本文選題：棉花(Gossypium + hirsutum)�。� 參考：《華中師范大學(xué)》2016年博士論文

【摘要】：棉花是世界上最重要的經(jīng)濟(jì)作物之一,能產(chǎn)生大量的天然紡織纖維。其中,四倍體陸地棉(Gossypium hirsutum)貢獻(xiàn)了全世界約90%的棉花纖維產(chǎn)量。茉莉素(JA)信號(hào)通路的抑制因子JAZ(Jasmonate ZIM-domain)蛋白在植物的生長(zhǎng)發(fā)育和抗性應(yīng)答中發(fā)揮重要的調(diào)控作用。然而,目前在四倍體陸地棉中很少有關(guān)于JAZ調(diào)控纖維發(fā)育的研究報(bào)道。本文分離鑒定了 30個(gè)非冗余的棉花JAZ基因,研究了它們的表達(dá)和互作模式。在此基礎(chǔ)上,對(duì)其中一個(gè)基因GhJAZ11-D進(jìn)行了深入研究,以期揭示該基因在棉花纖維伸長(zhǎng)發(fā)育中的調(diào)控作用。此外,本文也研究了棉花XND1對(duì)植物木質(zhì)部發(fā)育的負(fù)調(diào)控作用。獲得的主要研究結(jié)果如下:1.棉花JAZ家族基因的鑒定、進(jìn)化關(guān)系和表達(dá)模式分析利用已公布的四倍體陸地棉的基因組序列公共數(shù)據(jù)庫(kù),我們鑒定了 30個(gè)棉花JAZ家族基因,其中15個(gè)來(lái)自A基因組,15個(gè)來(lái)自D基因組。進(jìn)化樹(shù)分析表明,棉花JAZ與擬南芥JAZ具有較高的同源性,因此,我們根據(jù)系統(tǒng)命名法把棉花的30個(gè)JAZ分別命名為GhJAZ1a-A/D至GhJAZ12-A/D。GhJAZ家族成員可分為5個(gè)進(jìn)化亞枝(Ⅰ至V),處于同一進(jìn)化亞枝的成員都含有相對(duì)保守的基因結(jié)構(gòu)和結(jié)構(gòu)域分布。表達(dá)分析結(jié)果表明,大多數(shù)棉花JAZ基因都可以受JA誘導(dǎo),而且不同的JAZ基因在棉花各組織器官和纖維發(fā)育階段的表達(dá)存在明顯差異。其中,一些JAZ基因在棉花纖維起始期和伸長(zhǎng)期都有較高的表達(dá),暗示著這些GhJAZ基因可能參與調(diào)控棉花纖維的起始和伸長(zhǎng)發(fā)育。2.GhJAZ蛋白互作模式分析亞細(xì)胞定位實(shí)驗(yàn)表明,所有GhJAZ蛋白都定位在細(xì)胞核中。為了進(jìn)一步闡述GhJAZ在植物體內(nèi)發(fā)揮功能的形式,我們選取了 9個(gè)GhJAZ蛋白進(jìn)行酵母雙雜交實(shí)驗(yàn)。結(jié)果表明,GhJAZ1a-A,GhJAZ1b-D、GhJAZ2-D、GhJAZ5-A、GhJAZ11-D 和 GhJAZ12-D 可以與其他大多數(shù)GhJAZ蛋白發(fā)生相互作用,而GhJAZ3-D和GhJAZ7-A只能與1-2個(gè)GhJAZ蛋白發(fā)生互作,這說(shuō)明GhJAZ蛋白在植物體內(nèi)有選擇性地形成同源或異源二聚體,發(fā)揮其功能。此外,大多數(shù)GhJAZ蛋白都可以與JA信號(hào)通路的關(guān)鍵因子GhCO11、GhMYC2、GhMYC3和GhNINJA發(fā)生相互作用。這一結(jié)果暗示GhJAZ蛋白可以與GhMYC2和GhMYC3互作,同時(shí)招募GhNINJA,從而抑制JA信號(hào)通路;而GhJAZ蛋白可以被SCFCOII蛋白酶體降解,解除對(duì)JA應(yīng)答基因的抑制,激活JA信號(hào)通路。3.GhJA 參與調(diào)控棉花纖維起始選取了 10個(gè)在纖維起始時(shí)期表達(dá)量相對(duì)較高的GhJAZ基因,研究它們分別在野生型棉花(徐州142)和其無(wú)絨無(wú)絮突變體(fl)開(kāi)花當(dāng)天的胚珠中的表達(dá)情況。實(shí)驗(yàn)結(jié)果表明,有8個(gè)JAZ基因都表現(xiàn)出相對(duì)一致的差異變化趨勢(shì)。先前有報(bào)道表明,GhMYB2、GhMYB23(與GL1同源)、GhMYB25、GhMYB25-like和GhDEL65(與GL3同源)都可以正調(diào)控棉花纖維細(xì)胞的起始。為進(jìn)一步探索GhJAZ對(duì)棉花纖維起始的調(diào)控,檢測(cè)了 GhJAZ蛋白與這些纖維起始調(diào)控因子之間的互作情況。酵母雙雜交和雙分子熒光互補(bǔ)實(shí)驗(yàn)證實(shí)GhJAZ1a-A可以與GhMYB23互作,GhJAZ1b-D可以與GhMYB23和GhMYB25-like互作。以上結(jié)果表明,GhJAZ蛋白可以通過(guò)與纖維起始調(diào)控因子相互作用,參與調(diào)控棉花纖維的起始過(guò)程。4.GhJAZ11-促進(jìn)棉花纖維細(xì)胞伸長(zhǎng)發(fā)育為詳細(xì)研究GhJAZ在棉花纖維發(fā)育中的功能,我們選取GhJAZ11-D基因進(jìn)行深入研究。構(gòu)建了 GhJAZ11-D過(guò)量表達(dá)(OE)和基因沉默(RNAi)載體,轉(zhuǎn)化棉花,獲得轉(zhuǎn)基因棉花植株。掃描電鏡的結(jié)果顯示GhJAZ11-D不參與調(diào)控棉花纖維的起始。表型觀察和纖維長(zhǎng)度的統(tǒng)計(jì)學(xué)分析表明,過(guò)量表達(dá)GhJAZ11-D轉(zhuǎn)基因棉花株系的纖維長(zhǎng)度明顯長(zhǎng)于野生型,但GhJAZ11-D RNAi棉花植株的纖維長(zhǎng)度沒(méi)有明顯變化。纖維品質(zhì)分析表明,過(guò)量表達(dá)轉(zhuǎn)基因株系的纖維比強(qiáng)度和馬克隆值也發(fā)生了改變。體外胚珠培養(yǎng)實(shí)驗(yàn)也證實(shí)GhJAZ11-D促進(jìn)棉纖維伸長(zhǎng)發(fā)育。為揭示GhJAZ11-D促進(jìn)纖維伸長(zhǎng)的機(jī)制,以GhJAZ11-D作為誘餌蛋白,篩選棉花纖維的酵母雙雜交cDNA文庫(kù),篩選其互作蛋白。其中,發(fā)現(xiàn)GhEIN3是GhJAZ11-D的互作蛋白之一。此外,與野生型相比,GhJAZ11-D過(guò)量表達(dá)轉(zhuǎn)基因棉花中乙烯應(yīng)答基因GhERF和GhEXP1的表達(dá)量下調(diào)。由于GhJAZ11-D可以與GhEIN3互作,由此推測(cè),植物激素JA和ET可以協(xié)同調(diào)控棉花纖維伸長(zhǎng)發(fā)育。5.GhXND1負(fù)調(diào)控木質(zhì)部發(fā)育在棉花中分離鑒定了一個(gè)編碼NAC轉(zhuǎn)錄因子的GhXND1基因,序列分析表明GhD1含有2個(gè)內(nèi)含子。GhXND1蛋白定位在細(xì)胞核中,具有轉(zhuǎn)錄自激活活性。GhXND1在棉花的子葉、花瓣、根、下胚軸和莖中優(yōu)勢(shì)表達(dá),而在其他組織和纖維發(fā)育階段的表達(dá)量較低。在擬南芥中異源表達(dá)GhXND1導(dǎo)致轉(zhuǎn)基因植株木質(zhì)部維管細(xì)胞的數(shù)量相對(duì)野生型減少,維管束間纖維的細(xì)胞層數(shù)也相對(duì)減少,一些次生壁物質(zhì)的相關(guān)合成基因的表達(dá)量在異源表達(dá)GhXND1的轉(zhuǎn)基因擬南芥中也明顯下調(diào)。以上結(jié)果表明,GhXND1可能負(fù)調(diào)控植物木質(zhì)部發(fā)育。
