- 學位論文
野生稻抗病基因Pi-ta分子演化及其相關誘導防禦基因之研究
Molecular Evolution and Related Defense Response of Pi-ta Gene in Wild Rice (Oryza rufipogon)
摘要
Pi-ta為水稻抗病（R）蛋白質，可專一性的辨識稻熱病病原菌的致病因子AVRpita，啟動抗病反應。此研究分析36個亞洲野生稻O. rufipogon的Pi-ta基因序列，經排列比較的結果，可以分成兩類基因群：H1及H2；藉由與外群非洲野生稻O. barthii比較，可推論H1衍生自H2。已知Pi-ta的第918胺基酸為決定對AVRpita的辨識專一性，由最大似然樹的結果，H1與水稻的Pi-ta分在同一個支序內，在第918胺基酸同樣為具有抗病性的丙胺酸，而H2則全都為不具抗病性的絲胺酸，以此胺基酸的差異分成抗病/不抗病主要的兩群進行相關的分析。H1以稻熱病病原菌IK81-25（具有AVRpita）接種，全為抗病，而H2有部分呈現不符合預期的結果，可能有不同的抗病（R）基因及致病因子（Avr）基因交互作用所造成。 由Pi-ta序列建構的基因樹結構、序列的變異明顯減小、高πnon/πsyn值及變異的相關地理分佈，推測野生稻的Pi-ta可能受到重複的選汰清除（recurrent selective sweep）及功能限制（functional constraint）的作用，而非單純族群擴張的結果。我們推論H1的為近期受到選汰清除作用，可能發生於早期稻米馴化的時期，稻熱病病原菌由小米轉移至野生稻或水稻後，因具有抗病的功能而被保留下來。而H2為更早期的選汰清除作用的結果，並受到功能限制的作用，而呈現出其低序列變異的特徵。 另外，我們運用微陣列分析具有Pi-ta基因的野生稻，在接種稻熱病病原菌後早期表現及抑制的基因。似接受器激酶（receptor-like kinase），細胞色素P450及WRKY轉錄因子為主要調節的基因類別；其它與抗病功能相關的基因，例如參與活性氧累積，訊息傳遞鏈及色胺酸的合成的基因亦有明顯的變化。未來需再進一步的實驗，來驗証這些廣泛調節基因功能及交互作用，這將有助於我們更了解水稻對抗生物逆境的機制。
並列摘要
Rice blast disease is caused by the fungal pathogen Magnaporthe grisea. The resistant response is triggered by a physical interaction between the protein products of the host R (Resistance) gene Pi-ta and the pathogen Avr (Avirulence) gene AVRpita. The genotype variation and resistant/susceptible phenotype at the Pi-ta locus of wild rice (Oryza rufipogon), the ancestor of cultivated rice (O. sativa), was surveyed in 36 locations worldwide to study the molecular evolution and functional adaptation of the Pi-ta gene. The low nucleotide polymorphism in the Pi-ta gene of O. rufipogon was similar to that of O. sativa, but greatly differed from what has been reported for other O. rufipogon genes. The haplotypes can be subdivided into two divergent haplogroups named H1 and H2. H1 is derived from H2, with nearly no variation and at a low frequency. H2 is common and the ancestral form. The LRR domain has a high πnon/πsyn ratio and the low polymorphism of Pi-ta gene might cause primarily by recurrent selective sweep and constraint by other putative physiological function. Meanwhile, we provide data to show the amino acid, Ala-918, of H1 in the leucine-rich repeat (LRR) domain has close relationship with resistant phenotype. H1 might arise recently during rice domestication, and associate with the scenario of the blast pathogen host shift from Italian millet to rice. To identify early induced and repressed defense genes involved in broad-spectrum resistance to rice blast, microarray is used to compare differentially expressed in AVRpita-resistant and -susceptible wild rice O. rufipogon. Receptor-like kinase (RLK), cytochrome P450, and WRKY transcription factor are the majority gene families induced. In addition, some genes that participate in ROS, signal transduction, and tryptophan pathway are discussed. Further functional validation and analysis of these genes will enhance our understanding of the molecular mechanism of broad-spectrum resistance in rice.
參考文獻
延伸閱讀
- 翁子涵（2018）。利用限制酶切位連結核酸定序技術探討野生稻導入系統之基因體多型性〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU201800044
- 許龍欣、王聖善、陳榮坤（2017）。稻熱病抗性相關分子標誌之開發與臺灣水稻品種Pik基因座單倍型分析。臺南區農業改良場研究彙報，(70)，11-23。https://www.airitilibrary.com/Article/Detail?DocID=02556189-201712-201712180017-201712180017-11-23
- 廖大經、吳佩真、吳東鴻（2018）。針對稻熱病Pia及Piz基因座分子輔助選育秈稻新品系之研究。台灣農業研究，67(1)，54-72。https://doi.org/10.6156/JTAR.201803_67(1).0006
- 詹雅勛（2007）。水稻蠟質基因之DNA序列變異及F2族群之遺傳組成研究〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2007.02472
- Cheng, Z. Q., Liu, Y. P., Chen, R., Peng, B., Xiong, H. B., Zhang, C., Zhong, Q. F., & Huang, X. Q. (2010). Diversity of Waxy Gene Alleles in the Wild Rice Species of the Oryza Genus. Botanical Studies, 51(4), 403-411. https://www.airitilibrary.com/Article/Detail?DocID=1817406X-201010-201012160090-201012160090-403-411