水稻果胶裂解酶基因DEL1的克隆和功能研究
发布时间：2018-08-05 14:08
【摘要】：果胶是植物细胞壁的重要组成成份,也是植物细胞壁结构最为复杂的多糖。果胶裂解酶作为众多果胶降解酶之一,能够通过β-消除作用将去酯化的同聚半乳糖醛酸降解成一个短链的果胶分子和一个4,5-不饱和寡聚半乳糖醛酸。到目前为止,尽管许多植物中都报道了果胶裂解酶基因的功能,主要表现在参与花粉、花药和雌蕊的发育、果实软化及成熟、抗病和植物器官或组织发育等方面,但在水稻中功能还鲜有报道。本研究在水稻甲基磺酸乙酯(EMS)诱变突变体库中筛选到一个水稻矮秆、叶片早衰突变体,命名为del1(dwarf and early-senescence leaf 1)。我们通过生理学、细胞学、遗传学、分子生物学以及生物信息学分析等试验方法,剖析了其突变表型变化的分子机理。主要研究结果如下:1.突变体del1在整个生育期表现出发育缓慢、植株矮小、根长变短、分蘖数减少等表型。石蜡切片分析结果表明,造成突变体del1植株矮小的原因是细胞数目减少导致的。流式细胞仪结果表明突变体del1细胞数目减少是由于细胞周期G1期受到阻滞引起的;2.突变体del1同时也表现出叶片早衰的表型,在播种后5天叶尖及叶片边缘发生黄化,随着生长发育叶的进行,叶片衰老表型更加严重,到了成熟期叶尖甚至出现撕裂表型。透射电镜观察突变体del1中叶绿体出现部分降解,类囊体排列紊乱,片层排列不规则等现象。同时,突变体del1中叶绿素含量和光合作用都显著下降,衰老相关基因的表达显著上升。台盼蓝染色及TUNEL实验表明,突变体del1衰老的叶片中细胞程序化死亡增加;NBT和DAB染色观察发现,del1突变体中活性氧含量显著升高,且电解质渗透率也显著升高。同时,活性氧相关酶活性及清除活性氧相关基因也显著升高。这些结果表明,突变体del1叶片的衰老主要是由于活性氧的积累导致的;3.遗传分析表明,del1是由单一核基因控制的隐性性状。以del1为母本,TN1为父本杂交获得F2定位群体,选取30株突变表型的单株进行初定位,将DEL1定位在10号染色体长壁上位于标记M1和M14之间。进一步扩大群体(1081株突变表型单株)将DEL1定位于标记M7和M8之间约45kb的物理距离范围内,该区间包含8个开放阅读框。并进一步通过测序发现第五个开放阅读框的第三个外显子发生一个碱基替换(G替换成T),导致氨基酸由色氨酸(Trp)变成亮氨酸(Leu)。序列分析表明DEL1编码一个果胶裂解酶前体基因,cDNA全长为1476bp,编码491个氨基酸。遗传互补实验证明,DEL1基因能够恢复矮秆和叶片早衰的表型;4.生物信息学分析DEL1蛋白序列含有一个PelC结构域,且保守位点与植物同源性非常高。进化分析表明,DEL1与拟南芥业已报到的易感白粉病PMR6基因在进化上亲缘关系很近,但DEL1却没有表现出抗病的特性,表明果胶裂解酶基因在单子叶和双子叶植物中已出现功能分化;5.DEL1基因在水稻各个组织器官广泛表达。其中,在茎,根和鞘等伸长部位表达量很高;6.果胶酶活性实验表明,突变体del1的酶活性要较野生型显著降低。透射电镜观察发现,突变体del1细胞壁结构发生明显变化,胞间层和次生细胞壁厚度显著下降,而初生细胞壁显著升高。同时,果胶、纤维素、半纤维素及7种中性单糖都发生了显著的变化。免疫组织化学实验表明,突变体del1果胶甲酯化显著升高。7.转录组分析表明,突变体del1中许多细胞壁相关和衰老相关基因表达量发生变化,表明DEL1可能通过间接的方式参与水稻植物发育和叶片衰老相关的通路中。以上结果说明:突变体del1矮秆、早衰表型是由于果胶裂解酶基因的突变引起细胞壁组成及成分变化导致的。
[Abstract]:Pectin is an important component of plant cell wall and the most complex polysaccharide in plant cell wall. As one of the many pectin degrading enzymes, pectin lyase can degrade the deesterified homopolymuraluronic acid into a short chain pectin molecule and a 4,5- unsaturated oligopoly galaconic acid by beta elimination. Although the function of pectin lyase gene is reported in many plants, it is mainly manifested in the development of pollen, anther and pistil, fruit softening and maturation, disease resistance and plant organ or tissue development, but the function in rice is rarely reported. This study was screened in the mutant library of EMS. A rice dwarf, leaf premature senescence mutant, named DEL1 (dwarf and early-senescence leaf 1). We analyzed the molecular mechanism of mutant phenotypic changes through physiological, cytological, genetic, molecular biology and bioinformatics analysis. The main results are as follows: 1. mutant DEL1 in the whole growth period table The results of the paraffin section analysis showed that the decrease of the mutant DEL1 plant was caused by the decrease of the number of cells. The result of flow cytometry showed that the decrease of the number of mutant DEL1 cells was due to the block of cell cycle G1 phase; the 2. mutant DEL1 was at the same time. The phenotypes of premature senescence of leaves were also displayed. The leaf tip and leaf edge of the leaves were yellowed at 5 days after sowing. With the growth and development of leaves, the leaf senescence phenotype was more serious, and the leaf tip of the leaf was even tearing phenotype. The transmission electron microscope observed that the chloroplasts were partially degraded, the thylakoids were arranged in disorder, and the lamellar arrangement was irregular in the mutant DEL1. At the same time, the chlorophyll content and Photosynthesis of the mutant DEL1 decreased significantly, and the expression of senescence related genes increased significantly. Trypan blue staining and TUNEL experiments showed that the programmed cell death of the mutant DEL1 senescent leaves increased; NBT and DAB staining showed that the content of active oxygen in the DEL1 mutant was significantly higher and the electrolyte was increased. The permeability also increased significantly. At the same time, active oxygen related enzyme activity and scavenging ROS related genes also increased significantly. These results showed that the senescence of the mutant DEL1 leaves was mainly due to the