【摘要】：生長(zhǎng)素作為一種重要的植物激素,它在植物體內(nèi)的作用不僅與其生物合成密切相關(guān),而且受其信號(hào)轉(zhuǎn)導(dǎo)中的轉(zhuǎn)錄因子調(diào)控。ARF是一類關(guān)鍵的生長(zhǎng)素響應(yīng)因子,近年來(lái)其分子機(jī)理的研究逐漸成為熱點(diǎn),其在植物各組織器官和多種生理活動(dòng)中的功能探究取得了很大進(jìn)展。然而生長(zhǎng)素調(diào)控植物生長(zhǎng)發(fā)育是一個(gè)極其復(fù)雜的過(guò)程,存在著嚴(yán)密的調(diào)控機(jī)制,番茄中ARF家族參與生長(zhǎng)素調(diào)控作用研究多集中于果實(shí)發(fā)育與成熟方面,而在花器官脫落方面ARF參與生長(zhǎng)素的調(diào)控作用機(jī)制尚未明確,因此探討ARF在生長(zhǎng)素調(diào)控番茄花柄脫落的功能機(jī)制具有重要理論和現(xiàn)實(shí)價(jià)值。本文在此基礎(chǔ)上,主要探討了番茄花柄脫落過(guò)程中ARFs家族的表達(dá)譜,隨后挑選與脫落相關(guān)的兩個(gè)番茄SlARFs基因,獲得了其轉(zhuǎn)基因植株,從多個(gè)角度深入探究了其在番茄生長(zhǎng)發(fā)育中的功能,為研究生長(zhǎng)素介導(dǎo)的植物生理功能的作用機(jī)制奠定了一定的理論基礎(chǔ)。主要研究結(jié)果如下：1.通過(guò)乙烯和IAA處理對(duì)番茄花柄外植體脫落率影響的調(diào)查,結(jié)合離層脫落防御基因Cystein-type endopeptidase的表達(dá)信號(hào)分析,認(rèn)為番茄開(kāi)花后8h是花柄外植體脫落的關(guān)鍵節(jié)點(diǎn),在此之前為生長(zhǎng)素抑制脫落時(shí)期,而在此以后為乙烯誘導(dǎo)和促進(jìn)脫落時(shí)期。2.采用P5::GUS'ChicoⅢ'轉(zhuǎn)基因番茄的染色信號(hào)形象化定位不同時(shí)期生長(zhǎng)素在花柄離區(qū)的分布與強(qiáng)度,并通過(guò)橫切和縱切掃描觀察,在番茄花柄脫落過(guò)程中無(wú)論是離區(qū)還是兩端部位,其生長(zhǎng)素都在減少,但離區(qū)部位的生長(zhǎng)素含量始終最高,而維管組織中的含量也較高；皮層和髓部中的含量隨脫落進(jìn)行而快速下降。在第32 h脫落完成后整個(gè)花柄的生長(zhǎng)素也完全消耗殆盡。不同培養(yǎng)方向?qū)τ谏L(zhǎng)素的運(yùn)輸、分布與消耗影響較大。另外,對(duì)脫落過(guò)程中IAA含量以及GUS活性變化的檢測(cè)也驗(yàn)證了8h是花柄外植體脫落的關(guān)鍵節(jié)點(diǎn)。3.分別探討了脫落前期(0-8 h)以及脫落后期(8-32 h)兩個(gè)階段中ARFs的表達(dá)情況。綜合分析發(fā)現(xiàn),在生長(zhǎng)素抑制脫落的前期階段是通過(guò)調(diào)控ARF1,2,3,4,5,7,8-1,9,,11,12,13,13-1,14和17實(shí)現(xiàn)的；而在乙烯誘導(dǎo)脫落的后期作用相反。在前期,生長(zhǎng)素介導(dǎo)了ARF8-1,9,11,12,13,13-1和14的表達(dá),乙烯調(diào)控了ARF13-1的表達(dá)；在后期,ARF14,ARF16和ARF19對(duì)乙烯較為敏感。通過(guò)后期脫落和未脫落兩組的比較分析,篩選出ARF5,6,8,10,11,16以及19被脫落誘導(dǎo)上調(diào),說(shuō)明這些基因?qū)ν瓿擅撀溥M(jìn)程起重要作用。另一方面,對(duì)番茄花柄外植體中SlARFs基因的絕對(duì)表達(dá)量分析表明,在正常花柄離區(qū)中ARF6-1, ARF8-1, ARF13以及ARF19-1是4個(gè)表達(dá)量最高的基因,推測(cè)其可能對(duì)離區(qū)發(fā)育和分離有更為重要的作用。4.設(shè)計(jì)出涵蓋ARF蛋白N端的保守的DNA結(jié)合域(即DBD功能區(qū)域)引物,并通過(guò)特異引物PCR擴(kuò)增,分別得到預(yù)期435 bp (SIARF2:512～947 bp)和422 bp (SlARF14:562～984 bp)的片段。利用Gateway克隆技術(shù)構(gòu)建了SlARF2及SlARF14的RNAi載體,并通過(guò)測(cè)序和酶切方法均驗(yàn)證了其正確性,為通過(guò)阻斷基因的特異性結(jié)合來(lái)探究目的基因功能提供材料。5.利用改進(jìn)的“中蔬六號(hào)”番茄高效再生體系,采用農(nóng)桿菌介導(dǎo)遺傳轉(zhuǎn)化法將目的載體導(dǎo)入植株,整個(gè)過(guò)程始終保持0.5 mg/L PPT的抗性篩選壓力,并將生根培養(yǎng)基中瓊脂含量由原來(lái)的0.8%調(diào)整為0.7%,有利于在抑菌前提下根系的生長(zhǎng),提高了移栽的成活率。對(duì)轉(zhuǎn)化的SlARF2-RNAi PPTR及SlARF14-RNAi PPTR番茄抗性株進(jìn)行PCR擴(kuò)增,進(jìn)一步證明外源基因已成功導(dǎo)轉(zhuǎn)入了番茄基因組中,獲得了一部分陽(yáng)性轉(zhuǎn)化植株,轉(zhuǎn)化率均高于30%。6.對(duì)SlARF14的蛋白結(jié)構(gòu)及組織特異性表達(dá)進(jìn)行的分析表明,SlARF14蛋白缺失能夠與Aux/IAA異源二聚化結(jié)合的CTD區(qū)域,SlARF14基因在大部分組織器官中均有表達(dá),尤其是雌蕊,花蕾,葉片以及膨大期的果實(shí)中,遺傳進(jìn)化分析表明SlARF14與已被證明與葉片生長(zhǎng)發(fā)育有關(guān)的番茄SlARF10高度同源,推測(cè)其可能存在著功能上的相近。