水稻窄叶突变体nal12的鉴定与基因精细定位
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摘要
【目的】水稻叶片是理想株型的主要组成部分。筛选和鉴定新的叶形突变材料,可为研究叶发育调控机制和塑造叶片理想形态打下基础。【方法】由粳稻品种武运粳31经EMS(ethyl methane sulphonate)诱变获得窄叶突变体nal12(narrow leaf 12);通过表型测量分析剑叶至倒4叶的叶片长度、宽度、大脉数和小脉数,并进行组织切片和显微观察;将突变体nal12与籼稻品种台中本地1号(TN1)杂交,在F_2分离群体中选取了1709个具窄叶突变表型的单株,通过已有的SSR、STS和新开发的分子标记进行精细定位。【结果】nal12在幼苗期就表现出了窄叶性状,在成熟期植株较野生型矮小,分蘖增多,茎秆变细。经组织切片观察分析,nal12叶片变窄是由于大脉和小脉数量减少造成。遗传分析表明该窄叶表型受单一隐性核基因调控;最终将该基因定位在第10染色体上LC2-RF37与LC4R-RF39标记之间约64.7 kb的范围内。该区间内共有10个开放阅读框,其中并无已报道的窄叶相关基因。qRT-PCR结果表明,NAL12基因的突变会影响生长素合成与运输相关基因的表达。【结论】NAL12为一个新的叶形调控基因。这为进一步研究水稻叶片发育,丰富其分子调控网络打下了基础。
【Objective】Leaf is a vital component of ideal plant type in rice. Screening and identification of new leaf mutants can lay the foundation for studying leaf development and constructing ideal leaf morphology. 【Method】A novel rice mutant named narrow leaf 12(nal12) was identified from an EMS mutagenized population of Wuyunjing 31. The leaf length, leaf width, numbers of large and small veins from flag leaf to the 4 th leaf from the top were measured, and the histological section and microscopic observation were conducted at the maturity stage. The F_2 segregating population was constructed from a cross between nal12 and indica variety TN1, and 1709 narrow-leaf individuals were selected for fine mapping of NAL12 with existing SSR, STS and developed molecular markers. 【Result】The nal12 showed narrow leaves at seedling stage, and exhibited dwarf, increased tillers and thinner stems at maturity stage. According to the anatomy observation of tissue sections, the narrow leaf phenotype of nal12 was caused by the decrease in the number of large veins and small veins compared with the wild type. Genetic analysis indicated that the narrow leaf trait was regulated by a single recessive nuclear gene, and NAL12 was finally mapped to a range of 64.7 kb between LC2-RF37 and LC4R-RF39 markers on chromosome 10. A total of 10 ORFs(open reading frames) were found and there were no narrow-leaf related genes reported in this region. qRT-PCR analysis revealed that the mutation of NAL12 was involved in the expression level of auxin synthesis and transport-related genes. 【Conclusion】NAL12 is a new leaf-shape regulatory gene, which lay a foundation for further research on rice leaf development and enrich its molecular regulatory network.
【Objective】Leaf is a vital component of ideal plant type in rice. Screening and identification of new leaf mutants can lay the foundation for studying leaf development and constructing ideal leaf morphology. 【Method】A novel rice mutant named narrow leaf 12(nal12) was identified from an EMS mutagenized population of Wuyunjing 31. The leaf length, leaf width, numbers of large and small veins from flag leaf to the 4 th leaf from the top were measured, and the histological section and microscopic observation were conducted at the maturity stage. The F_2 segregating population was constructed from a cross between nal12 and indica variety TN1, and 1709 narrow-leaf individuals were selected for fine mapping of NAL12 with existing SSR, STS and developed molecular markers. 【Result】The nal12 showed narrow leaves at seedling stage, and exhibited dwarf, increased tillers and thinner stems at maturity stage. According to the anatomy observation of tissue sections, the narrow leaf phenotype of nal12 was caused by the decrease in the number of large veins and small veins compared with the wild type. Genetic analysis indicated that the narrow leaf trait was regulated by a single recessive nuclear gene, and NAL12 was finally mapped to a range of 64.7 kb between LC2-RF37 and LC4R-RF39 markers on chromosome 10. A total of 10 ORFs(open reading frames) were found and there were no narrow-leaf related genes reported in this region. qRT-PCR analysis revealed that the mutation of NAL12 was involved in the expression level of auxin synthesis and transport-related genes. 【Conclusion】NAL12 is a new leaf-shape regulatory gene, which lay a foundation for further research on rice leaf development and enrich its molecular regulatory network.
