岷江百合多聚半乳糖醛酸酶抑制蛋白基因及其启动子的克隆与分析

Cloning and Analysis of a Polygalacturonase Inhibiting Protein Gene and Its Promoter from Lilium regale Wilson

多聚半乳糖醛酸酶抑制蛋白(PGIPs)在植物抵御病原菌侵染的过程中具有重要的作用。从尖孢镰刀菌抗性百合野生种岷江百合中分离得到一个PGIP基因及其启动子。LrPGIP的开放阅读框长度为1008 bp,编码一个含有335个氨基酸残基的蛋白质,并具有7个富含亮氨酸重复结构域。LrPGIP与油棕、椰子、林烟草的PGIP同源性较高,分别为91%,94%,86%,且与单子叶植物海枣、粗柄象腿蕉、油棕、椰子的PGIP蛋白亲缘关系更近。LrPGIP在根、茎、叶、花、鳞片中均有表达,在根中的表达量最高,在鳞片中的表达量最低。LrPGIP的表达水平受尖孢镰刀菌的诱导,在接种尖孢镰刀菌后72 h表达量最高。植物信号分子水杨酸、茉莉酸甲酯、乙烯利、过氧化氢均诱导LrPGIP表达。LrPGIP受乙烯利诱导后的表达量最高,茉莉酸甲酯次之。LrPGIP启动子片段的长度为706 bp,具有激素以及胁迫响应等多个顺式作用元件。将LrPGIP启动子驱动β-葡萄糖苷酸酶(GUS)基因的表达框转入烟草表达,G US活性明显受尖孢镰刀菌、交链格孢侵染以及氯化汞、氯化钠胁迫的诱导,同时还响应茉莉酸、水杨酸等防卫相关植物激素。氯化汞处理后的GUS活性上调幅度最大,其次是交链格孢和乙烯利。可见,LrPGIP启动子活性受植物激素、生物及非生物胁迫的诱导。上述结果表明,LrPGIP可能是岷江百合抵御尖孢镰刀菌侵染的重要抗病基因。

Polygalacturonase-inhibiting proteins(PGIPs)play an important role during response to pathogen infection.A PGIP gene and its promoter were isolated from Lilium regale Wilson,which has strong resistance to Fusarium oxysporum.The open reading frame of LrPGIP was 1008 bp in length,which encoded a protein with a length of 335 amino acid residues and contained 7 leucine-rich repeat domains.LrPGIP had high homology with PGIP of Elaeis guineensis,Cocos nucifera and Nicotiana sylvestris,which was 91%,94%and 86%,respectively.The LrPGIP showed closer relation with Phoenix dactylifera,Ensete ventricosum,E.guineensis and C.nucifera.LrPGIP was expressed in roots,stems,leaves,flowers and scales.It had the highest expression level in roots and the lowest expression level in scales.The expression level of LrPGIP was induced by F.oxysporum,and the highest expression level was at 72 h after F.oxysporum infection.The plant signal molecules including salicylic acid(SA),jasmonic acid(JA),ethephon(ETH)and hydrogen peroxide induced the expression of LrPGIP.Among them,the expression level of LrPGIP induced by ETH was the highest,which was followed by JA.The length of LrPGIP promoter fragment was 706 bp and had some cis-acting elements,such as hormone and stress responding elements.The expression cassette of theβ-glucuronidase(GUS)driven by the LrPGIP promoter was transferred into tobacco for expression,and the GUS activity was obviously induced by the infection of F.oxysporum and Alternaria alternata as well as the stress of HgCl_(2) and NaCl.It also responded to defense-related plant hormones including JA,SA and ETH.The up-regulation of GUS activity by HgCl_(2) treatment was the greatest,followed by A.alternata and ETH.These data showed that the LrPGIP promoter activity was induced by plant hormones,biotic and abiotic stresses.The above results indicated that LrPGIP might be an important disease resistance gene in L.regale against F.oxysporum.