水稻白条纹叶突变体wsl1的生理特性分析及基因定位
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摘要
水稻叶绿素缺失突变是一种常见的突变类型,对叶绿素缺失突变体基因进行定位克隆和功能分析,可进一步丰富叶绿素代谢的分子机理研究。在辽粳371的60Co-γ射线辐射诱变后代中筛选到1个白条纹叶突变体wsl1,经多代自交,其白条纹性状能稳定遗传。利用图位克隆方法定位该基因,同时在孕穗期进行光合色素含量测定和光合参数测定,成熟期考察其穗部农艺性状。该突变体从4叶期开始表现白条纹性状,在茎部、叶鞘、叶缘等不同部位均表现白条纹性状且该性状一直持续到成熟期。与野生型相比,突变体结实率极显著下降,仅为野生型的75.33%;千粒重极显著的增加,比野生型增加7.97%;粒长极显著下降,是野生型的90.15%,粒宽和粒厚极显著增加,分别增加11.51%和7.58%。在孕穗期,突变体wsl1的叶绿素b含量比野生型下降23.20%,差异达到极显著水平。突变体的叶绿素a和类胡萝卜素含量与野生型的无明显差异。与野生型相比,突变体的净光合速率、气孔导度、胞间二氧化碳浓度都显著下降,分别为野生型的85%、72%和95%。而突变体的光能转化效率极显著高于野生型,比野生型高11.88%。透射电镜观察发现,突变体植株叶肉细胞类囊体片层减少,结构模糊。遗传分析表明,水稻白条纹叶突变体wsl1突变表型受1对隐性核基因控制,该基因被定位到第1染色体短臂末端,是一个控制水稻叶色的新基因。该基因的发现有助于深入了解水稻叶色形成和调控的机制,同时丰富了水稻特异种质资源。
Rice leaf color mutation is a common type.The localization and cloning and functional analysis of chlorophyll-deficient mutant genes can further enrich the molecular mechanism of chlorophyll metabolism.A white stripe leaf mutant wsl1 was isolated from the offspring of60Co-γ irradiation treatment with onliaogeng 371. After several generations of self-crossing, its white-striped traits could be stably inherited. The gene was mapped by map-based cloning method, the light and pigment content and photosynthetic parameters were measured at the booting stage, and the agronomic traits of the ear were investigated at the maturity stage. The mutant showed white stripe traits in different parts such as stem, leaf sheath and leaf margin, and the trait persistedfrom the 4-leaf stage to maturity. The experimental data showed that the seed setting rate was significantly lower than wild type, which was only 75.33%; the 1000-grain weight increased significantly, which was 7.97% higher than wild type; the grain length decreased significantly, which was 90.15% of the wild type. The grain width and grain thickness increased significantly, increasing by 11.51% and 7.58%, respectively. At the booting stage, the chlorophyll b content of the mutant wsl1 was decreased by 23.20% compared with the wild type, and the difference reached a very significant level. The chlorophyll a and carotenoid contents of the mutants were not significantly different from the wild type. At the same time, the photosynthetic physiological characteristics data of the mutants showed that the net photosynthetic rate, stomatal conductance and intercellular carbon dioxide concentration of the mutants were significantly decreased, which were 85%, 72% and 95% of the wild type,respectively.But the light energy conversion efficiency of the mutant was significantly higher than that of the wild type, which was11.88% higher than that of the wild type. Transmission electron microscopy showed that the mesophyll cell thylakoid ofthe mutant plant had reducedlamellar layers and fuzzy structure. In the last, the genetic analysis showed that the wsl1 mutant wascontrolled by a pair of recessive nuclear genes. The gene was mapped to the end of the short arm of chromosome 1 and was a new gene controlling leaf color of rice. The discovery of this gene enriched and understood the mechanism of leaf color formation and regulation in rice, and enrichedrice-specific germplasm resources.