[Abstract]:Cotton is one of the most important economic crops in the world and produces a large number of natural textile fibers. Among them, tetraploid terrestrial cotton (Gossypium hirsutum) contributes about 90% of the world's cotton fiber production. The inhibitory factor JAZ (Jasmonate ZIM-domain) of jasmonin (JA) signaling pathway plays a heavy role in the growth and response of plants. However, there are few reports on the development of JAZ control fiber in tetraploid upland cotton. In this paper, 30 non redundant JAZ genes were isolated and identified, and their expression and interaction patterns were studied. On this basis, a gene GhJAZ11-D was deeply studied in order to reveal the gene in cotton. In addition, the negative regulation effect of cotton XND1 on the development of plant xylem was also studied. The main results obtained are as follows: 1. identification of JAZ family gene, evolution relationship and expression pattern analysis using the public database of genome sequence of tetraploid terrestrial cotton. 30 cotton JAZ family genes were determined, of which 15 were from the A genome and 15 from the D genome. The phylogenetic tree analysis showed that cotton JAZ and Arabidopsis JAZ had higher homology. Therefore, we named the 30 JAZ of cotton from GhJAZ1a-A/D to GhJAZ12-A/D.GhJAZ family members according to the system naming method to be divided into 5 evolutionary sub branches (I to 5) V), the members of the same evolutionary branch contain relatively conservative gene structure and domain distribution. The results of expression analysis show that most cotton JAZ genes can be induced by JA, and the expression of different JAZ genes in different tissues and organs and fiber development stages of cotton is distinct. Some of the JAZ genes are in cotton fiber. Both initial and elongation stages have high expression, suggesting that these GhJAZ genes may participate in the regulation of the initiation and elongation of cotton fiber and the interaction pattern analysis of.2.GhJAZ protein interaction. The subcellular localization experiment shows that all GhJAZ proteins are located in the nucleus. In order to further elaborate the form of function of GhJAZ in the plant, we select it. 9 GhJAZ proteins were used in yeast two hybrid experiments. The results showed that GhJAZ1a-A, GhJAZ1b-D, GhJAZ2-D, GhJAZ5-A, GhJAZ11-D and GhJAZ12-D can interact with most of the other GhJAZ proteins, and GhJAZ3-D and GhJAZ7-A can only interact with 1-2 GhJAZ proteins, which indicates that the GhJAZ protein is selective in the formation of homology in the plant. In addition, most GhJAZ proteins interact with the key factors of JA signaling pathway GhCO11, GhMYC2, GhMYC3 and GhNINJA. This result suggests that the GhJAZ protein can interact with GhMYC2 and GhMYC3, simultaneously recruiting GhNINJA, and inhibiting the JA signaling pathway; and GhJAZ protein can be protease. Body degradation, relieving the inhibition of the JA response gene and activating the JA signaling pathway.3.GhJA to participate in the regulation of cotton fiber initiation, select 10 GhJAZ genes that have relatively high expression in the initial period of fiber, and study their expression in the ovules of the wild type cotton (Xuzhou 142) and its non cashmere mutants (FL) on the day of flowering, respectively. The results showed that 8 JAZ genes showed a