【作者】 戴良英；

【导师】 李栒； 罗宽； John.G.Turner；

【作者基本信息】 湖南农业大学 ， 作物遗传育种， 2001， 博士

【摘要】 茉莉酸及其衍生物（统称为茉莉素）是一类新的植物生长调节剂。茉莉素广泛存在于高等植物中，其生物合成由亚麻酸经脂肪氧合酶途径合成，它对植物的生长发育如根的生长、果实成熟、块茎增大以及花粉形成等有重要影响，它作为伤反应所必须的信号分子来调控植物对逆境、伤害，病原物侵染以及害虫侵袭的反应。 尽管茉莉素对植物的影响已有广泛的研究，但对其作用分子机理研究极少。COI1基因是从拟南芥中分离的茉莉素信号传递途径中的唯一基因，它介导茉莉素所调控的植物育性及抗性。coil突变体对茉莉素不敏感，表现雄性不育，抗性完全丧失。根据由COI1序列所得到的蛋白质氨基酸序列分析表明COI1含有2个重要的功能域，即16个富含亮氨酸重复序列和一个F-box。人和酵母等许多生物中都含有F-box蛋白，它与SKP1和cdc53（cullin）形成泛素连接酶复合体，称为SCF复合体，其作用是选择性结合降解蛋白参与泛素蛋白降解途径，而泛素蛋白降解系统可以调控许多重要过程如细胞循环，信号传导，基因转录以及免疫反应，雄性不育和细胞程序化死亡。对生物的生长发育及生物和非生物逆境的反应有重要作用。由于COI1含有F-box功能域，因此，COI1也很可能是SCF组成成分，介导茉莉酸信号传导途径。本研究以COI1为诱饵采用酵母双杂交分析体系筛选拟南芥cDNA文库，分离和鉴定COI1互作基因，得到COI1互作基因12个，其中ASK1与SKP1基因高度同源，并从遗传学、分子生物学等方面，首次证明ASK1和COI1形成复合体，并一起调控植物育性，进一步分离得到ASK1互作基因24个，完成了序列分析，为阐明茉莉素调控植物生长发育的分子机理提供了重要线索，并为作物抗性和雄性不育基因工程提供了新思路。 主要研究结果如下： 1、酵母双杂交试验 获得ASK1、COAP2、COAP3、COAP4等12个重要COI1互作基因。 首次以COil为诱饵利用酵母双杂交分析体系筛选拟南芥cDNA文库，得到 300多个阳性克隆，经PCR、Rsalk切PCR产物以后归为12群，从代表菌落提 取吓42＊D C＊＊A重组质粒进行了测序，完成了序列分析，得到＊SK！、＊0＊＊2、 COAP3、COAP4等12个基因，其中ASKI、COAPZ、COAP3等基因与SKPI等 重要基因有较高的同源性。证明了COil与ASKI、COAPZ和COAP3能强烈互作。 2、COnk作基因功能分析 （！）T—DNA筛选 筛选T CDS—7和CD6—7 2个T—DNA株系DNA库，但未得到供试基因 ASKI、COAPZ、COAP3和COAP4的阳性结果。 （）首次利用反义RNA技术，从邀传上、分于生物学、生物化学等方面证明 了ASK！词控植物曹性 构建了ASKI、COAPZ、COAP3和COAP4等反义玉因，完成了拟甫芥的转 化，获得36株转反义ASKI植株，其中27株表现雄性不育，6株育性降低，获 得转反义COAPZ，转反义COAP3、转反义COAP4植株，分别为57株、96株和 ！02株，都表现可育。证明了所有转反义基因植株在对McJA的反应以及抗虫性、 杭病、抗干早等方面与野生型植株没有差异。以野生型植株花沏和转反义ASKI 基因雄性不育植株杂交，FI代和BCI代可育与不育株比例坏为1：！。完成了转反 义 ASKI基因雄性不育植株的 PCR、RT—PCR、Western blot等分子检测，结果表 明转ASKI反义基因雄性不育植株中ASKI的表达受到显著抑制。从而证明ASKI 橱控植物育性。 3、完成了GST—ASKI融合蛋白的表达与纯化。建立了ASKI在酵母中的表 达及纯化体系。获得GST—ASKI t合蛋白纯化产物10mg左右。 4、建立了COil在大肠杆茵中的表达及纯化体系。完成了COil蛋白的表达 及纯化，获得COil蛋白纯化产物！sing左右。 5、分别建立了ASKI、COil抗血清制备体系，获得了效价高、专一性强的 。一ASKI、a—COil抗血清。 6、完成了a—ASKI、a—COil 抗血清的专一性检测。采用ELISA 以及 ＿． Western blot分析体系证明 a—ASKI、a—COil具有很强的专化性，只和其相应 二 的蛋白专一结合． 7、创建了COil和ASKI的共免疫沉淀试验体系，国际上首次证明ASKI和 COil在植物体内形成蛋白复合体，并一起记控植物育性。 8、获得ASKI互作基因24个。 以ASKI为诱饵，继续筛选了拟南芥酵母双杂交。DNA文库，获得24个ASKI 互作基因。序列分析表明，其中有很多重要基因，PLYI57和PLYB49含F—b。X， PLYI57可能与硫吸收代谢有关。 9、首次提出拟南芥中茉莉酸信号传递途径与拟南芥中SCF复合体及作用等 模型。更多还原