引文
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[3]Cho S H,Yoo S C,Zhang H T,Pandeya D,Koh H J,Hwang J Y,Kim G T,Paek N C.The rice narrow leaf2and narrow leaf3 loci encode WUSCHEL-related homeobox 3A(OsWOX3A)and function in leaf,spikelet,tiller and lateral root development.New Phytol,2013,198(4):1071-1084.
[4]Fujino K,Matsuda Y,Ozawa K,Nishimura T,Koshiba T,Fraaije M W,Sekiguchi H.NARROW LEAF 7 controls leaf shape mediated by auxin in rice.Mol Genet Genom,2008,279(5):499-507.
[5]Li W,Wu C,Hu G C,Xing L,Qian W J,Si H M,Sun ZX,Wang X C,Fu Y P,Liu W Z.Characterization and fine mapping of a novel rice narrow leaf mutant nal9.J Integr Plant Biol,2013,55(11):1016-1025.
[6]Jiang H,Li Z,Zeng D L,Gao Z Y,Guo L B,Fang Y X,Zhang G H,Dong G J,Yan M X,Liu J,Qian Q.Identification and characterization of NARROW ANDROLLED LEAF 1,a novel gene regulating leaf morphology and plant architecture in rice.Plant Mol Biol,2010,73(3):283-292.
[7]Li M,Xiong G Y,Li R,Cui J J,Tang D,Zhang B C,Pauly M,Cheng Z K,Zhou Y H.Rice cellulose synthase-like D4 is essential for normal cell-wall biosynthesis and plant growth.Plant J,2009,60(6):1055-1069.
[8]Wu C,Fu Y P,Hu G C,Si H M,Cheng S H,Liu W Z.Isolation and characterization of a rice mutant with narrow and rolled leaves.Planta,2010,232(2):313-324.
[9]Zhao S S,Zhao L,Liu F X,Wu Y Z,Zhu Z F,Sun C Q,Tan L B.NARROW AND ROLLED LEAF 2 regulates leaf shape,male fertility,and seed size in rice.J Integr Plant Biol,2016,58(12):983-996.
[10]Liu X F,Li M,Liu K,Tang D,Sun M F,Li Y F,Shen Y,Du G J,Cheng Z K.Semi-Rolled Leaf2 modulates rice leaf rolling by regulating abaxial side cell differentiation.J Exp Bot,2016,67(8):2139-2150.
[11]Ma L,Sang X C,Zhang T,Yu Z Y,Li Y F,Zhao F M,Wang Z W,Wang Y T,Yu P,Wang N,Zhang C W,Ling YH,Yang Z L,He G H.ABNORMAL VASCULARBUNDLES regulates cell proliferation and procambium cell establishment during aerial organ development in rice.New Phytol,2017,213(1):275-286.
[12]Xu J,Wang L,Zhou M Y,Zeng D L,Hu J,Zhu L,Ren DY,Dong G J,Gao Z Y,Guo L B,Qian Q,Zhang W Z,Zhang G H.Narrow albino leaf 1 is allelic to CHR729,regulates leaf morphogenesis and development by affecting auxin metabolism in rice.Plant Growth Regul,2017,82(1):175-186.
[13]Ma X D,Ma J,Zhai H H,Xin P Y,Chu J F,Qiao Y L,Han L Z.CHR729 is a chd3 protein that controls seedling development in rice.PLoS One,2015,10(9):e0138934.
[14]Zhang G H,Li S Y,Wang L,Ye W J,Zeng D L,Rao Y C,Peng Y L,Hu J,Yang Y L,Xu J,Ren D Y,Gao Z Y,Zhu L,Dong G J,Hu X M,Yan M X,Guo L B,Li C Y,Qian Q.LSCHL4,from japonica cultivar,which is allelic to nal1,increases yield of indica super rice 93-11.Mol Plant,2014,7(8):1350-1364.
