植物耐溫度逆境主要轉錄因子及所調節基因之搜尋與探討

Abstract

全球氣候急遽變遷下，氣候因子嚴重地影響植物生長發育及面對逆境時存活的能力，在非生物逆境中，溫度逆境對作物影響最為廣泛。許多基因調控植物在逆境下的反應，其中轉錄因子已被證實為植物面對溫度逆境時調控之要角，近年來，關於轉錄因子對植物在溫度逆境之調控的單一種植物或個別基因之研究甚多，但缺乏跨物種或多種近緣基因之研究，基於此本研究蒐集植物中不同的調控溫度逆境之轉錄因子及轉錄因子所調控基因之資料，進行綜合性的探討。 本研究於NCBI、Brassica database、JGI、MaizeGDB及Phytozome等資料庫搜尋與溫度逆境相關之植物轉錄因子或相關基因的核苷酸及胺基酸序列，結果獲得最多數的HSF、WRKY、DREB/CBF及DREB/CBF所調控之COR15基因之資料。結果顯示多數HSF轉錄因子僅受高溫之影響，亦有均能受高溫及低溫影響者；除了在小麥TaWRKY33轉錄因子受高溫及低溫影響外，其餘WRKY轉錄因子均受低溫影響；而DREB/CBF轉錄因子則因植物之不同而有高溫或低溫影響之差異；轉錄因子所調節之COR15基因則主要受低溫影響。 研究結果顯示單子葉與雙子葉植物間的WRKY轉錄因子的全基因序列長度、高度相似性片段G/C所佔比率均有明顯的差異，但是在HSF、DREB/CBF轉錄因子及COR15基因則均未有明顯差異。 針對核苷酸序列進行多重序列比較，發現三大轉錄因子及COR15基因在物種間具有兩區間之連續高度相似性片段，在單子葉及雙子葉植物中皆具有高度相似性，因此可針對連續高度相似性片段設計引子，作為篩檢三大轉錄因子及COR15基因在物種間之通用工具。 依據胺基酸序列高度相似性序列進行分群，結果可分為兩大群，且群間具特有的高度相似性序列。三大轉錄因子中轉譯起始點－甲硫胺酸 (methionine) 至DNA binding domain之前，物種間胺基酸序列歧異度較高，而DNA binding domain在單子葉及雙子葉植物中均具有高度的保守性；而在三大轉錄因子之胺基酸連續高度相似性片段，即位於轉錄因子之DNA binding domain上，證實核苷酸與胺基酸序列的保守序列分別所對應的轉錄及轉譯位置的正確性，而此區域之胺基酸序列較核苷酸序列有較高之保守性，則是因有少數遺傳字碼簡併性的發生。在阿拉伯芥的COR15基因之99 bp – 103 bp區間的核苷酸序列亦為DREB/CBF element的核心保守序列 (CCGAC)，說明DREB/CBF轉錄因子可結合COR15基因之核心保守序列，進而調控COR15在低溫逆境下之表現，更加驗證COR15基因與DREB/CBF轉錄因子間之調控關係。 本研究剖析HSF、WRKY、DREB/CBF轉錄因子及COR15基因之核苷酸及胺基酸序列在物種間之保守性，並辨識出在植物物種間連續高度相似性之片段，並利用此片段設計引子，作為篩檢物種間三大轉錄因子及COR15基因之利用工具，期望結果能作為各種作物選育耐溫度逆境品系之參考。

Under the dramatic change of climate, the climatic factors seriously affect the growth and the survival rate of plants. Extreme temperature is the most diverse abiotic stress to the crops. Many genes regulating the reactions of plant under stress were reported. Transcription factors (TF) have been proven to the most important regulating factors of plant under temperature stress. However, only single plant species or particular gene related to TF for plants under temperature stress were studied in the previous studies. Studies on the TFs involving in multiple species or correlated genes are needed. Therefore, DNA or amino acid sequences of TFs related to temperature stress from various plant species were collected and analyzed in this study to obtain a comprehensive result. Information of TFs related to temperature stress was searched from NCBI, Brassica database, JGI, MaizeGDB and Phytozome databases. Among the investigated data, TFs HSF, WRKY, and DREB/CBF as well as COR15 gene are the most predominant. The investigated data indicate that most HSF TFs are affected only by high temperature stress, and only a few HSF TFs are affected by both high and low temperatures. Except for wheat’s TaWRKY33 impacted by both high and low temperatures, all WRKY TFs are affected by low temperature. Nevertheless, DREB/CBF TFs affected by high or low temperature are varied according to different plant species. And TF regulating genes - COR15 are affected by cold stress. The results indicated that obvious differences between monocot and dicot plant species on whole genomic sequence length, G/C content, high similarity region, high similarity region of G/C content were observed in WRKY TFs, but not found in HSF, WRKY, DREB/CBF TFs and COR15 gene. Due to the incessantly conservative DNA sequences of above three TF families and COR15 gene were identified by multiple sequence alignments, universal primer pairs were designed to screen the TFs or genes for diverse plant species. Two groups of plant species in each TF family were found in the cluster analysis according to amino acid sequences, and the main divergent regions were located between the translation start point (methionine) and the downstream of binding domain. The conservative amino acid sequences were traced on the DNA binding domain, indicating the accurate corresponding regions for transcription and translation. Owing to the existence of degenerate codons, higher conservative rates were found in the amino acid sequences than the nucleic acid sequences. The finding that the DNA sequence of 99 bp -103 bp region on COR15 gene was the same as the core conserved sequence (CCGAC) of DREB/CBF element in Arabidopsis thaliana, indicating the possible regulation system for cold stress through the binding of the two conserved sequences. In this study, we dissected the analogous sequences of nucleic acid and amino acid of three TF families and identified high conservative regions among plant species. Based on the identified sequences, universal primer pairs were designed as a tool to screen these TF families and regulated gene COR15 for diverse plant species. The results of this study could provide basic information for selecting superior lines of various plant species with tolerance or resistance to temperature stress.