【摘要】：植物在抵抗生物和非生物脅迫時(shí),往往限制其生長。盡管在自然環(huán)境與農(nóng)業(yè)生態(tài)系統(tǒng)中生長與防御的平衡對植物的產(chǎn)量起到重要影響,且茉莉酸(jasmonic acid,JA)信號途徑被認(rèn)為在調(diào)節(jié)這個(gè)平衡過程中起到重要作用,但是對調(diào)節(jié)植物生長與防御的分子機(jī)制的了解非常貧乏。本研究分析了受JA信號途徑影響的棉花兩個(gè)基因的功能。首先,從陸地棉發(fā)育早期的纖維中克隆得到一個(gè)DFR同源基因,編碼355個(gè)氨基酸,命名為GhDFR1。通過病毒介導(dǎo)的基因沉默技術(shù)發(fā)現(xiàn)在棉花中沉默GhDFR1能夠顯著的減少花青素、原花青素的積累�；贚C-MS分析,GhDFR1沉默植株相比對照植株纖維中兩個(gè)原花青素單體,( )-epicatachin表兒茶素和( )-epigallocatachin表沒食子兒茶素的含量顯著的下降,卻出現(xiàn)兩種新的單體( )-catachin兒茶素和( )-gallocatachin沒食子兒茶素。GhDFR1在擬南芥突變體TT3中異源表達(dá)能夠重建花青素生物合成途徑,回復(fù)原花青素在擬南芥種皮中積累。同時(shí),本研究分離獲得一個(gè)棉花NINJA(novel interactor of JA ZIM-domain)基因,其包含一個(gè)1305 bp的開放閱讀框,將其命名為GhNINJA。GhNINJA基因編碼434個(gè)氨基酸。根據(jù)qRT-PCR分析,GhNINJA在根中優(yōu)勢表達(dá),且表達(dá)水平受到大麗輪枝菌侵染誘導(dǎo)。通過病毒介導(dǎo)的基因沉默技術(shù),獲得GhNINJA棉花沉默植株,靶基因表達(dá)水平僅相當(dāng)于對照的6%。表型觀察發(fā)現(xiàn)沉默植株側(cè)根生長受到顯著抑制。通過顯微觀察分析,結(jié)果表明GhNINJA沉默植株根組織分化區(qū)細(xì)胞伸長受到顯著抑制,其細(xì)胞長度顯著短于對照。同時(shí),接種大麗輪枝菌分析,結(jié)果表明沉默植株的抗病性顯著提高,進(jìn)一步抗病標(biāo)記基因分析表明,沉默植株中抗病標(biāo)記基因PDF1.2和PR4均上調(diào)表達(dá),可能造成沉默植株抗病性提高�？傊�,GhDFR1是棉花花青素、原花青素生物合成途徑的重要功能基因,決定花青素和原花青素的積累水平和類型,也是JA信號途徑調(diào)控的下游靶基因。GhNINJA是棉花JA信號途徑中一個(gè)重要的抑制子,通過植物間激素的信號網(wǎng)絡(luò)不僅調(diào)控側(cè)根的生長,而且在植物抗病響應(yīng)中起著重要作用。這兩個(gè)基因的功能分析為棉花生長和抗性之間的調(diào)控關(guān)系研究提供了基礎(chǔ)數(shù)據(jù),也為選育優(yōu)良的抗逆基因以及相關(guān)性狀品種的植物改良和分子育種提科學(xué)依據(jù)。
[Abstract]:Plants tend to limit their growth when they resist biotic and abiotic stresses. Although the balance between growth and defense in the natural environment and agro-ecosystem plays an important role in plant yield, the jasmonic acido JA signaling pathway is thought to play an important role in regulating this balance. But little is known about the molecular mechanisms that regulate plant growth and defense. This study analyzed the function of two genes in cotton affected by JA signaling pathway. Firstly, a homologous gene of DFR was cloned from the early fiber of Upland cotton, encoding 355 amino acids and named GhDFR1. Viral mediated gene silencing technique showed that silencing GhDFR1 in cotton could significantly reduce the accumulation of anthocyanins and procyanidins. The contents of catechin, epicatachin and epigallocatachin in two proanthocyanidins monomers in the fiber of control plants were significantly decreased based on LC-MS analysis. However, two new monomers, catachin catechin and catechin, were expressed in Arabidopsis mutants TT3. The heterologous expression of catechin. GhDFR1 could reconstruct the biosynthesis pathway of anthocyanin and restore the accumulation of proanthocyanidins in the seed coat of Arabidopsis thaliana. At the same time, a cotton NINJA (novel interactor of JA ZIM-domain) gene was isolated, which contained a 1305 BP open reading frame and was named GhNINJA.GhNINJA gene encoding 434 amino acids. According to qRT-PCR analysis, GhNINJA was expressed preferentially in root, and the expression level was induced by Rhizoctonia dahliensis. GhNINJA cotton silencing plants were obtained by virus-mediated gene silencing. The target gene expression level was only 6% of that of the control. Phenotypic observation showed that the lateral root growth of silencing plants was significantly inhibited. The results of microscopic observation and analysis showed that the cell elongation in the root tissue differentiation zone of GhNINJA silencing plants was significantly inhibited and the cell length was significantly shorter than that in the control group. At the same time, the results showed that the resistance of the silencing plants was significantly improved, and the further analysis of the resistance marker genes showed that the expression of PDF1.2 and PR4 were up-regulated in the silencing plants. The disease resistance of silencing plants may be increased. GhDFR1 is an important functional gene of anthocyanin and procyanidin biosynthesis pathway in cotton, which determines the accumulation level and type of anthocyanins and procyanidins. GhNINJA, a downstream target gene regulated by JA signaling pathway, is an important inhibitor of JA signaling pathway in cotton. It not only regulates the growth of lateral roots but also plays an important role in plant disease resistance response through interplant hormone signaling network. The functional analysis of the two genes provides basic data for the study of the regulatory relationship between cotton growth and resistance, and also provides scientific basis for the selection of excellent stress resistance genes and for plant improvement and molecular breeding of related traits.
【學(xué)位授予單位】：吉首大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S562

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本文編號：2154617