Functional Evolution of a Cold-induced Gene, CBF3, in Arabidopsis thaliana Ecotypes

我們以28個生態型的阿拉伯芥研究受冷誘導基因，CBF3的功能性演化。 CBF3是受冷調控的訊息傳導路徑上重要的“開關”。我們以不同的軟體分析了1297-bp長的啟動子區域序列以及909-bp轉錄區域序列。經過4oC，1.5小時冷處理後，我們以real-rime PCR測量不同生態型之CBF3表現差異，並將此表現差異和以啟動子序列建構之親緣樹互相對照，試圖找出決定該基因表現之重要序列。大部分啟動子及轉錄區域的序列變異符合中性演化，然而藉由sliding window method of Tajima’s D test，我們偵測到不同形式的天擇存在。雖然啟動子上一段區域可能受到balancing selection，然而此一結果之生物上的意義仍未知。我們也在transcriptional activation domain的C端偵測到以低頻率存在之alleles，此一現象也可能由天擇決定。在蛋白質序列的演化上，CBF3大致受到purifying selection作用。然而跟高度保守的AP2 domain相較，Ka/Ks的比值在transcriptional activation domain有劇烈的波動。我們在此區域找到Ka/Ks比值超過1的區段。經由codeml程式(PAML package)的檢測，我們在此區段中偵測到ㄧ個可能是受到positive selection作用的胺基酸(151E)。此胺基酸在7個生態型中被置換為alanine。根據前人對於CBF1之transcriptional activation domain的研究，此一胺基酸(151E)若被取代為alanine，則其活化下游基因表現之能力會增強。我們因此推斷positive selection選擇此一突變，以增加阿拉伯芥植株對於冷的耐受性。經由研究對偶基因特異性表現(allele-specific expression)的策略，我們在啟動子上找到2個核苷酸序列與CBF3低表現量相關。我們也找到ㄧ個天然的CBF3 knockout line，Kas-2。此一發現可供我們進一步釐清CBF3在植物抗冷中扮演的角色。We are interested in functional evolution of CBF3, one of the “master switches” in cold-regulated pathways, in 28 ecotypes of Arabidopsis thaliana. We analyzed sequence polymorphism of 1297-bp promoter and 909-bp transcriptional unit (TU) regions with the aids of different software. After 1.5h chilling treatment (4oC), the CBF3 expression levels were measured by real-time PCR, and the expression variations were mapped to the phylogeny tree constructed according to promoter sequences polymorphisms. The promoter and TU evolution are compatible with neutral evolution while heterogeneity of natural selection was also detected by the sliding window method of Tajima’s D test. A region in promoter was possibly subject to balancing selection, however, the meaning of this is not clear at present. We also found selection against low frequency alleles in C-terminal transcriptional activation domain. In protein sequence evolution, the CBF3 is under strong purifying selection which is against deleterious substitution. However, in contrast to highly conserved AP2 domain, the Ka/Ks ratio fluctuated in transcriptional activation domain in paired comparisons among CBF paralogs. A region with ω more than one was found. The amino acid residue within this region, 151E, was identified by codeml (PAML package) as positively selected site. 151E was substituted by 151A in seven ecotypes we have studied. According to a previous report of trans-activation assay in the transcriptional activation domain, this substitution led to stronger trans-activity. We believe this substitution was favored by positive selection to acquire better cold-tolerance for Arabidopsis in the wild. Using the allele-specific expression approach, we identified two nucleotide sites that are related to low expression of CBF3 after chilling treatment. Kas-2, a CBF3 natural knockout line, was found in this study and will be useful to evaluate the roles of CBF3 in freezing tolerance in Arabidopsis.Abstract 1 中文摘要 2 INTRODUCTION 3 Molecular evolution and statistics to detect evolutionary process 3 Advantages of using natural variation to predict functional sequences 4 The necessity of searching the link of genotype-phenotype 5 In protein evolution 5 Allele-specific expression approach 6 CBF gene family in Arabidopsis thaliana 7 Divergence of CBF paralogs 8 Strategies and goals of CBF functional evolution research 10 Objectives that have been obtained in this report: 10 MATERIALS AND METHODS 12 Arabidopsis thaliana ecotypes 12 DNA extraction, PCR, and Sequencing 12 Molecular evolution and population genetic analysis 13 Cold-treatment and sampling procedure 14 RNA extraction and cDNA synthesis 14 Real-time PCR analysis 15 RESULTS 17 Sequence polymorphism in CBF3 (AT4G25480) 17 The evolutionary distances among CBF paralogs 18 CBF3 nucleotide diversity is compatible with neutral evolution while heterogeneity of selection strength was also detected 19 A probable selective sweep occurred in both promoter and coding sequence but no significant heterogeneity of polymorphism to divergence ratios was identified 19 The CBF3 protein is under strong purifying selection but significant differences of selection constraints were detected in different functional domains 20 A positively selected site was identified within transcriptional activation domain 21 CBF3 expressions in ecotypes after chilling treatment 22 Real-time PCR data calibration 22 Variation of CBF3 expression levels in different ecotypes 23 DISCUSSION 25 The technical bias in real-time PCR and sampling 25 The sequence polymorphism in CBF3 gene 25 The link of coding sequence diversity to previous functional assay 27 Positive selection in CBF3 protein sequence 28 Matching the expression levels with phylogeny constructed according to promoter sequences 29 FIGURES and TABLES 31 REFERENCES 46 Appendix 5