對(duì)12個(gè)Aux/IAAs基因進(jìn)行表達(dá)檢測(cè)結(jié)果顯示,大部分Aux/IAAs基因在SlARF14-RNAi與對(duì)照植株中的表達(dá)無(wú)顯著差異,暗示對(duì)于結(jié)構(gòu)缺失(Ⅲ Ⅳ)的SlARF14,僅有個(gè)別Aux/IAAs存在某種形式的調(diào)控,相對(duì)其它ARFs家族成員,S1ARF14可能具有相對(duì)獨(dú)立的作用。7. SlARF14-RNAi植株葉片邊緣更加圓滑卷翹,缺裂變淺或減少,葉色深綠,葉片加厚且側(cè)生葉和小葉間出現(xiàn)一定程度的融合,隨葉片生長(zhǎng)呈現(xiàn)傾向于橫向的擴(kuò)展膨大的趨勢(shì),對(duì)葉片發(fā)育相關(guān)的LeT6和LeEXPA10基因有一定的調(diào)控作用；在光合水平的調(diào)控上,其在發(fā)育早期提高了光合速率,增強(qiáng)了葉綠素積累以及激發(fā)了Rubisco酶活的釋放,同時(shí)上調(diào)了RBCL,RBCS和CAB的表達(dá)；在激素水平的調(diào)控上,其葉片發(fā)育涉及了ZR的參與以及IAA的早期調(diào)控。8. SlARF14-RNAi植株花器官在發(fā)育中易衰敗死亡,剝?nèi)セò昙靶廴?露出子房,發(fā)現(xiàn)雌蕊發(fā)育不良,柱頭出現(xiàn)彎曲,子房發(fā)育異常,正常授粉受精受到影響,難以坐果,即便坐果,亦容易形成畸形果實(shí)。授粉前后雌蕊和雄蕊中IAA含量普遍偏低,授粉后雌蕊中IAA含量未見(jiàn)顯著升高。9. SlARF14-RNAi植株花柄在第8 h,脫落信號(hào)尚未完全啟動(dòng)時(shí)已有脫落發(fā)生,說(shuō)明前期生長(zhǎng)素對(duì)其的抑制作用有所減弱；而在后期其脫落速率與對(duì)照持平,在第36h全部完成了脫落；在乙烯誘導(dǎo)的脫落過(guò)程中,SlARF14-RNAi植株花柄在脫落啟動(dòng)后速率稍慢于對(duì)照,表現(xiàn)對(duì)乙烯欠敏感。與前文探討的ARF14基因在脫落不同時(shí)期表達(dá)模式所反映的結(jié)果基本一致。10.對(duì)S1ARF2的蛋白結(jié)構(gòu)及組織特異性表達(dá)進(jìn)行的分析表明,S1ARF2具有ARF蛋白完整結(jié)構(gòu),SIARF2基因在大部分組織器官中均有表達(dá),尤其在根系,花器官以及幼苗中表達(dá)較高,遺傳進(jìn)化分析表明S1ARF2與已被證明與葉片衰老及花器官脫落有關(guān)的擬南芥AtARF2高度同源,推測(cè)其可能存在著功能上的相近。對(duì)12個(gè)Aux/IAAs基因進(jìn)行表達(dá)檢測(cè)結(jié)果顯示,大部分Aux/IAAs基因在SlARF2-RNAi與對(duì)照植株中的表達(dá)無(wú)顯著差異,S1ARF2的功能調(diào)控可能是通過(guò)與IAA1,IAA3,IAA7以及IAA26等發(fā)生特異性相互作用來(lái)進(jìn)行的。11.通過(guò)對(duì)PrOARF2:GUS的幼苗染色檢測(cè)SIARF2在不同發(fā)育時(shí)期植株體內(nèi)不同部位的分布情況發(fā)現(xiàn),SIARF2基因主要集中新生葉基部、生長(zhǎng)點(diǎn)、葉腋、一級(jí)主莖部位(尤其是表皮毛)、側(cè)根基部、須根發(fā)生部位以及完全開(kāi)放的花的雄蕊中強(qiáng)烈表達(dá)。證明S1ARF2集中在幼嫩組織或生長(zhǎng)發(fā)育活躍部位表達(dá),影響雄蕊成熟。12. SIARF2-RNAi植株葉片葉色鮮綠,側(cè)生葉間小葉數(shù)量減少。在衰老過(guò)程中,與對(duì)照植株葉片在老化時(shí)大規(guī)�？焖傥S死亡相比,SlARF2-RNAi植株葉片延緩了老化進(jìn)程,作為衰老上調(diào)基因,SENU1和SENU5基因的延遲表達(dá)證明了這一點(diǎn)；在光合水平上,同一時(shí)期其葉片可維持相對(duì)較高的存活率,個(gè)體葉片光合速率,最大光化學(xué)效率,葉綠素殘留量,Rubisco酶活性下降均慢于對(duì)照,同時(shí)RBCL,RBCS基因的下調(diào)相對(duì)滯后；抗氧化酶活性下降速率大大慢于對(duì)照；在激素水平上,其葉片衰老獨(dú)立于ZR途徑,涉及IAA的調(diào)控。13.SIARF2-RNAi植株花器官的花梗及萼片部位的表皮毛明顯減少,花器官(包括萼片,花瓣,雄蕊,雌蕊)長(zhǎng)度均小于對(duì)照。授粉前后雌蕊和雄蕊中IAA含量普遍偏低,其雄蕊對(duì)于IAA的敏感性有所降低。14. SIARF2-RNAi植株花器官在自然脫落的第28 h以前均小于對(duì)照,而關(guān)鍵時(shí)期在16～20h之間,這期間脫落率上升非常緩慢,脫落有所延遲,而在20 h以后恢復(fù)正常；在乙烯誘導(dǎo)的脫落過(guò)程中,SlARF2-RNAi植株花柄脫落率僅在8h時(shí)處于極低水平,這時(shí)雖在乙烯誘導(dǎo)下脫落亦難以發(fā)生,其他時(shí)間點(diǎn)與對(duì)照近乎一致。
[Abstract]:As an important plant hormone, auxin is not only closely related to its biosynthesis, but also regulated by transcription factors in its signal transduction. ARF is a kind of key auxin-responsive factors. In recent years, the study of its molecular mechanism has gradually become a hot spot in plant tissues and organs and a variety of physiological activities. However, auxin regulation of plant growth and development is a very complex process, and there is a rigorous regulatory mechanism. Research on the role of ARF family in the regulation