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National Chung Hsing University Institutional Repository - NCHUIR > 農業暨自然資源學院 > 生物科技學研究所 > 依資料類型分類 > 碩博士論文 >  阿拉伯芥中調節植物發育之一群NAC-like基因的特性與功能性分析

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/152590

標題: 阿拉伯芥中調節植物發育之一群NAC-like基因的特性與功能性分析
Characterization and Functional Analysis of NAC-like Genes in Regulating Plant Development in Arabidopsis thaliana
作者: 施靜芳
Shih, Ching-Fang
Contributors: 楊長賢
Chang-Hsien Yang
生物科技學研究所
關鍵字: 阿拉伯芥;NAC;茉莉酸;雄不稔;分生組織
Arabidopsis thaliana;NAC;Jasmonic Acid;Fertility;Meristem
日期: 2012
Issue Date: 2013-11-18 10:44:23 (UTC+8)
Publisher: 生物科技學研究所
摘要: 第一章
本研究主要在探討阿拉伯芥中一個NAC-like基因AIF (ANTHER INDEHISCENCE FACTOR) 在植物發育上扮演的功能性分析。首先,本實驗利用啟動子分析(promoter assay) 方法觀察該基因的表現部位,結果顯示在AIF::GUS轉基因植物中,GUS活性較強表現於花器上,尤其在年輕花苞之花絲頂端與花粉中,隨著花器的成熟其GUS活性慢慢減弱。在細胞定位 (cellular localization) 實驗發現,YFP:AIF-C (此為AIF蛋白質缺少一段穿膜區) 之融合蛋白質主要累積於阿拉伯芥細胞核內,而具全長含穿膜區之YFP:AIF融合蛋白質則表現於胞器膜上,且未在細胞核內看見蛋白質的累積。進一步利用西方墨點法 (western blot) 分析,發現花朵的樣本中,有一截切型式的AIF蛋白質被偵側到,此結果顯示出AIF蛋白質必需經過一道截切加工過程,方可從胞器膜上釋放出來,進而進入細胞核內調控其下游基因表現。而在性狀觀察上,異位表現AIF-C轉基因植物發生雄不稔 (male-sterile) 現象,主要為花朵發育過程中其雄蕊之花藥不開裂所導致。分析AIF胺基酸序列發現此蛋白質具有一段抑制區 (repressor domain),並藉由chimeric repressor silence technology (CRES-T) 技術 (將基因序列構築至帶有抑制子SRDX序列之載體上,進一步轉殖至阿拉伯芥中,藉由其表現之性狀來分析NAC-like基因之功能) 之實驗結果發現,大量表現AIF-C與AIF-C+SRDX出現花藥不開裂的相似性狀,因此推測AIF的功能主要作為抑制子 (repressor) 的角色以調控花藥的開裂。進一步實驗得知,主要引起其花藥不開裂的原因,是由於合成茉莉酸 (jasmonic acid) 之相關基因,如DAD1/LOX3/AOS/OPR3/OPCL1/AOCs等基因被降低表現所導致。若進一步利用人為外加處理茉莉酸 (JA) 荷爾蒙於花藥不開裂之異位表現AIF-C或AIF-C+SRDX轉基因植物花器上,即可成功使其花藥正常開裂。此外,實驗也同時構築AIF-C+VP16載體,亦即於AIF序列後融合一段VP16-AD強的活化子序列 (activator),目的為創造出顯性抑制突變 (dominant-negative mutant) 株。在AIF-C+VP16顯性抑制突變植株中,發現花藥明顯提早開裂,進一步分析JA合成相關基因DAD1/LOX3/AOS/OPR3/OPCL1,皆有明顯提高表現。因此由本研究的數據顯示,AIF的功能主要藉由調控JA合成相關基因進一步控制花藥的開裂。

