水稻蜡质合成代谢基因WSL3及WSL4的克隆和功能分析

发布时间：2018-01-14 14:32

本文关键词：水稻蜡质合成代谢基因WSL3及WSL4的克隆和功能分析　出处：《中国农业科学院》2016年博士论文　论文类型：学位论文

【摘要】：陆地植物表面覆盖着一层蜡质层,蜡质作为植物表面与环境之间的第一层屏障,具有重要的功能。蜡质能防止植物水分的流失,能屏蔽过量的紫外线,能形成自洁式的表面抵御外来的灰尘,能作为物理屏障来抵挡害虫和病原菌的侵害,还能在器官边界确定上起到重要的作用。水稻作为重要的模式作物,对水稻蜡质控制基因进行研究,进而利用其改良水稻的抗逆能力,具有重要的意义。本文以两个水稻叶片蜡质减少突变体Wax crystal-sparse leaf 3(wsl3)和Wax crystal-sparse leaf 4(wsl4)为材料,在表型鉴定的基础上,利用图位克隆技术对目标基因进行了分离,进一步对其功能进行了初步的研究,主要内容如下:1.蜡质突变体wsl3和wsl4的表型分析通过对日本晴组培突变体库的筛选,获得了水稻叶片蜡质减少的突变体wsl3和wsl4。两个突变体均呈现叶片沾水的表型,且wsl4突变体比野生型株高略矮,分蘖数减少。透射电镜(TEM)观察发现wsl3和wsl4突变体叶片表皮增厚。扫描电镜(SEM)观察发现突变体叶片表面蜡质晶体显著性降低。GC-MS测定发现尽管突变体叶片表面角质含量与野生型相当,但是,突变体叶片表面蜡质各成分含量相对于野生型则显著性下降。生理学实验表明,wsl3和wsl4突变体的细胞膜通透性都有一定程度的升高。2.蜡质代谢合成基因WSL3和WSL4的图位克隆和功能验证利用图位克隆技术,我们将WSL3定位于4号染色体InDel标记M-2396和M-2404之间83kb区间内,对该区间的基因测序发现LOC_Os04g40730第一个外显子的736和737的两个连续的碱基GC突变为TT,导致了所编码的蛋白质第207位的氨基酸由丙氨酸突变为苯丙氨酸。WSL4基因定位于3号染色体InDel标记M-6283和M-6341的58 kb区间内,对该区间基因测序发现Loc_Os03g12030基因的1281位的碱基由G突变为A,位于第二个外显子上,导致了所编码的蛋白质第312位的氨基酸由缬氨酸突变为甲硫氨酸。生物信息学分析结果表明LOC_Os04g40730和Loc_Os03g12030分别编码KCR和KCS类蛋白,都是蜡质合成途径的基因。进一步的遗传互补和RNAi干扰实验表明这两个基因即为目标基因。3.WSL3和WSL4基因的时空表达分析及亚细胞定位利用实时荧光定量RT-PCR和GUS表达实验对WSL3和WSL4两个基因的表达模式进行了分析。结果表明,WSL3和WSL4在根、茎、叶、花等各个组织中均有表达,属于组成型表达基因。构建WSL3和WSL4基因的亚细胞定位GFP载体,在烟草中瞬时表达,发现它们都能与ER marker共定位,表明WSL3和WSL4蛋白都定位于内质网上。4.WSL3蛋白具有脂肪酸延长酶活性在酵母ybr159wΔ突变体中异源表达水稻的WSL3基因,其生长缺陷的表型能得到恢复。当WSL3基因与拟南芥的FAE1基因在ybr159wΔ突变体中共表达的时候,能在酵母ybr159wΔ突变体检测到20:0,22:0,20:1Δ11以及22:1Δ13四种脂肪酸产物,这些结果表明WSL3具有KCR酶的脂肪酸延长酶活性。
[Abstract]:The surface of terrestrial plants is covered with a waxy layer which acts as the first barrier between the plant surface and the environment and has important functions. Wax can prevent the loss of plant water and shield from excessive ultraviolet rays. It can form a self-cleaning surface to resist foreign dust, can act as a physical barrier against pests and pathogens, but also plays an important role in the determination of organ boundaries. Rice is an important model crop. In order to improve the stress resistance of rice, the waxy controlling gene of rice was studied. In this paper, two rice leaf waxy reduction mutants, Wax crystal-sparse leaf 3 and wsl3, were used. And Wax crystal-sparse leaf 4wsl4). On the basis of phenotypic identification, the target gene was isolated by map cloning, and its function was studied preliminarily. The main contents are as follows: 1. Phenotypic analysis of waxy mutants wsl3 and wsl4 through screening of Japanese clear tissue culture mutants library. The two mutants, wsl3 and wsl4. both showed the phenotype of water soaking in leaves, and the wsl4 mutants were slightly shorter than wild type plants. The number of tillers decreased and the leaf epidermis of wsl3 and wsl4 mutants were observed by TEM. It was observed that the waxy crystals on the leaf surface of the mutant decreased significantly. GC-MS showed that the keratinine content on the leaf surface of the mutant was similar to that of the wild type. However, the contents of waxy components on the leaf surface of the mutant decreased significantly compared with the wild type. The membrane permeability of both wsl3 and wsl4 mutants increased to a certain extent. 2. Map cloning and functional verification of waxy metabolic synthesis genes WSL3 and WSL4 using map cloning technology. We mapped WSL3 in the 83kb region between M-2396 and M-2404 on chromosome 4 InDel marker. Sequencing of the gene in this region revealed that two consecutive base GC mutations of 736 and 737 of the first exon of LOC_Os04g40730 were TT. The amino acid at position 207 of the encoded protein was mutated from alanine to phenylalanine. WSL4 gene was mapped to 58 labeled M-6283 and M-6341 on chromosome 3 by InDel. Within the kb interval. Sequencing of the interval gene revealed that the 1281 base of the Loc_Os03g12030 gene mutated from G to A, and was located on the second exon. The amino acid at position 312 of the encoded protein mutated from valine to methionine. Bioinformatics analysis showed that LOC_Os04g40730 and Loc_Os03g1203. 0 encodes KCR and KCS class proteins respectively. Further genetic complementation and RNAi interference experiments show that these two genes are the target genes. 3. Spatio-temporal expression analysis and subcellular localization of WSL3 and WSL4 genes. The expression patterns of WSL3 and WSL4 genes were analyzed by real-time fluorescence quantitative RT-PCR and GUS expression experiments. WSL3 and WSL4 are expressed in root, stem, leaf, flower and other tissues, which belong to the constitutive expression gene. Subcellular localization GFP vector of WSL3 and WSL4 gene was constructed. Transient expression in tobacco showed that they could co-locate with ER marker. The results showed that both WSL3 and WSL4 proteins were located in the endoplasmic reticulum. 4. WSL3 protein had fatty acid prolongation enzyme activity and heterologous expression of WSL3 gene in yeast ybr159w 螖 mutant. The phenotype of growth defect could be recovered when WSL3 gene and Arabidopsis FAE1 gene were co-expressed in ybr159w 螖 mutant. Four fatty acid products, 20: 20: 22: 20: 1 螖 11 and 22: 1 螖 13, could be detected in yeast ybr159w 螖 mutants. These results indicated that WSL3 had the activity of fatty acid prolongation enzyme of KCR enzyme.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2

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