【摘要】：生長(zhǎng)素響應(yīng)因子(Auxin response factor,ARF)作為一類(lèi)轉(zhuǎn)錄因子,通過(guò)調(diào)控下游基因表達(dá)參與植物諸多生理生化進(jìn)程。而ARF的轉(zhuǎn)錄水平卻受miRNA的調(diào)控�；谇叭撕驼n題組的研究基礎(chǔ),初步明確了番茄miRNA160可調(diào)控靶基因SIARF10、SIARF16和SIARF17,miRNA167可調(diào)控slARF6和slARF8。本研究為進(jìn)一步明確miRNA160和miRNA167調(diào)控靶基因AREs參與番茄生長(zhǎng)發(fā)育的功能,利用35S::mSIARF10-6(抗miRNA160降解的形式)轉(zhuǎn)基因材料探討了miRNA160調(diào)控SIARF10對(duì)番茄葉片失水速率的影響,并明確了ARF10通過(guò)直接影響氣孔發(fā)育和水通道蛋白表達(dá)而增加番茄葉片的水分導(dǎo)度；而利用dgt,gib-3突變體研究了ARFs/miRNAs在番茄果實(shí)發(fā)育早期果皮發(fā)育的作用,初步探討了受miRNA160和miRNA167調(diào)控的ARFs可能協(xié)同生長(zhǎng)素和赤霉素調(diào)控果皮的細(xì)胞分裂和膨大：根據(jù)構(gòu)建并獲得的超表達(dá)miRNA160的轉(zhuǎn)基因植株及T1代,初步發(fā)現(xiàn)miRNA160調(diào)控的ARFs對(duì)番茄葉片有重要的調(diào)控作用。這些研究將為進(jìn)一步明確ARF/miRNA的生物學(xué)調(diào)控機(jī)制和生長(zhǎng)素調(diào)控植物生長(zhǎng)發(fā)育的作用機(jī)理奠定基礎(chǔ)。主要研究結(jié)果如下：1.采用mSIARF10過(guò)表達(dá)轉(zhuǎn)基因番茄(抗miR160降解)葉片進(jìn)行表型分析發(fā)現(xiàn),其葉片顯著變窄(長(zhǎng)/寬),氣孔變大,氣孔密度變小。通常窄葉片具有較小的失水速率,然而與野生型相比,35S:mSIARF10-6的離體葉片表現(xiàn)出更大的葉片失水速率。在水分脅迫處理過(guò)程中,35S:mSIARF10-6積累了更高的ABA含量,而對(duì)于外源ABA影響其氣孔的敏感度顯著高于野生型。但進(jìn)一步分析表明35S:mSIARF10-6實(shí)際的葉片失水率與依靠氣孔水蒸氣散失的計(jì)算失水率不同,這表明了還有其他方式影響35S:mSIARF10-6葉片失水。進(jìn)一步利用水通道蛋白(aquaporins,AQPs)抑制劑HgCl2處理證實(shí)35S:mSIARF10-6有較高的AQP活性,具有更高的水力導(dǎo)度。以上結(jié)果表明35S:mSIARF10-6失水率的提高是氣孔和水通道蛋白活性共同作用的結(jié)果。2.根據(jù)RNA-sequencing表明在35S:mSIARF10-6中有5個(gè)AQP家族基因,14個(gè)ABA合成與信號(hào)轉(zhuǎn)導(dǎo)相關(guān)基因以及3個(gè)氣孔發(fā)育相關(guān)基因表達(dá)水平發(fā)生顯著變化,其中SIABI5具有ABA依賴(lài)的轉(zhuǎn)錄因子活性。對(duì)上調(diào)表達(dá)的AQP基因的啟動(dòng)子分析表明,上調(diào)的AQP基因啟動(dòng)子均包含ABRE或AuxRE啟動(dòng)子元件。挑選上調(diào)表達(dá)最顯著的三個(gè)基因(SITIP1-1,SIPIP2;4和SINIP-type like)進(jìn)行啟動(dòng)子活性分析試驗(yàn)發(fā)現(xiàn),包含AuxRE元件的SIPIP2;4和SINIP-like的啟動(dòng)子區(qū)域以及包含ABRE元件的SIPIP2;4和SITIP1;1啟動(dòng)子區(qū)域在35S:mSIARF10-6轉(zhuǎn)基因材料中GUS酶活性顯著增強(qiáng)。EMSA和酵母單雜交試驗(yàn)證實(shí),SIARF10可以與AQP基因啟動(dòng)子中的AuxRE啟動(dòng)子元件結(jié)合。而將35S:mSIARF10-6中表達(dá)量顯著提高的轉(zhuǎn)錄因子SIAB15進(jìn)行酵母單雜交,證實(shí)可與AQP基因啟動(dòng)子中的ABRE啟動(dòng)子元件結(jié)合。因此35S:mSIARF10-6的AQP表達(dá)增強(qiáng)可能是ARF10和ABI5上調(diào)表達(dá)的結(jié)果。然而,通過(guò)SIARF10沉默與SIABI5超表達(dá)的瞬時(shí)表達(dá)發(fā)現(xiàn),下調(diào)SIARF10可以降低番茄葉片失水率,超表達(dá)SIABI5在水分脅迫處理前6h顯著下調(diào)失水率,因此35S:mSIARF10-6的高失水率不是由于SIABI5的作用,而是SIARF10直接作用的結(jié)果。本研究表明,盡管SIARF10通過(guò)增加ABA合成與信號(hào)響應(yīng)通過(guò)調(diào)控氣孔開(kāi)度來(lái)降低水分缺失,但SIARF10通過(guò)影響氣孔發(fā)育和水通道蛋白活性來(lái)加速水分散失。因此miR160調(diào)控的SIARF10對(duì)于維持葉片的水分平衡有著重要意義。3.利用外源IAA噴施番茄GA缺失突變體(gib-3)以及外源GA噴施番茄IAA信號(hào)途徑受阻突變體(dgt)果實(shí)材料。解剖結(jié)果表明IAA導(dǎo)致果皮增厚,果皮細(xì)胞層數(shù)增加,而GA并沒(méi)有增加果皮細(xì)胞層數(shù)；RT-PCR研究發(fā)現(xiàn),IAA處理gib-3突變體后SIARF6,SIARF8,SIARF10和SIARF16表達(dá)水平顯著下調(diào),而miR160和miRl67顯著上調(diào)；而dgt突變體中SIARF6,SIARF8,SIARF10和SIARF16表達(dá)水平受到GA影響顯著上調(diào),而miRNAs表達(dá)水平顯著下調(diào)。這些結(jié)果表明了在番茄果實(shí)發(fā)育初期,SIARF6, SIARF8,SIARF10和SIARF16的表達(dá)與果皮細(xì)胞層數(shù)呈負(fù)相關(guān)。進(jìn)一步利用35S:mSIARF10-6轉(zhuǎn)基因番茄植株進(jìn)行果實(shí)果皮分析,結(jié)果表明mSIARF10過(guò)表達(dá)使坐果期果皮細(xì)胞層數(shù)顯著降低。以上結(jié)果表明GA和IAA介導(dǎo)的miRNAs及其靶基因ARFs可能參與番茄果實(shí)發(fā)育早期的果皮細(xì)胞增殖過(guò)程。4.利用Gateway技術(shù)成功構(gòu)建了miRNA160超表達(dá)載體。將番茄miRNA160前體連入具有強(qiáng)啟動(dòng)子的pB7WG2D,1載體。通過(guò)優(yōu)化的番茄遺傳轉(zhuǎn)化體系成功獲得番茄轉(zhuǎn)基因植株。并通過(guò)熒光檢測(cè),分子及蛋白水平等鑒定方法成功驗(yàn)證了T1代轉(zhuǎn)基因植株番茄。對(duì)轉(zhuǎn)基因番茄植株進(jìn)行表型分析,發(fā)現(xiàn)35S::SImiR160轉(zhuǎn)基因植株的表型：葉片寬度增大,葉裂減少,葉面積增大等。暗示了miR160對(duì)番茄葉片發(fā)育的重要調(diào)控作用。為一進(jìn)步探究miRNA160對(duì)番茄的生長(zhǎng)發(fā)育機(jī)制提供了優(yōu)良試材。
