PGIPs) are multifunctional proteins related to plant autoimmunity and belong to the plant extracellular leucine-rich repeat (eLRR) protein superfamily. PGIPs play a role in host defense in many plants. In the present study, a novel PGIP gene,PpPGIPwas isolated fromPyrus pyrifoliaNakai cv Huobali. The nucleotide sequence ofPpPGIPwas highly homologous withPGIPsfrom other plant species and the protein encoded byPpPGIPhas several conserved LRR domains. The putative protein PpPGIP was closely clustered with several PGIPs from horticultural plants on the phylogenetic tree. The constructed homology model of PpPGIP indicated that the main-chain conformation and the folding patterns of PpPGIP were highly similar to structural features of PvPGIP2 fromPhaseolus vulgaris. The expression levels ofPpPGIPin healthy tissue and organ of ‘Huobali’ were analyzed with RT-PCR, andPpPGIPaccumulated a little in young leaves, butPpPGIPwas expressed abundantly in the pericarp of ‘Huobali’ fruits. Furthermore, in order to verify the function ofPpPGIP, the constitutive plant expression vector ofPpPGIPwas constructed and transferred into tobacco (Nicotiana tabacumL. cv Xanthi). The Southern blot and real-time PCR analyses demonstrated that thePpPGIPgene was integrated into the genome of the tobacco transformants and highly expressed in the transgenic lines. The antifungal activity ofPpPGIPwas detected in vitro plates, and the crude protein extract of transgenic tobacco plants inhibited the hyphal growth ofPhomopsissp.,Alternariasp.,Penicilliumsp., andAspergillus nigerin different degrees.