of auxin in tomato mainly focuses on fruit development and maturation, while in the aspect of flower organ abscission, ARF is involved in the regulation mechanism of auxin. Therefore, it is of great theoretical and practical value to study the functional mechanism of ARF in auxin regulation of petiole abscission in tomato. On this basis, the expression profiles of ARFs family in the process of petiole abscission in tomato were mainly discussed. Then two genes related to abscission were selected and transgenic plants were obtained. The main results are as follows: 1. Investigation on the effect of ethylene and IAA treatment on the explant abscission rate of tomato flower stalk, combined with the abscission defense gene Cystein-type endopeptida. Se expression signal analysis showed that 8 h after flowering was the key node of explant abscission, before which was the period of auxin inhibition abscission, and after that was the period of ethylene induction and promotion abscission. The content of auxin in the detached area and the two ends of the petiole was decreased, but the content of auxin in the detached area was always the highest, and that in the vascular tissue was also higher. The content of cortex and pith decreased rapidly with the detached process. In addition, the changes of IAA content and GUS activity in the process of abscission also confirmed that 8 h was the key node for the abscission of petiole explants. 3. The early abscission stage (0-8 h) and the late abscission stage (8-3) were discussed respectively. 2 h) The expression of ARFs in the two stages. It was found that at the early stage of auxin inhibiting abscission, ARF1, 2, 3, 4, 5, 7, 8-1, 9, 11, 12, 13, 13-1, 14 and 17 were regulated by regulating the expression of ARF13-1, 9, 11, 12, 13-1 and 14, while at the later stage of ethylene-induced abscission, auxin mediated the expression of ARF8-1, 9, 11, 12, 13-1 and 14. The results showed that ARF5, 6, 8, 10, 11, 16 and 19 were up-regulated by abscission induction, indicating that these genes played an important role in the process of abscission. The results showed that ARF6-1, ARF8-1, ARF13 and ARF19-1 were the four most expressed genes in the normal petiole exfoliation region. It was speculated that ARF6-1, ARF13 and ARF19-1 might play a more important role in the exfoliation development and isolation. 4. The primers encompassing the conserved DNA binding domain (that is, the DBD functional region) of the N-terminal of ARF protein were designed and amplified by PCR with specific primers. 5 BP (SIARF2:512-947 bp) and 422 BP (SlARF14:562-984 bp) fragments were constructed by Gateway cloning technique. The RNAi vectors of SlARF2 and SlARF14 were sequenced and digested to verify their correctness, which provided materials for exploring the function of the target gene by blocking the specific binding of genes. 