[Abstract]:Auxin response factor (ARF), as a class of transcription factors, participates in many physiological and biochemical processes in plants by regulating downstream gene expression, and the transcriptional level of ARF is regulated by miRNA. Based on the research basis of previous and subject groups, the target gene SIARF10 of tomato miRNA160, SIARF16, SIARF17, miRN, is preliminarily clarified. A167 can regulate slARF6 and slARF8. in this study to further clarify the function of miRNA160 and miRNA167 regulating target gene AREs to participate in tomato growth and development. Using 35S:: mSIARF10-6 (the form of anti miRNA160 degradation), the effect of SIARF10 on the rate of water loss in tomato leaves was investigated, and the direct influence of ARF10 on the gas was determined. The water conductivity of tomato leaves was increased by pore development and aquaporin expression, and the effect of ARFs/miRNAs on the development of fruit peel in the early stage of tomato fruit development was studied by using DGT and gib-3 mutants. The cell division and expansion of ARFs regulated by miRNA160 and miRNA167 was preliminarily discussed in the regulation of auxin and gibberellin. The transgenic plants and T1 generation of the overexpression of miRNA160 have been obtained. It is found that the ARFs regulated by miRNA160 plays an important role in regulating the leaves of tomato. These studies will lay a foundation for further clarifying the biological regulation mechanism of ARF/miRNA and the mechanism of the growth and development of plants by auxin. The main results are as follows: 1. the use of mS The phenotypic analysis of IARF10 overexpressed transgenic tomato (anti miR160 degradation) leaves showed that the leaves were significantly narrower (long / wide), the stomata became larger, and the stomatal density became smaller. Usually narrow leaves had smaller water loss rates. However, compared with the wild type, the leaves of the 35S:mSIARF10-6 leaves showed greater leaf loss rate. During the process, 35S:mSIARF10-6 accumulated a higher ABA content, but the sensitivity of the exogenous ABA to the stomata was significantly higher than that in the wild type. However, the further analysis showed that the actual loss rate of the 35S:mSIARF10-6 leaves was different from that of the calculated water vapor loss depending on the pore water vapor loss. This indicates that there are other ways to influence the water loss of the 35S:mSIARF10-6 leaves. One step using aquaporins (AQPs) inhibitor HgCl2 treatment confirmed that 35S:mSIARF10-6 had higher AQP activity and had higher hydraulic conductivity. The above results showed that the increase of 35S:mSIARF10-6 water loss rate was the result of the co action of stomata and aquaporin activity,.2. based on RNA-sequencing showed that there were 5 AQP in 35S:mSIARF10-6. Family genes, 14 ABA synthesis and signal transduction related genes and 3 stomatal development related genes have significant changes, in which SIABI5 has ABA dependent transcription factor activity. The up-regulated AQP promoter analysis showed that the up regulation of AQP gene promoter contained ABRE or AuxRE promoter components. The promoter activity analysis test of the most significant three genes (SITIP1-1, SIPIP2; 4 and SINIP-type like) found that SIPIP2 containing AuxRE elements, the promoter region of 4 and SINIP-like, and SIPIP2, 4 and SITIP1 containing ABRE elements; 1 promoter region significantly enhanced the activity of the GUS enzyme activity in the 35S:mSIARF10-6 transgenic material and the yeast single The hybridization test confirmed that SIARF10 could be