Rice leaf color mutation is a common type.The localization and cloning and functional analysis of chlorophyll-deficient mutant genes can further enrich the molecular mechanism of chlorophyll metabolism.A white stripe leaf mutant wsl1 was isolated from the offspring of60Co-γ irradiation treatment with onliaogeng 371. After several generations of self-crossing, its white-striped traits could be stably inherited. The gene was mapped by map-based cloning method, the light and pigment content and photosynthetic parameters were measured at the booting stage, and the agronomic traits of the ear were investigated at the maturity stage. The mutant showed white stripe traits in different parts such as stem, leaf sheath and leaf margin, and the trait persistedfrom the 4-leaf stage to maturity. The experimental data showed that the seed setting rate was significantly lower than wild type, which was only 75.33%; the 1000-grain weight increased significantly, which was 7.97% higher than wild type; the grain length decreased significantly, which was 90.15% of the wild type. The grain width and grain thickness increased significantly, increasing by 11.51% and 7.58%, respectively. At the booting stage, the chlorophyll b content of the mutant wsl1 was decreased by 23.20% compared with the wild type, and the difference reached a very significant level. The chlorophyll a and carotenoid contents of the mutants were not significantly different from the wild type. At the same time, the photosynthetic physiological characteristics data of the mutants showed that the net photosynthetic rate, stomatal conductance and intercellular carbon dioxide concentration of the mutants were significantly decreased, which were 85%, 72% and 95% of the wild type,respectively.But the light energy conversion efficiency of the mutant was significantly higher than that of the wild type, which was11.88% higher than that of the wild type. Transmission electron microscopy showed that the mesophyll cell thylakoid ofthe mutant plant had reducedlamellar layers and fuzzy structure. In the last, the genetic analysis showed that the wsl1 mutant wascontrolled by a pair of recessive nuclear genes. The gene was mapped to the end of the short arm of chromosome 1 and was a new gene controlling leaf color of rice. The discovery of this gene enriched and understood the mechanism of leaf color formation and regulation in rice, and enrichedrice-specific germplasm resources.
引文
[1]初志战,刘小林,陈远玲,等.一个水稻白化致死突变基因的精细定位和遗传研究[J].中国水稻科学,2016,30(2):136-142.
[2]郭龙彪,程式华,钱前.水稻基因组测序和分析的研究进展[J].中国水稻科学,2004,18(6):557-562.
[3]张力科,高用明.水稻叶色突变体及其基因定位和克隆的研究进展[J].作物杂志,2009(2):12-16.
[4]刘聪利,魏祥进,邵高能,等.水稻叶色突变分子机制的研究进展[J].中国稻米,2012,18(4):15-21.
[5]刘胜,魏祥进,邵高能,等.一个水稻“斑马叶“叶色突变体基因zebra leaf2(zl2)的图位克隆[J].中国水稻科学,2013,27(3):231-239.
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[7] HAN S H,SAKURABA Y,KOH H J,et al.Leaf variegation in the rice zebra2 mutant is caused by photoperiodic accumulation of tetra-Cis-lycopene and singlet oxygen[J].Mol Cells,2012,33:87-97.
[8] YIN C C,MA B,DEREK P C,et al.Ethylene responses in rice roots and coleoptiles are differentially regulated by a carotenoid isomerase-mediated abscisic acid pathway[J].The Plant Cell,2015,27(4):1061-1081.
[9] FANG J,CHAI C L,QIAN Q,et al.Mutations of genes in synthesis of the carotenoid precursors of ABA lead to preharvest sprouting and photo-oxidation in rice[J].The Plant Journal,2008,54(2):177-189.
[10]李燕群,钟萍,高志艳,等.水稻斑马叶突变体zebra524的表型鉴定及候选基因分析[J].中国农业科学,2014,47(15):2907-2915.
[11]谭振华.水稻白条纹叶基因OsPPR3的图位克隆及功能分析[D].长沙:湖南大学,2012.
[12] YOO S C,CHO S H,SUGIMOTO H,et al.Rice virescent 3 and stripe 1 encoding the large and small subunits of ribonucleotide reductase are required for chloroplast biogenesis during early leaf development[J].Plant Physiol,2009,150(1):388-401.
[13] NAGAO S.Genie analysis and linkage relationship of characters in rice[J].Adv Genet,1951,4(1):188-211.
[14] MAEKAWA M,INUKAI T,SHINBASHI N.A new gene for leaf stripe(st-6)fund in linkage group 3[J].Rice Genet Newsl,1990,7(1):108-109.
[15]成钦淑,叶邦全,袁灿,等.水稻白条纹叶突变体st11的遗传分析与基因定位[J].中国水稻科学,2015,29(1):14-21.
[16] WANG L W,WANG C M,WANG Y H,et al.WSL3,a component of the plastid-encoded plastid RNA polymerase,is essential for early chloroplast development in rice[J].Plant MolBiol,2016, 92:581-595.
[17] GE C W,WANG L,YE W J,et al.Single-point mutation of an histidine-aspartic domain-containing gene involving in chloroplast ribosome biogenesis leads to white fine stripe leaf in rice[J].Scientific Reports,2017,7(1):3298.