accumulation of active oxygen; 3. genetic analysis showed that DEL1 was a recessive trait controlled by a single nuclear gene. DEL1 was the parent, and TN1 was paternal hybridization. F2 locational population was selected for the first location of 30 mutant phenotypes, and DEL1 was located between the long wall of chromosome 10 and between the marker M1 and M14. Further enlargement group (1081 mutant phenotypes) located DEL1 in the physical distance of about 45KB between M7 and M8, which included 8 open reading frames. After sequencing, the third exons of fifth open reading frames were found to have a base replacement (G substitution T), resulting in amino acid from tryptophan (Trp) into leucine (Leu). Sequence analysis showed that DEL1 encodes a pectin lyase precursor gene, cDNA full length was 1476bp, and coded 491 amino acids. Genetic complementarity experiments proved that DEL1 gene could be restored. The phenotype of the dwarf and the premature senescence of leaves; 4. bioinformatics analysis of the DEL1 protein sequence contains a PelC domain, and the conserved loci are very homologous to plants. Evolutionary analysis shows that DEL1 and Arabidopsis are reported to be susceptible to powdery mildew of the PMR6 gene which is closely related to evolution, but DEL1 does not show the characteristics of disease resistance, indicating fruit. The gene in the monocotyledon and the dicotyledonous plants had functional differentiation, and the 5.DEL1 gene was widely expressed in various tissues and organs of the rice. Among them, the expression in the elongated parts, such as stem, root and sheath, was very high. The activity of 6. pectinase showed that the enzyme activity of the mutant DEL1 was significantly lower than that of the wild type. Transmission electron microscope observation found that the mutant del The wall structure of 1 cells changed obviously, the thickness of intercellular and secondary cell wall decreased significantly, while the wall of primary cells increased significantly. At the same time, the pectin, cellulose, hemicellulose and 7 kinds of neutral monosaccharides had been significantly changed. The immuno histochemical experiments showed that the mutation of the mutant DEL1 was significantly increased by the.7. transcriptional analysis. The expression of cell wall related and senescence related genes in the body DEL1 changes, indicating that DEL1 may participate in the pathway related to plant development and leaf senescence in an indirect way. The above results indicate that the mutant DEL1 dwarf, the phenotype of the premature senescence is due to the mutation of the pectin lyase gene, which leads to the changes in the composition and composition of the cell wall. It is.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S511
本文编号：2166022
[Abstract]:Pectin is an important component of plant cell wall and the most complex polysaccharide in plant cell wall. As one of the many pectin degrading enzymes, pectin lyase can degrade the deesterified homopolymuraluronic acid into a short chain pectin molecule and a 4,5- unsaturated oligopoly galaconic acid by beta elimination. Although the function of pectin lyase gene is reported in many plants, it is mainly manifested in the development of pollen, anther and pistil, fruit softening and maturation, disease resistance and plant organ or tissue development, but the function in rice is rarely reported. This study was screened in the mutant library of EMS. A rice dwarf, leaf premature senescence mutant, named DEL1 (dwarf and early-senescence leaf 1). We analyzed the molecular mechanism of mutant phenotypic changes through physiological, cytological, genetic, molecular biology and bioinformatics analysis. The main results are as follows: 1. mutant DEL1 in the whole growth period table The results of the paraffin section analysis showed that the decrease of the mutant DEL1 plant was caused by the decrease of the number of cells. The result of flow cytometry showed that the decrease of the number of mutant DEL1 cells was due to the block of cell cycle G1 phase; the 