combined with the AuxRE promoter element in the AQP gene promoter, and the yeast single hybridization was carried out with the transcriptional factor SIAB15, which was significantly increased in the 35S:mSIARF10-6 expression, and proved to be associated with the ABRE promoter element in the AQP gene promoter. Therefore, the enhancement of 35S: mSIARF10-6 AQP expression may be on ARF10 and ABI5. However, through the transient expression of SIARF10 silencing and SIABI5 overexpression, it was found that down-regulation of SIARF10 could reduce the water loss rate of tomato leaves, and the overexpression of SIABI5 was significantly down regulated by 6h before water stress treatment. Therefore, the high water loss rate of 35S:mSIARF10-6 was not due to the effect of SIABI5, but the result of SIARF10's direct effect. It is shown that, although SIARF10 reduces water loss by regulating stomatal opening by increasing ABA synthesis and signal response, SIARF10 accelerates water loss by affecting the stomatal development and aquaporin activity. Therefore, miR160 regulated SIARF10 is of great significance for maintaining the water balance of leaves.3. using exogenous IAA to spray tomato GA deficiency. The loss of mutant (gib-3) and exogenous GA spraying tomato IAA signal pathway blocked mutant (DGT) fruit material. The anatomical results showed that IAA resulted in the thickening of the peel and the increase of the number of cells in the pericarp, while GA did not increase the number of cell layers. The RT-PCR study found that IAA treated gib-3 mutants as SIARF6, SIARF8, SIARF10, and expression levels decreased significantly. The expression level of SIARF6, SIARF8, SIARF10 and SIARF16 in DGT mutants was significantly up-regulated by GA, and the expression level of miRNAs decreased significantly in DGT mutants. These results showed that the expression of SIARF6, SIARF8, SIARF10, and SIARF6 was negatively correlated with the number of peel cell layers in the early stage of tomato fruit development. The fruit peel analysis of mSIARF10-6 transgenic tomato plants showed that over expression of mSIARF10 resulted in a significant decrease in the number of cell layers in fruit peels. The above results showed that miRNAs and the target gene ARFs mediated by GA and IAA may participate in the proliferation of fruit cells in the early stage of tomato fruit development.4. using Gateway technology to successfully construct miRNA160 super. The expression vector. The tomato miRNA160 precursor was joined with the strong promoter pB7WG2D, 1 carrier. The tomato transgenic plants were successfully obtained through the optimized tomato genetic transformation system. The transgenic tomato plants of the T1 generation were successfully verified by the fluorescence detection, molecular and protein level and other identification methods. The phenotype of 35S:: SImiR160 transgenic plants: the widen leaf width, the decrease of leaf cleft and the increase of leaf area. It suggests that miR160 plays an important role in regulating the development of tomato leaves. It provides a good test for the research of the mechanism of miRNA160 for the growth and development of tomato.
【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】：2016
【分類(lèi)號(hào)】：S641.2

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4 保定定州市農(nóng)牧局 王虎;棚室番茄卷葉為哪般[N];河北農(nóng)民報(bào);2009年

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8 河北省農(nóng)科院植保所 孫茜　徐文亭;雨后暴晴警惕番茄遭高溫燙傷[N];河北科技報(bào);2013年

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