Transcript analysis of GH3-like gene family, with special emphasis on GH3-20 in Arabidopsis

FIN219是cop1-6的一個基因外抑制體(extragenic suppressor)，參與遠紅光接受體光敏素A的訊息傳遞；它產生一個575胺基酸的蛋白質，屬於GH3-like基因家族的成員。BLAST search的結果顯示，在阿拉伯芥中存在至少20個GH3-like的基因，但他們的功能尚未清楚；為了進一步瞭解它們是否參與訊息傳遞及在阿拉伯芥生長發育過程中所扮演的角色，決定利用光訊息突變體及T-DNA插入的突變株，研究他們基因的表現。 從這20個基因當中選取九個做為研究的對象，分別為GH3-1、GH3-2、GH3-3、GH3-4、GH3-5、GH3-6、GH3-9、GH3-18及GH3-20。我們發現這九個基因的蛋白質有1-2個可以與其他蛋白質作用的coiled-coil domain，並且在promoter含有許多與光調控相關的cis-elements，如GATA box、IBOX等。利用T-DNA插入的四個GH3-like基因突變株(GH3-2、GH3-4、GH3-5、及GH3-17)，檢測在白光、藍光、紅光、遠紅光及黑暗中阿拉伯芥幼苗的外表型，發現這些GH3-like基因的T-DNA插入突變株具有在遠紅光及藍光中較不敏感的外表型。進一步利用RT-PCR方法檢測他們在不同光訊息突變體中，在不同光源處理下的基因表現情況，結果顯示這些基因可能分別參與不同的光訊息傳遞中。 另外針對GH3-20的研究發現，大量表現和抑制 GH3-20基因在Col.、phyA、phyB、fin219及cry1xcry2的轉殖株中，只有大量表現在野生型阿拉伯芥的轉殖株，其幼苗在遠紅光中會有較長的下胚軸，而其他則無明顯的外表型，暗示GH3-20可能與其他家族成員有基因功能的重複性(gene redundancy)，並且需要PHYA、PHYB、FIN219、CRY1及CRY2均正常的情況下才能發揮負調控下胚軸受遠紅光抑制的角色。 利用RT-PCR的方法檢測GH3-20的組織專一性表現，結果顯示GH3-20主要表現在根部的器官，其他組織部位則無法偵測到它的mRNA，因此暗示GH3-20可能參與根的生長發育。另外，為了觀察GH3-20在細胞中表現的位置，利用基因槍的技術將GH3-20與GFP及GUS兩者的融合體送到洋蔥表皮細胞，結果顯示此基因的蛋白質不論是在黑暗或光照下，均存在細胞質與細胞核內，並不會因為光照而有蛋白質轉移的現象。FIN219 is an extragenic suppressor of cop1-6 and involved in phytochrome A-mediated far-red light signal transduction. It encodes a 575 amino-acid protein that belongs to the GH3-like gene family in Arabidopsis. BLAST search result indicated that there are 20 GH3-like genes present in the Arabidopsis genome. However, their function remains elusive. To further understand weather the members are all involved in light signal transductions and regulate various aspects of Arabidopsis development, several light signaling mutants and T-DNA insertion lines are utilized for the analysis of their gene expression. Nine GH3-like genes, including GH3-1, GH3-2, GH3-3, GH3-4, GH3-5, GH3-6, GH3-9, GH3-18, and GH3-20, were chosed for studies of the functional roles in light signaling. Motif-searching analysis indicated that each of the primary sequences predicted from these nine genes contained 1-2 coiled-coil domains that were believed to be responsible for protein-protein interactions. As well, it was found that there are several light-regulated cis-elements such as GATA box and IBOX present in the promoters of these GH3-like genes. Consistently, the results of RT-PCR by using gene specific primers have revealed that the expression of the GH3-like genes was actually affected by different photoreceptors and some light signaling components. Furthermore, T-DNA insertion lines for some of these GH3-like members exhibited a long hypocotyl phenotype under FR, Blue (B), and red (R) light conditions, and this phenotype was light fluence-dependent. Taken together, these data indicate that the GH3-like gene family in Arabidopsis was indeed involved in light signaling pathway and may play an important role in FR, R and B-mediated inhibition of the hypocotyl elongation. In addition, we also overexpressed or antisensed the GH3-20 gene encoding 190 amino acids in wild type Arabidopsis and various light responsive mutants to investigate the effect on plant development. Preliminary results indicated that the transgenic seedlings overexpressing the GH3-20 gene only in wild-type background