分析蝴蝶蘭PeMADS6轉錄因子參與病程相關基因表現之交互作用

Abstract

一般植物在幼苗時期較易感病，隨著時間成長其抗病性也會增加，此稱為發育型抗性 (Developmental Resistance)。而植物在發育過程中，與發育相關之轉錄因子會調控基因的開啟以及關閉，目前已知花部發育主要受MADS-box family 轉錄因子調控。實驗室先前已建構東亞蘭嵌紋病毒 (Cymbidium mosaic virus, CymMV) virus-induced gene silencing (VIGS) 基因靜默載體能夠在蝴蝶蘭(Phalaenopsis amabilis)上進行功能性分析，並發現當花部器官之PeMADS6下降時會造成CymMV鞘蛋白 (coat protein, CP) 基因大量累積，因此推測PeMADS6可能參與抗病基因之調控。為研究此現象，先由蝴蝶蘭EST資料庫中尋找病程相關 (pathogenesis-related, PR) 基因作為分析抗病途徑之標記，並且瞭解PR基因是否受PeMADS6影響。在病毒感染的花部器官中PR1、PR10大量表現，然而在PeMADS6表現量降低時則無法偵測；PR7則是在病毒感染的葉部組織中能夠大量表現，但在花部器官中卻無法表現；PR16不論PeMADS6表現量是否下降，在病毒感染的情況可以在葉部組織以及花部組織中被偵測。發現PR1、PR7、PR10以及PR16會受PeMADS6影響，因此利用genome walking延伸這些PR基因之上游區域序列，目前已有PR1、PR10以及PR16的上游區域，藉由順向調控核酸序列分析，可在PR1和PR10上游區域中找出大量與抗病相關之核酸序列，並且發現在PR1上游區域中含有7個CArG核酸區域，此區域為MADS轉錄因子結合區域，但PR10上游區域則不含CArG核酸區域。因此進一步以DNA與PeMADS6蛋白質的結合，結果可知PeMADS6轉錄因子能夠直接與PR1上游區域結合，而無法與PR10之上游區域結合。本實驗初步瞭解花部發育之轉錄因子可能直接與間接調控抗病基因的表現。

Generally, plant is more susceptible during seedling stage and gradually become more resistance to pathogen infection. This type of resistance is named developmental resistance. Several transcription factors are reported to be involved in plant development. The most known transcription factors that involved in plant development belong to the MADS-box gene family. In our previous study, we knockdowned a Phalaenopsis orchid MADS-box gene, PeMADS6, in Phalaenopsis amabilis by Cymbidium mosaic virus- (CymMV) induced gene silencing vector for functional analysis. Accidentially, we found the accumulation of CymMV coat protein (CP) gene was greatly increased in PeMADS6 silenced floral organ. We speculated that PeMADS6 may involve in the Phalaenopsis orchid developmental resistance. To explore the possibility, we identified pathogen-related (PR) genes from orchid EST library, and analyzed the PR genes expression in response to PeMADS6 expression. We found that PR1 and PR10 can be induced by CymMV both in leaves and flowers; however, both PR1 and PR10 can not be induced in PeMADS6 silenced flowers. In addition, PR7 can be induced by CymMV in leaves but not flowers, and PR16 can be induced by CymMV both in leaves and flowers regardless the expression of PeMADS6. To further analyze if the PeMADS6 involved in the regulation of PR genes, we extended the upstream region of these PR genes by genome walking and analyzed the cis-acting elements in those regions. Notably, 7 CArG motifs were found in the upstream region of PR1 but not in PR10. Protein-DNA binding assay was conducted to assay wheather PeMADS6 can directly bind the upstream region of PR1. The results indicate that PeMADS6 can directly bind to upstream region of PR1 but not PR10, and suggeste that PeMADS6 may regulate PR1 in a directly mannor and PR10 in an indirectly mannor.