5. The improved "Zhongshu VI" was used. "Tomato high-efficiency regeneration system, using Agrobacterium tumefaciens-mediated genetic transformation method to introduce the target vector into plants, the whole process has always maintained 0.5 mg/L PPT resistance screening pressure, and the agar content in rooting medium from 0.8% to 0.7%, is conducive to the growth of roots under the premise of bacteriostasis, improve the survival rate of transplantation. SlARF2-RNAi PPTR and SlARF14-RNAi PPTR resistant tomato plants were amplified by PCR. It was further proved that foreign genes had been successfully transfected into tomato genome, and some positive transgenic plants were obtained. The transformation rate was higher than 30%. 6. The analysis of SlARF14 protein structure and tissue-specific expression showed that the deletion of SlARF14 protein could be associated with Aux/I. SlARF14 gene was expressed in most tissues and organs, especially in pistils, buds, leaves and fruits during enlargement. Genetic evolution analysis showed that SlARF14 was highly homologous to SlARF10, which had been proved to be related to leaf growth and development. It was speculated that there might be functional similarities between SlARF14 gene and SlARF10 in 12 tomatoes. The results of Aux/IAAs gene expression test showed that most of the Aux/IAAs gene expression in SlARF14-RNAi was not significantly different from that in the control plants, suggesting that only a few Aux/IAAs had some form of regulation on SLARF14 with structural deletion (III IV). Compared with other ARFs family members, S1ARF14 may have a relatively independent role. The edge of leaves of RNAi plants was more smooth and curly, the absence of fission was lighter or less, the color of leaves was dark green, the leaves were thickened and the lateral leaves and interlobular fusion appeared to a certain extent. With the growth of leaves tended to expand laterally, the genes of LeT6 and LeEXPA10 related to leaf development were regulated to some extent at photosynthetic level. On the other hand, it increased the photosynthetic rate, enhanced the accumulation of chlorophyll and stimulated the release of Rubisco enzyme activity, and up-regulated the expression of RBCL, RBCS and CAB. On the regulation of hormone level, the leaf development involved the participation of ZR and the early regulation of IAA. It was found that the pistil was undeveloped, the stigma was curved, the ovary was abnormal, normal pollination and fertilization were affected, it was difficult to set fruit, even fruit setting, it was easy to form deformed fruit. The petiole shedding occurred at the 8th hour before the shedding signal