【Abstract】 Isolation and Characterization of Arabidopsis Genes Interacting with COIl Abstract Plant hormones are involved in all aspects of plaiit growth and development. Jasmonic acid and its derivates (here referred as to jasmonates (IA,)) are a new 鑜ass of plant regulators that are synthesized from lipids through the octadecanoid pathway and widely distributed in plant kingdom. They influence many plant developmental processes including root growth, fruit ripening, tuberization and pollen formation. Jasmonates also act as a wounding signal to regulate plant response to stress, wounding, and pathogen infection and insect attack. Although the effects of jasmonates on plants are well studied, the molecular basis of JA action remains unknown. COIl gene was the first isolated regulatory gene in IA signal pathway and found to be required for plant fertility and defense. The coil mutant plants are insensitive to lAs and extremely susceptible to insects ,fungal pathogens and drought. They also display male sterile phenotype. The deduced COIl amino acid sequence showed that COIl has two important motifs: F-box domain and leucine rich repeats domain. The F-box domain was present in many yeast, C. elegan and human proteins which interact with Skpl and Cdc53 (cullin) to assemble ubiquitin ligase complexes, designated SCF. The function of SCF complex is to selectively recruit many short-lived proteins for degradation. The ubiquitin-rnediated degradation of regulatory proteins plays important roles in control of numerous processes, including cell cycle control, signal transduction, transcription regulation, receptor-down regulation, and endocytosis. The ubiquitin protein degradation system has been implicated in the immune response and programmed cell death. Because COIl contains this important F-box motif, we speculate that COIl might be a component of SCF complex that is involved in IA signal pathway. In this study, we drew on yeast two-hybrid system to identify the 4 proteins that interact with COIl. As a result, we have identified several proteins, one of COIl interacting proteins was found to be Arabidopsis SKPI-like protein ASK1. ASKI was expressed in yeast and purified for raising antibody in rabbit. We have demonstrated for the first time that COIl interacts with ASK 1, to form a complex that is required for iA action in planta. Furthermore, we showed that expression of antisense ASKI results in male sterility. 24 ASK1 associated genes were isolated and sequenced. The result of the study gave an important clue to study in molecular basis of IA action. The main results of this study are as follows: 1. Yeast two-hybrid system We used COIl as bait to screen Arabidopsis cDNA library in yeast two-hybrid system. More than 300 putative positive colonies have been isolated. They belonged to 12 distinct groups. Representative cDNAs from each group were sequenced. DNA database searching showed that one of these genes, represented by 59 positive colony, was identical to the previously identified Arabidopsis SKPI like protein ASK1 (formerly named as AtSkpl). Other putative COIl Associated Proteins were designated as COAP2, 3 to 12, respectively. COIl interacted strongly with ASK 1, COAP2 and COAP3. Presence of the F-box motif in COIl led us to p更多还原