第二章
NAC-like基因是在其蛋白質N端帶有約160個胺基酸之NAC domain的基因群,被認為參與多種植物發育過程的調控。本研究從阿拉伯芥中,另選殖出分屬兩個不同次群 (subgroup) 的NAC-like (AtNACL) 基因,包括AtNACL1和AtNACL2,並進一步分析其特性及功能性。首先,利用啟動子分析 (promoter assay),將此兩次群基因之啟動子序列構築至帶有GUS報導基因之載體上,並轉殖至阿拉伯芥中,觀察分析這些AtNACL基因表現位置及情況。在性狀分析方面,AtNACL1及AtNACL2 T-DNA插入或反義股 (antisense) 突變株中,莖、葉、花及根的形成上皆有明顯變異,如鋸齒葉 (serrated leaves),叢生花序及叢根等性狀。此外,為了解不同AtNACL基因群之間的交互關係及功能,進一步利用遺傳雜交 (genetic crosses) 及RNAi策略進行分析。在遺傳雜交分析上,atnacl1/atnacl2雙基因突變株 (double mutants) 中,胚與種子的發育明顯出現致死現象。同時亦利用RNAi策略,抑制同群基因的表現,產生多基因突變株,包括atnacl2/4/6以及atnacl1/5/7/9。此兩群多基因突變株中,出現嚴重的莖與葉的變異。這些結果顯示,相同群 (subgroup) 的NACL基因在調節阿拉伯芥的莖及花的發育上,具有功能重疊 (redundant) 特性。綜合上述分析結果,本研究對植物中NAC-like基因的功能提供了更進一步的了解。
第一章
ANTHER INDEHISCENCE FACTOR (AIF), a NAC-like gene, was identified in Arabidopsis. In AIF::GUS flowers, strong GUS activity was detected in the anther, the upper parts of the filaments and in the pollen of young flower buds; GUS activity was reduced in mature flowers. YFP:AIF-C (lacking a transmembrane domain) fusion proteins accumulated in the nuclei of the Arabidopsis cells whereas the YFP:AIF fusion proteins accumulated in the membrane and absent in the nuclei. Further detection of a cleaved AIF protein by western blot analysis in flowers revealed that AIF needs to be processed and released from the membrane to function. The ectopic expression of AIF-C caused a male-sterile phenotype with indehiscent anthers throughout flower development in transgenic Arabidopsis. The presence of a repressor domain in AIF and the similar phenotype of indehiscent anthers in AIF-C+SRDX transgenic plants indicate that AIF acts as a repressor. The defect in anther dehiscence was due to the down-regulation of genes that participate in jasmonic acid (JA) biosynthesis, such as DAD1/LOX3/AOS/OPR3/OPCL1/AOCs. Further analysis indicated that the external application of JA rescued the indehiscence of anthers in flowers that ectopically expressed AIF-C or AIF-C+SRDX. AIF in the AIF-C+VP16 transgenic dominant-negative mutant plants was converted to a potent activator by fusion to a VP16-AD motif, the dehiscence of the anthers in the transgenic plants was significantly promoted, and the expression of DAD1/AOS/AOC/OPR3/OPCL1 was up-regulated. Our data suggest a role for AIF in controlling anther dehiscence by suppressing the expression of JA biosynthesis genes in Arabidopsis.

第二章
NAC-domain proteins are a class of plant-specific transcription factors that contained a conserved 160 amino acid NAC domain at their N-terminus of proteins have been considered to be involved in the regulation of many processes of plant development. To investigate their function, NAC-like (AtNACL) genes in two subgroups (AtNACL1 and AtNACL2) were cloned and characterized in Arabidopsis. The expression of these AtNACL genes was analyzed and further confirmed by promoter assay by transforming constructs fusing the promoter of these genes with GUS reporter gene in Arabidopsis. Shoot, leaf, flower and root formation was altered in T-DNA insertional or antisense mutants of AtNACL1 and AtNACL2 by producing serrated leaves, generating a bushy phenotype with numerous inflorescences and branches of roots. To further investigate the function and the interaction among different AtNACL genes, multiple mutants for different AtNACL genes were generated by genetic crosses and RNAi approach. For example, genetic analysis revealed that the development of embryo and seed were aborted in atnacl1/ atnacl2 double mutants. Severed shoot and leaf alteration was observed in triple mutants of atnacl2/4/6 or triple mutants of atnacl5/7/9 by RNAi approach for each subgroups of NACL genes. These results revealed that genes in same subgroups of NACL genes might have redundant function in the regulation of both shoot and flower development in Arabidopsis. These results provided new insight of the function for NAC-like genes in plants.
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