[18] ARNOND I.Copper enzymes in islolated chloroplasts poly phenoloxidase in beta vulagarislant[J].Plant Physiol,1949,24:1-15.
[19] DOYLE J J,DOYLE J L.A rapid DNA isolation procedure for small quatities of fresh leaf tissue[J].Phytochem Bull,1987,19(1):11-15.
[20] MICHELMORE R W,PARAN I,KESSELIR V.Idenificationof markers linked to disease-resistance genes by bulked segregantanalysis:a rapid method to detect markersin specific genomic region by using segregating populations[J].Proc Natl Acad Sci USA, 1991,88:9828-9832.
[21]李建武.生物化学实验原理和方法[M].北京:北京大学出版社,1997:157,275.
[22]刘喜,周春雷,任雅琨,等.水稻叶色白化转绿突变体WGL的遗传分析与基因定位[J].南京农业大学学报,2015,38(5):712-719.
[23]刘进.三个水稻叶绿素代谢相关基因的鉴定与功能分析[D].沈阳:沈阳农业大学,2016.
[24] NAGATA N,TANAKA R,SOTOH S,et al.Indentification of a vinyl reductase gene for chlorophyll synthesis in Arabidopsis thaliana and implications for the evotionofprochlorococcus species[J].Plant Cell,2005,17(1):233-240.
[25] LEE S,KIM J H,YOO E S,et al.Different regulation of chlorophyll a oxygenase genes in rice[J].Plant Mol Biol,2005,57(6):805-818.
[26] BEALE S I.Green genes gleaned[J].Trends in Plant Science,2005,10(7):309-312.
[27] SUGIMOTO H,KUSUMI K,NOGUCHI K,et a1.The rice nuclear gene,VIRESCENT 2,is essential for chloroplast development and encodes a novel type of guanylate kinase targeted to plastids and mitochondria[J].Plant J,2007,52(3):512-527.
[28] ZHANG H T,LI J J,YOO J H,et a1.Rice Chlorina-1 and Chlorina-9 encode ChlD and ChlI subunits of Mg-chelatase,a key enzyme for chlorophyll synthesis and chloroplast development[J].Plant Mol Biol,2006,62(3):325-337.
[29]何蔷薇.水稻斑马叶突变体zl11的图位克隆和功能分析[D].扬州:扬州大学,2017.
[30]左西龙.水稻斑马叶表型突变性状的发生规律与遗传生理基础研究[D].武汉:华中农业大学,2017.
[31]朱丽,刘文真,吴超,等.水稻着丝粒附近一个淡绿叶突变相关基因的定位分析[J].中国水稻科学,2007,21(3):228-234.
[32]吕典华,宗学凤,王三根,等.两个水稻叶色突变体的光合特性研究[J].作物学报,2009,35(12):2304-2308.
[33]欧立军.水稻叶色突变体的高光合特性[J].作物学报,2011,37(10):1860-1867.
[2]郭龙彪,程式华,钱前.水稻基因组测序和分析的研究进展[J].中国水稻科学,2004,18(6):557-562.
[3]张力科,高用明.水稻叶色突变体及其基因定位和克隆的研究进展[J].作物杂志,2009(2):12-16.
[4]刘聪利,魏祥进,邵高能,等.水稻叶色突变分子机制的研究进展[J].中国稻米,2012,18(4):15-21.
[5]刘胜,魏祥进,邵高能,等.一个水稻“斑马叶“叶色突变体基因zebra leaf2(zl2)的图位克隆[J].中国水稻科学,2013,27(3):231-239.
[6] CHAI C L,FANG J,LIU Y,et al.ZEBRA2,encoding a carotenoid isomerase,is involved in photoprotection in rice[J].Plant Molecular Biology,2011,75(3):211-221.
[7] HAN S H,SAKURABA Y,KOH H J,et al.Leaf variegation in the rice zebra2 mutant is caused by photoperiodic accumulation of tetra-Cis-lycopene and singlet oxygen[J].Mol Cells,2012,33:87-97.
[8] YIN C C,MA B,DEREK P C,et al.Ethylene responses in rice roots and coleoptiles are differentially regulated by a carotenoid isomerase-mediated abscisic acid pathway[J].The Plant Cell,2015,27(4):1061-1081.
[9] FANG J,CHAI C L,QIAN Q,et al.Mutations of genes in synthesis of the carotenoid precursors of ABA lead to preharvest sprouting and photo-oxidation in rice[J].The Plant Journal,2008,54(2):177-189.