was fully activated, indicating that the inhibitory effect of auxin on the petiole shedding was weakened, and the shedding rate was the same as that of the control at the later stage, and all the petioles shedding was completed at the 36th hour. In the process of ethylene-induced shedding, the petiole shedding rate of SlARF14-RNAi plants was slightly slower than that of the control. The results showed that S1ARF2 had an intact structure of ARF protein and SIARF2 gene was expressed in most tissues and organs, especially in roots and floral organs. Genetic evolution analysis showed that S1ARF2 was highly homologous to Arabidopsis AtARF2, which had been proved to be associated with leaf senescence and floral organ abscission. There was no significant difference in the expression of SIARF2. The function of S1ARF2 may be regulated by specific interaction with IAA1, IAA3, IAA7 and IAA26. 11. The distribution of SIARF2 in different parts of plants at different developmental stages was detected by PrOARF2: GUS seedling staining. Strong expression of S1ARF2 in axillary, primary stem (especially epidermal hairs), lateral root base, fibrous roots and stamens of fully open flowers showed that S1ARF2 was concentrated in young tissues or active parts of growth and development, affecting stamen maturation. Compared with the control plants, the leaves of SlARF2-RNAi plants delayed the aging process, which was proved by the delayed expression of SENU1 and SENU5 genes as the up-regulated genes of senescence. The decreases of photochemical efficiency, chlorophyll residue and Rubisco enzyme activity were slower than those of the control, while the decreases of RBCL and RBCS genes were relatively lagged; the decreases of antioxidant enzyme activity were much slower than those of the control; at the hormone level, leaf senescence was independent of ZR pathway and involved in IAA regulation. 13. SIARF2-RNAi plant pedicels and sepals of floral organs. The length of flower organs (including sepals, petals, stamens and pistils) was less than that of the control. The content of IAA in pistils and stamens was generally low before and after pollination, and the sensitivity of stamens to IAA was decreased. 14. The flower organs of SIARF2-RNAi plants were less than that of the control before 28 h of natural abscission, and the critical period was between 16 and 20 h. In the process of ethylene-induced abscission, the rate of petiole abscission of SlARF2-RNAi plants was only at a very low level at 8 h, which was difficult to occur even under ethylene-induced abscission, and the other time points were almost the same as the control.
【學(xué)位授予單位】：沈陽(yáng)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：S641.2

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