水稻果膠裂解酶基因DEL1的克隆和功能研究
發(fā)布時間：2018-08-05 14:08
【摘要】：果膠是植物細胞壁的重要組成成份,也是植物細胞壁結構最為復雜的多糖。果膠裂解酶作為眾多果膠降解酶之一,能夠通過β-消除作用將去酯化的同聚半乳糖醛酸降解成一個短鏈的果膠分子和一個4,5-不飽和寡聚半乳糖醛酸。到目前為止,盡管許多植物中都報道了果膠裂解酶基因的功能,主要表現(xiàn)在參與花粉、花藥和雌蕊的發(fā)育、果實軟化及成熟、抗病和植物器官或組織發(fā)育等方面,但在水稻中功能還鮮有報道。本研究在水稻甲基磺酸乙酯(EMS)誘變突變體庫中篩選到一個水稻矮稈、葉片早衰突變體,命名為del1(dwarf and early-senescence leaf 1)。我們通過生理學、細胞學、遺傳學、分子生物學以及生物信息學分析等試驗方法,剖析了其突變表型變化的分子機理。主要研究結果如下:1.突變體del1在整個生育期表現(xiàn)出發(fā)育緩慢、植株矮小、根長變短、分蘗數(shù)減少等表型。石蠟切片分析結果表明,造成突變體del1植株矮小的原因是細胞數(shù)目減少導致的。流式細胞儀結果表明突變體del1細胞數(shù)目減少是由于細胞周期G1期受到阻滯引起的;2.突變體del1同時也表現(xiàn)出葉片早衰的表型,在播種后5天葉尖及葉片邊緣發(fā)生黃化,隨著生長發(fā)育葉的進行,葉片衰老表型更加嚴重,到了成熟期葉尖甚至出現(xiàn)撕裂表型。透射電鏡觀察突變體del1中葉綠體出現(xiàn)部分降解,類囊體排列紊亂,片層排列不規(guī)則等現(xiàn)象。同時,突變體del1中葉綠素含量和光合作用都顯著下降,衰老相關基因的表達顯著上升。臺盼藍染色及TUNEL實驗表明,突變體del1衰老的葉片中細胞程序化死亡增加;NBT和DAB染色觀察發(fā)現(xiàn),del1突變體中活性氧含量顯著升高,且電解質滲透率也顯著升高。同時,活性氧相關酶活性及清除活性氧相關基因也顯著升高。這些結果表明,突變體del1葉片的衰老主要是由于活性氧的積累導致的;3.遺傳分析表明,del1是由單一核基因控制的隱性性狀。以del1為母本,TN1為父本雜交獲得F2定位群體,選取30株突變表型的單株進行初定位,將DEL1定位在10號染色體長壁上位于標記M1和M14之間。進一步擴大群體(1081株突變表型單株)將DEL1定位于標記M7和M8之間約45kb的物理距離范圍內,該區(qū)間包含8個開放閱讀框。并進一步通過測序發(fā)現(xiàn)第五個開放閱讀框的第三個外顯子發(fā)生一個堿基替換(G替換成T),導致氨基酸由色氨酸(Trp)變成亮氨酸(Leu)。序列分析表明DEL1編碼一個果膠裂解酶前體基因,cDNA全長為1476bp,編碼491個氨基酸。遺傳互補實驗證明,DEL1基因能夠恢復矮稈和葉片早衰的表型;4.生物信息學分析DEL1蛋白序列含有一個PelC結構域,且保守位點與植物同源性非常高。進化分析表明,DEL1與擬南芥業(yè)已報到的易感白粉病PMR6基因在進化上親緣關系很近,但DEL1卻沒有表現(xiàn)出抗病的特性,表明果膠裂解酶基因在單子葉和雙子葉植物中已出現(xiàn)功能分化;5.DEL1基因在水稻各個組織器官廣泛表達。其中,在莖,根和鞘等伸長部位表達量很高;6.果膠酶活性實驗表明,突變體del1的酶活性要較野生型顯著降低。透射電鏡觀察發(fā)現(xiàn),突變體del1細胞壁結構發(fā)生明顯變化,胞間層和次生細胞壁厚度顯著下降,而初生細胞壁顯著升高。同時,果膠、纖維素、半纖維素及7種中性單糖都發(fā)生了顯著的變化。免疫組織化學實驗表明,突變體del1果膠甲酯化顯著升高。7.轉錄組分析表明,突變體del1中許多細胞壁相關和衰老相關基因表達量發(fā)生變化,表明DEL1可能通過間接的方式參與水稻植物發(fā)育和葉片衰老相關的通路中。以上結果說明:突變體del1矮稈、早衰表型是由于果膠裂解酶基因的突變引起細胞壁組成及成分變化導致的。
[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.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S511
本文編號：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.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S511
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