MiR396 調控 SVP 基因表現造成花器葉化及其與植物菌質體效應蛋白 SAP54 之關係

MiR396-mediated SVP gene expression in floral transition reprogramming and its involvement with phytoplasma effector SAP54

黃禹馨(Yu-Hsin Huang)

指導教授 ： 林長平

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10583

摘要

植物菌質體為一群侷限於植物維管束中的絕對寄生菌，感染花生簇葉病植物菌質體 (peanuts witches’ broom phytoplasma, PnWB) 之日日春 (Catharanthus roseus) 植株產生簇葉、花器綠化及花器葉化等典型病徵。根據本實驗室建立之日日春全轉錄體與小分子 RNA 資料庫中發現 Cr_miR396a 可能會抑制 SHORT VEGETATIVE PHASE (CrSVP1, CrSVP2) 基因表現，利用農桿菌滲透注射法將 miR396a 與 CrSVPs 共同表現於菸草中並偵測到 CrSVPs 表現量下降。在 Cr_MIR396a 轉基因阿拉伯芥中，其 AtSVP 基因低度表現而 AtFT 基因高度表現，此結果皆顯示 miR396a 確實會抑制 CrSVP1 與 CrSVP2 基因表現，並影響下游基因表現。許多報導指出高度表現 SVP 之轉基因植物將會產生花器葉化之表型，另有研究指出，翠菊黃萎病植物菌質體 (aster yellows witches’ broom phytoplasma, AYWB) 之 SAP54AY 是主要造成植物花器葉片化之效應蛋白 (effector)， SAP54AY 轉基因阿拉伯芥展現出嚴重之花器葉化表型。此外， SAP54AY 抑制 At_miR396 之生合成，造成 AtSVP 表現量上升而發展葉狀花器，進一步將 SAP54AY 轉基因植物進行全轉錄體與小分子 RNA 之次世代定序分析，將其結果與日日春資料庫進行開花相關基因與微型核酸 (microRNA) 之表現量趨勢分析，發現在兩種植物樣本中皆有 miR396 調控 SVP 基因表現之現象，且大部分開花相關基因具有相同之表現趨勢。本研究推測植物菌質體的效應蛋白 SAP54AY 干擾了 miR396 所控制之 SVP 基因表現，進而使 SVP 基因表現量上升造成花器葉化之病徵。

關鍵字

花生簇葉病植物菌質體 ； 翠菊黃萎病植物菌質體 ； 花器葉化 ； SAP54 ； SVP ； miR396

並列摘要

Phytoplasmas are special bacteria that are obligated parasites on plants and cause floral transition. The significant symptoms of peanuts witches’ broom (PnWB) phytoplasma-infected Catharanthus roseus plants are witches’ broom, virescence, and phyllody. In previous studies, the transcriptome and small RNA profiles of PnWB -infected C. roseus plants showed that Cr_miR396a might target SHORT VEGETATIVE PHASE (CrSVP1 and CrSVP2) genes. The transient expression analysis showed that Cr_miR396a specifically down regulation of CrSVP1 and CrSVP2 in vivo. In transgenic Arabidopsis expressing Cr_MIR396a, the AtSVP showed low expression level, but high level of AtFT compared to non-transgenic Arabidopsis plant, confirming the results of transient expression analysis. Moreover, many reports indicated that over-expressing SVP genes causes leafy flower phenotype, implying that high expression levels of SVP trigger leafy flower development. SAP54 is an important phytoplasma effector, which causes leafy flower phenotype in plant host. The transcriptome and miRNA profiles of transgenic Arabidopsis expressing SAP54AY were analyzed by next generation sequencing (NGS). The expression levels of flowering related genes of SAP54AY plants are consistent with PnWB-infected C. roseus plants. Moreover, SAP54AY repressed At_miR396 biogenesis, causing AtSVP over-expression in SAP54AY plants. We hypothesized that SAP54AY triggers leafy flower development through negative regulation of the miR396, resulting over-expression of SVP gene and abnormal floral development.

並列關鍵字

PnWB phytoplasma ； AYWB phytoplasma ； SAP54 ； SVP ； miR396 ； leafy flower

參考文獻

Abe, M., Kobayashi, Y., Yamamoto, S., Daimon, Y., Yamaguchi, A., Ikeda, Y., Ichinoki, H., Notaguchi, M., Goto, K., Araki, T. 2005. FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex. Science 309: 1052-1056.

Allen, R. S., Li, J., Stahle, M. I., Dubroue, A., Gubler, F., Millar, A. A. 2007. Genetic analysis reveals functional redundancy and the major target genes of the Arabidopsis miR159 family. Proc. Natl. Acad. Sci. USA. 104: 16371-16376.

Aukerman, M. J., Sakai, H. 2003. Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-like target genes. Plant Cell 15: 2730-2741.

Azevedo, J., Garcia, D., Pontier, D., Ohnesorge, S., Yu, A., Garcia, S., Braun, L., Bergdoll, M., Hakimi, M. A., Lagrange, T., Voinnet, O. 2010. Argonaute quenching and global changes in Dicer homeostasis caused by a pathogen-encoded GW repeat protein. Genes Dev. 24: 904-915.

Bai, X., Correa, V. R., Toruno, T. Y., Ammar El, D., Kamoun, S., Hogenhout, S. A. 2009. AY-WB phytoplasma secretes a protein that targets plant cell nuclei. Mol. Plant-Microbe Interact. 22: 18-30.