[10]李燕群,钟萍,高志艳,等.水稻斑马叶突变体zebra524的表型鉴定及候选基因分析[J].中国农业科学,2014,47(15):2907-2915.
[11]谭振华.水稻白条纹叶基因OsPPR3的图位克隆及功能分析[D].长沙:湖南大学,2012.
[12] YOO S C,CHO S H,SUGIMOTO H,et al.Rice virescent 3 and stripe 1 encoding the large and small subunits of ribonucleotide reductase are required for chloroplast biogenesis during early leaf development[J].Plant Physiol,2009,150(1):388-401.
[13] NAGAO S.Genie analysis and linkage relationship of characters in rice[J].Adv Genet,1951,4(1):188-211.
[14] MAEKAWA M,INUKAI T,SHINBASHI N.A new gene for leaf stripe(st-6)fund in linkage group 3[J].Rice Genet Newsl,1990,7(1):108-109.
[15]成钦淑,叶邦全,袁灿,等.水稻白条纹叶突变体st11的遗传分析与基因定位[J].中国水稻科学,2015,29(1):14-21.
[16] WANG L W,WANG C M,WANG Y H,et al.WSL3,a component of the plastid-encoded plastid RNA polymerase,is essential for early chloroplast development in rice[J].Plant MolBiol,2016, 92:581-595.
[17] GE C W,WANG L,YE W J,et al.Single-point mutation of an histidine-aspartic domain-containing gene involving in chloroplast ribosome biogenesis leads to white fine stripe leaf in rice[J].Scientific Reports,2017,7(1):3298.
[18] ARNOND I.Copper enzymes in islolated chloroplasts poly phenoloxidase in beta vulagarislant[J].Plant Physiol,1949,24:1-15.
[19] DOYLE J J,DOYLE J L.A rapid DNA isolation procedure for small quatities of fresh leaf tissue[J].Phytochem Bull,1987,19(1):11-15.
[20] MICHELMORE R W,PARAN I,KESSELIR V.Idenificationof markers linked to disease-resistance genes by bulked segregantanalysis:a rapid method to detect markersin specific genomic region by using segregating populations[J].Proc Natl Acad Sci USA, 1991,88:9828-9832.
[21]李建武.生物化学实验原理和方法[M].北京:北京大学出版社,1997:157,275.
[22]刘喜,周春雷,任雅琨,等.水稻叶色白化转绿突变体WGL的遗传分析与基因定位[J].南京农业大学学报,2015,38(5):712-719.
[23]刘进.三个水稻叶绿素代谢相关基因的鉴定与功能分析[D].沈阳:沈阳农业大学,2016.
[24] NAGATA N,TANAKA R,SOTOH S,et al.Indentification of a vinyl reductase gene for chlorophyll synthesis in Arabidopsis thaliana and implications for the evotionofprochlorococcus species[J].Plant Cell,2005,17(1):233-240.
[25] LEE S,KIM J H,YOO E S,et al.Different regulation of chlorophyll a oxygenase genes in rice[J].Plant Mol Biol,2005,57(6):805-818.
[26] BEALE S I.Green genes gleaned[J].Trends in Plant Science,2005,10(7):309-312.
[27] SUGIMOTO H,KUSUMI K,NOGUCHI K,et a1.The rice nuclear gene,VIRESCENT 2,is essential for chloroplast development and encodes a novel type of guanylate kinase targeted to plastids and mitochondria[J].Plant J,2007,52(3):512-527.
[28] ZHANG H T,LI J J,YOO J H,et a1.Rice Chlorina-1 and Chlorina-9 encode ChlD and ChlI subunits of Mg-chelatase,a key enzyme for chlorophyll synthesis and chloroplast development[J].Plant Mol Biol,2006,62(3):325-337.
[29]何蔷薇.水稻斑马叶突变体zl11的图位克隆和功能分析[D].扬州:扬州大学,2017.
[30]左西龙.水稻斑马叶表型突变性状的发生规律与遗传生理基础研究[D].武汉:华中农业大学,2017.
[31]朱丽,刘文真,吴超,等.水稻着丝粒附近一个淡绿叶突变相关基因的定位分析[J].中国水稻科学,2007,21(3):228-234.
[32]吕典华,宗学凤,王三根,等.两个水稻叶色突变体的光合特性研究[J].作物学报,2009,35(12):2304-2308.
[33]欧立军.水稻叶色突变体的高光合特性[J].作物学报,2011,37(10):1860-1867.