relatively consistent variation trend. Previously, it was reported that GhMYB2, GhMYB23 (and GL1 homologous), GhMYB25, GhMYB25-like and GhDEL65 (with GL3 homologous) could regulate the initiation of cotton fiber cells. The regulation of GhJAZ on the initiation of cotton fibers was further explored, and GhJAZ proteins were detected and this was detected. The interaction between the initial regulatory factors of some fibers. Yeast two and double molecular fluorescence complementation tests confirmed that GhJAZ1a-A can interact with GhMYB23, and GhJAZ1b-D can interact with GhMYB23 and GhMYB25-like. The above results show that GhJAZ protein can interact with the initial regulator of fiber and participate in the regulation of the initiation of cotton fiber. Process.4.GhJAZ11- promoted the elongation of cotton fiber cells to study the function of GhJAZ in the development of cotton fiber. We selected GhJAZ11-D gene for in-depth study. We constructed GhJAZ11-D overexpression (OE) and gene silencing (RNAi) vector, transformed cotton, and obtained transgenic cotton plants. The results of scanning electron microscopy showed that GhJAZ11-D was not involved. The morphological observation and fiber length analysis showed that the fiber length of overexpressed GhJAZ11-D transgenic cotton lines was significantly longer than that in the wild type, but the fiber length of GhJAZ11-D RNAi cotton plants did not change obviously. Fiber quality analysis showed that the fiber strength and horse of the transgenic lines were overexpressed. The clone value also changed. In vitro ovule culture experiment also confirmed that GhJAZ11-D promoted the elongation development of cotton fiber. In order to reveal the mechanism of GhJAZ11-D to promote fiber elongation, the yeast two hybrid cDNA Library of cotton fiber was screened by GhJAZ11-D as bait protein, and the interaction protein of the cotton fiber was screened. It was found that GhEIN3 was one of the interaction proteins of GhJAZ11-D. In addition, compared with the wild type, the expression of ethylene responsive gene GhERF and GhEXP1 in GhJAZ11-D overexpressed transgenic cotton was downregulated. Because GhJAZ11-D can interact with GhEIN3, it is suggested that plant hormone JA and ET can be used to regulate the development of the negative regulatory wood part of cotton fiber elongation and identify a code in cotton. The sequence analysis of the GhXND1 gene of NAC transcriptional factor indicates that GhD1 contains 2 introns.GhXND1 protein located in the nucleus, and the transcriptional active.GhXND1 is dominant in the cotyledon, petals, roots, hypocotyls and stems of cotton, but low in other tissues and fibrous development stages. The heterologous expression of GhXND1 in Arabidopsis leads to GhXND1 The number of vascular cells in the xylem of the transgenic plants decreased relative to the wild type, and the number of cell layers between the vascular bundles also decreased relatively. The expression of some secondary wall material related synthetic genes also decreased obviously in the transgenic Arabidopsis thaliana with the heterologous expression of GhXND1. The results showed that GhXND1 may negatively regulate the development of the xylem in the plant.
【學(xué)位授予單位】：華中師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S562

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本文編號(hào)：1983314