棉花（Gossypium hirsutum）JAZ11-D和XND1基因的生物学功能及调控研究

发布时间：2018-06-05 20:31

本文选题：棉花(Gossypium + hirsutum)　；参考：《华中师范大学》2016年博士论文

【摘要】：棉花是世界上最重要的经济作物之一,能产生大量的天然纺织纤维。其中,四倍体陆地棉(Gossypium hirsutum)贡献了全世界约90%的棉花纤维产量。茉莉素(JA)信号通路的抑制因子JAZ(Jasmonate ZIM-domain)蛋白在植物的生长发育和抗性应答中发挥重要的调控作用。然而,目前在四倍体陆地棉中很少有关于JAZ调控纤维发育的研究报道。本文分离鉴定了 30个非冗余的棉花JAZ基因,研究了它们的表达和互作模式。在此基础上,对其中一个基因GhJAZ11-D进行了深入研究,以期揭示该基因在棉花纤维伸长发育中的调控作用。此外,本文也研究了棉花XND1对植物木质部发育的负调控作用。获得的主要研究结果如下:1.棉花JAZ家族基因的鉴定、进化关系和表达模式分析利用已公布的四倍体陆地棉的基因组序列公共数据库,我们鉴定了 30个棉花JAZ家族基因,其中15个来自A基因组,15个来自D基因组。进化树分析表明,棉花JAZ与拟南芥JAZ具有较高的同源性,因此,我们根据系统命名法把棉花的30个JAZ分别命名为GhJAZ1a-A/D至GhJAZ12-A/D。GhJAZ家族成员可分为5个进化亚枝(Ⅰ至V),处于同一进化亚枝的成员都含有相对保守的基因结构和结构域分布。表达分析结果表明,大多数棉花JAZ基因都可以受JA诱导,而且不同的JAZ基因在棉花各组织器官和纤维发育阶段的表达存在明显差异。其中,一些JAZ基因在棉花纤维起始期和伸长期都有较高的表达,暗示着这些GhJAZ基因可能参与调控棉花纤维的起始和伸长发育。2.GhJAZ蛋白互作模式分析亚细胞定位实验表明,所有GhJAZ蛋白都定位在细胞核中。为了进一步阐述GhJAZ在植物体内发挥功能的形式,我们选取了 9个GhJAZ蛋白进行酵母双杂交实验。结果表明,GhJAZ1a-A,GhJAZ1b-D、GhJAZ2-D、GhJAZ5-A、GhJAZ11-D 和 GhJAZ12-D 可以与其他大多数GhJAZ蛋白发生相互作用,而GhJAZ3-D和GhJAZ7-A只能与1-2个GhJAZ蛋白发生互作,这说明GhJAZ蛋白在植物体内有选择性地形成同源或异源二聚体,发挥其功能。此外,大多数GhJAZ蛋白都可以与JA信号通路的关键因子GhCO11、GhMYC2、GhMYC3和GhNINJA发生相互作用。这一结果暗示GhJAZ蛋白可以与GhMYC2和GhMYC3互作,同时招募GhNINJA,从而抑制JA信号通路;而GhJAZ蛋白可以被SCFCOII蛋白酶体降解,解除对JA应答基因的抑制,激活JA信号通路。3.GhJA 参与调控棉花纤维起始选取了 10个在纤维起始时期表达量相对较高的GhJAZ基因,研究它们分别在野生型棉花(徐州142)和其无绒无絮突变体(fl)开花当天的胚珠中的表达情况。实验结果表明,有8个JAZ基因都表现出相对一致的差异变化趋势。先前有报道表明,GhMYB2、GhMYB23(与GL1同源)、GhMYB25、GhMYB25-like和GhDEL65(与GL3同源)都可以正调控棉花纤维细胞的起始。为进一步探索GhJAZ对棉花纤维起始的调控,检测了 GhJAZ蛋白与这些纤维起始调控因子之间的互作情况。酵母双杂交和双分子荧光互补实验证实GhJAZ1a-A可以与GhMYB23互作,GhJAZ1b-D可以与GhMYB23和GhMYB25-like互作。以上结果表明,GhJAZ蛋白可以通过与纤维起始调控因子相互作用,参与调控棉花纤维的起始过程。4.GhJAZ11-促进棉花纤维细胞伸长发育为详细研究GhJAZ在棉花纤维发育中的功能,我们选取GhJAZ11-D基因进行深入研究。构建了 GhJAZ11-D过量表达(OE)和基因沉默(RNAi)载体,转化棉花,获得转基因棉花植株。扫描电镜的结果显示GhJAZ11-D不参与调控棉花纤维的起始。表型观察和纤维长度的统计学分析表明,过量表达GhJAZ11-D转基因棉花株系的纤维长度明显长于野生型,但GhJAZ11-D RNAi棉花植株的纤维长度没有明显变化。纤维品质分析表明,过量表达转基因株系的纤维比强度和马克隆值也发生了改变。体外胚珠培养实验也证实GhJAZ11-D促进棉纤维伸长发育。为揭示GhJAZ11-D促进纤维伸长的机制,以GhJAZ11-D作为诱饵蛋白,筛选棉花纤维的酵母双杂交cDNA文库,筛选其互作蛋白。其中,发现GhEIN3是GhJAZ11-D的互作蛋白之一。此外,与野生型相比,GhJAZ11-D过量表达转基因棉花中乙烯应答基因GhERF和GhEXP1的表达量下调。由于GhJAZ11-D可以与GhEIN3互作,由此推测,植物激素JA和ET可以协同调控棉花纤维伸长发育。5.GhXND1负调控木质部发育在棉花中分离鉴定了一个编码NAC转录因子的GhXND1基因,序列分析表明GhD1含有2个内含子。GhXND1蛋白定位在细胞核中,具有转录自激活活性。GhXND1在棉花的子叶、花瓣、根、下胚轴和茎中优势表达,而在其他组织和纤维发育阶段的表达量较低。在拟南芥中异源表达GhXND1导致转基因植株木质部维管细胞的数量相对野生型减少,维管束间纤维的细胞层数也相对减少,一些次生壁物质的相关合成基因的表达量在异源表达GhXND1的转基因拟南芥中也明显下调。以上结果表明,GhXND1可能负调控植物木质部发育。
[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.
【学位授予单位】：华中师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S562

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