探讨苦瓜核醣体失活蛋白基因家族分子演化之研究.doc

探討苦瓜核醣體失活蛋白基因家族分子演化之研究 The molecular evolution of ribosome-inactivating protein gene family in Momordica charantia L. 研究生：王薇琍 Wang , Wei-Li 指導教授：江友中 Chiang , Yu-Chung 【摘要】 核醣體失活蛋白 Ribosome-inactivating proteins 是一種植物對抗病原菌的保護性蛋白質，現今研究顯示具有抑制蛋白質生合成能力。現今研究多為蛋白質至基因階層，在分子演化方面並不清楚。因此，本研究的目的是以苦瓜為模式植物，測試RIP基因家族的核酸變異，在分子演化與基因功能上的意義。我們利用栽培、雜交品系苦瓜和野生苦瓜的基因序列資料，計算分析其基因變異和分子演化的關聯，苦瓜基因組中RIP基因家族共選殖3不同基因座，利用3種不同基因座的RIP序列進行分析，此3型RIP的基因型 haplotype 數目分別為34、37和40。此3型的非同義性取代值大於同義性取代值。在核苷酸分歧度 θ 和核苷酸歧異度 π 的數值方面，Locus 1分別為0.01006和0.00321；Locus 2分別為0.01421和0.00373；Locus 3分別為0.01406和0.00412。利用中性假說來測試天擇現象，我們利用Tajima’s D方法來分析DNA序列是否有受到影響。所得到結果顯示此3型RIP基因皆呈現為顯著偏離現象差，數值分別為locus 1 D -2.299, p 0.01 ； locus 2 D -2.416, p 0.01 和 locus 3 D -2.367, p 0.01 ，而在葉綠體DNA的atpB-rbcL的區間則呈現中性結果。由中性假說測試的數據呈現顯著偏差和同義非同義性取代的比值來看，我們可推測出苦瓜之核醣體失活蛋白基因家族受到天擇效應和馴化作用的影響。 核糖體失活蛋白、Tajima’s D、同義性取代、非同義性取代、馴化作用 Ribosome-inactivating proteins RIP can inhibit protein synthesis and protect plants against pathogens. Molecular variations of the RIP gene family, however, are still unclear. In this study, we estimate the nucleotide variations and construct the pattern of molecular evolution in RIP gene family using bitter melon as a model plant. We identify the genetic diversity and molecular evolutionary history within and among wild, hybrid, and cultivated varieties of bitter melons. Samples were analyzed using PCR and cloning to sequence three different loci of the RIP genes locus 1-3 and one neutral chloroplast spacer. We analysis 72 sequences in each locus of RIP genes; 34 haplotypes in locus 1, 37 haplotypes in locus 2, and 40 hyplotypes in locus 3 were identified. In each type of RIP genes, the numbers of nonsynonymous mutations are more than synonymous mutations. Nucleotide diversity and divergence were detected 0.01006 θ and 0.00321 π in locus 1, 0.01421 and 0.00373 in locus 2, and 0.01406 and 0.00412 in locus 3. Test of selection and neutrality Tajima’s D were performed to assess the evolutionary proces