2. mutant DEL1 was at the same time. The phenotypes of premature senescence of leaves were also displayed. The leaf tip and leaf edge of the leaves were yellowed at 5 days after sowing. With the growth and development of leaves, the leaf senescence phenotype was more serious, and the leaf tip of the leaf was even tearing phenotype. The transmission electron microscope observed that the chloroplasts were partially degraded, the thylakoids were arranged in disorder, and the lamellar arrangement was irregular in the mutant DEL1. At the same time, the chlorophyll content and Photosynthesis of the mutant DEL1 decreased significantly, and the expression of senescence related genes increased significantly. Trypan blue staining and TUNEL experiments showed that the programmed cell death of the mutant DEL1 senescent leaves increased; NBT and DAB staining showed that the content of active oxygen in the DEL1 mutant was significantly higher and the electrolyte was increased. The permeability also increased significantly. At the same time, active oxygen related enzyme activity and scavenging ROS related genes also increased significantly. These results showed that the senescence of the mutant DEL1 leaves was mainly due to the accumulation of active oxygen; 3. genetic analysis showed that DEL1 was a recessive trait controlled by a single nuclear gene. DEL1 was the parent, and TN1 was paternal hybridization. F2 locational population was selected for the first location of 30 mutant phenotypes, and DEL1 was located between the long wall of chromosome 10 and between the marker M1 and M14. Further enlargement group (1081 mutant phenotypes) located DEL1 in the physical distance of about 45KB between M7 and M8, which included 8 open reading frames. After sequencing, the third exons of fifth open reading frames were found to have a base replacement (G substitution T), resulting in amino acid from tryptophan (Trp) into leucine (Leu). Sequence analysis showed that DEL1 encodes a pectin lyase precursor gene, cDNA full length was 1476bp, and coded 491 amino acids. Genetic complementarity experiments proved that DEL1 gene could be restored. The phenotype of the dwarf and the premature senescence of leaves; 4. bioinformatics analysis of the DEL1 protein sequence contains a PelC domain, and the conserved loci are very homologous to plants. Evolutionary analysis shows that DEL1 and Arabidopsis are reported to be susceptible to powdery mildew of the PMR6 gene which is closely related to evolution, but DEL1 does not show the characteristics of disease resistance, indicating fruit. The gene in the monocotyledon and the dicotyledonous plants had functional differentiation, and the 5.DEL1 gene was widely expressed in various tissues and organs of the rice. Among them, the expression in the elongated parts, such as stem, root and sheath, was very high. The activity of 6. pectinase showed that the enzyme activity of the mutant DEL1 was significantly lower than that of the wild type. Transmission electron microscope observation found that the mutant del The wall structure of 1 cells changed obviously, the thickness of intercellular and secondary cell wall decreased significantly, while the wall of primary cells increased significantly. At the same time, the pectin, cellulose, hemicellulose and 7 kinds of neutral monosaccharides had been significantly changed. The immuno histochemical experiments showed that the mutation of the mutant DEL1 was significantly increased by the.7. transcriptional analysis. The expression of cell wall related and senescence related genes in the body DEL1 changes, indicating that DEL1 may participate in the pathway related to plant development and leaf senescence in an indirect way. The above results indicate that the mutant DEL1 dwarf, the phenotype of the premature senescence is due to the mutation of the pectin lyase gene, which leads to the changes in the composition and composition of the cell wall. It is.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S511
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