rather than in other mutants exhibited a long hypocotyl phenotype under FR. However, the transgenic seedlings inhibiting the GH3-20 gene expression did not show any obvious phenotype under FR, which suggests that GH3-20 may have a redundant function with other members of the GH3-like gene family in Arabidopsis. That transgenic seedlings overexpressing the GH3-20 gene in phyA, phyB, fin219, cry1 and cry2 mutants did not show obvious phenotypes is a good implication that its expression may require these proteins to function correctly in FR. By using RT-PCR, GH3-20 displays a tissue-specific manner with high levels of the transcripts in roots, but is not detectable in other tissues, which implies that GH3-20 may have physiological functions in root development. Moreover, the transient assay of subcellular localizations of the GH3-20 by particle bombardment in onion cells showed that GH3-20 exists in both the cytosol and the nucleus without changes by light.中文摘要………………………………………………………………………….…I 英文摘要……………………………………………………………………..……..III 第一章 前言 一、導讀…………………………………………………………….…..…1 二、植物的光訊息傳遞…..…………………………….……………....…2 三、阿拉伯芥的GH3-like基因…………..…………………..…..……...10 四、植物生長素對植物的生理調控………………………...……..….…12 五、研究目標…………………………………………………...…...……15 第二章 材料與方法 一、植物材料與生長條件……………………………..…………….…..16 二、GH3-like gene clones……..………………………………..…….….17 三、GH3-20基因construct的構築……………..………………….…...18 四、基因轉殖與轉殖株篩選……………………..…………………..….18 五、RNA的粹取與RNA表現量分析……………..…………………..…19 六、組織專一性的表現…………………………………….……...….....20 七、細胞層次座落位置與基因槍法………………..……….………..…20 第三章 結果 一、九個GH3-like基因所轉譯的蛋白質結構有coiled-coil domain可以與其他蛋白質產生交互作用……………………………………..21 二、九個GH3-like基因的promoter上有許多與光調控相關的cis-elements…………….…….……………………………..……..22 三、九個GH3-like基因中調取了七個，並完成clone…………………………………………………………….....23 四、GH3-2、GH3-4、GH3-5及GH3-17的T-DNA插入突變株造成阿拉伯芥幼苗在藍光、紅光與遠紅光的不敏感………………………23 五、在白光、遠紅光及藍光下，部分GH3-like基因表現的確受到光訊息突變株的調控…………………………………………………...…..24 六、大量表現GH3-20基因於野生型阿拉伯芥中造成轉殖株對遠紅光的敏感度下降…..…………………………………...………..………..25 七、抑制及大量表現GH3-20基因於phyA、phyB、fin219及cry1xcry2阿拉伯芥突變體中，並沒有得到與突變體相異的外表型……….27 八、GH3-20在不同光源下會受到紅光、遠紅光或藍光的訊息調控基因所調控……………………………………………………………….28 九、GH3-20不論在黑暗或是光照的情況下，其蛋白質同時表現於細胞質與細胞核中…………………………………………………...…..28 十、GH3-20蛋白質專一表現在成熟植株的根部…….……….…….…29 第四章 討論 一、GH3-like基因可以受到光的調控，並且可能與其他蛋白質產生交互作用…………………..….………………………………………..….50 二、GH3-like基因家族在阿拉伯芥感受藍光、紅光與遠紅光的訊息傳遞路徑中扮演著重要角色…………………………...…….…………..51 三、RT-PCR結果顯示在不同光源下，GH3-2、GH3-4及GH3-5可能受到紅光或遠紅光的光訊息傳遞基因調控……………….………….53 四、大量表現GH3-20基因正譯股(sense)於野生型阿拉伯芥中造成轉殖株對遠紅光的敏感度下降…………………….……………….……54 五、抑制及大量表現GH3-20基因於phyA、phyB、fin219及cry1xcry2阿拉伯芥突變體中，並沒有得到與突變體相異的外表型……..…57 六、RT-PCR的結果顯示GH3-20在不同光源下會受到紅光、遠紅光或藍光的訊息調控基因所調控…………………….……………….…58 七、GH3-20不論在黑暗或是光照的情況下，其蛋白質同時表現於細胞質與細胞核中………..………………………………………………60 八、GH3-20蛋白質的表現具有組織專一性，專一表現在成熟植株的根部…………………………………………………………….……….62 參考文獻……………………………………….………………………….….…….64 附錄一、光訊息傳遞路徑…………………………………………………………73 附錄二、實驗中所用表現載體……………………………………………………74 附錄三、阿拉伯芥之無菌栽培…………………………………….………..…….75 附錄四、總RNA的抽取………………………………………………………….76 附錄五、Quantitative RT-PCR……………………………….….……………….77 附錄六、Gene cloning…………………………….….……………………..……..80 附錄七、Genomic DNA粹取……….….……………………………………..…..84 附錄八、基因槍法（particle bombardment）…………………………………..85 附錄九、表現載體之構築….….…………………………………………….……..87 附錄十、阿拉伯芥之轉殖及篩選………………………………………….………8