[15]桑贤春,林婷婷,何沛龙,王晓雯,廖红香,张孝波,马玲,何光华.水稻显性窄叶突变体Dnal1的鉴定与基因定位.中国农业科学,2014,47(9):1819-1827.Sang X C,Lin T T,He P L,Wang X W,Liao H S,Zhang X B,Ma L,He G H.Identification and gene mapping of a dominant narrow leaf mutant Dnal1 in rice(Oryza sativa).Sci Agric Sin,2014,47(9):1819-1827.(in Chinses with English abstract)
[16]方云霞,朱丽,潘江杰,余海平,薛大伟,饶玉春,王小虎,石珍源,张栋,祝阳舟,董国军,曾大力,张光恒,郭龙标,胡江,钱前.水稻窄叶突变体nal10的鉴定与基因精细定位.中国水稻科学,2015,29(6):587-594.Fang Y X,Zhu L,Pan J J,Yu H P,Xue D W,Rao Y C,Wang X H,Shi Z Y,Zhang D,Zhu Y Z,Dong G J,Zeng D L,Zhang G H,Guo L B,Hu J,Qian Q.Identification and fine mapping of a narrow leaf mutant nal10 in rice.Chin J Rice Sci,2015,29(6):587-594.(in Chinses with English abstract)
[17]Kim C M,Park S H,Je B I,Park S H,Park S J,Piao H L,Eun M Y,Dolan L,Han C D.OsCSLD1,a cellulose synthase-like D1 gene,is required for root hair morphogenesis in rice.Plant Physiol,2007,143(3):1220-1230.
[18]Takahisa Y,Kenji S,Naoki S,Akio M,Hirohiko H,Masahiko I,Taketa S.Root hairless 2(rth2)mutant represents a loss-of-function allele of the cellulose synthase-like gene OsCSLD1 in rice.Breeding Sci,2011,61(3):225-233.
[19]Vanneste S,Friml J.Auxin:A trigger for change in plant development.Cell,2009,136(6):1005-1016.
[20]Zhao Y.Auxin biosynthesis and its role in plant development.Annu Rev Plant Biol,2010,61:49-64.
[2]Qi J,Qian Q,Bu Q Y,Li S Y,Chen Q,Sun J Q,Liang WX,Zhou Y H,Chu C C,Li X G,Ren F G,Palme K,Zhao B G,Chen J F,Chen M S,Li C Y.Mutation of the rice narrow leaf1 gene,which encodes a novel protein,affects vein patterning and polar auxin transport.Plant Physiol,2008,147(4):1947-1959.
[3]Cho S H,Yoo S C,Zhang H T,Pandeya D,Koh H J,Hwang J Y,Kim G T,Paek N C.The rice narrow leaf2and narrow leaf3 loci encode WUSCHEL-related homeobox 3A(OsWOX3A)and function in leaf,spikelet,tiller and lateral root development.New Phytol,2013,198(4):1071-1084.
[4]Fujino K,Matsuda Y,Ozawa K,Nishimura T,Koshiba T,Fraaije M W,Sekiguchi H.NARROW LEAF 7 controls leaf shape mediated by auxin in rice.Mol Genet Genom,2008,279(5):499-507.
[5]Li W,Wu C,Hu G C,Xing L,Qian W J,Si H M,Sun ZX,Wang X C,Fu Y P,Liu W Z.Characterization and fine mapping of a novel rice narrow leaf mutant nal9.J Integr Plant Biol,2013,55(11):1016-1025.
[6]Jiang H,Li Z,Zeng D L,Gao Z Y,Guo L B,Fang Y X,Zhang G H,Dong G J,Yan M X,Liu J,Qian Q.Identification and characterization of NARROW ANDROLLED LEAF 1,a novel gene regulating leaf morphology and plant architecture in rice.Plant Mol Biol,2010,73(3):283-292.
[7]Li M,Xiong G Y,Li R,Cui J J,Tang D,Zhang B C,Pauly M,Cheng Z K,Zhou Y H.Rice cellulose synthase-like D4 is essential for normal cell-wall biosynthesis and plant growth.Plant J,2009,60(6):1055-1069.
[8]Wu C,Fu Y P,Hu G C,Si H M,Cheng S H,Liu W Z.Isolation and characterization of a rice mutant with narrow and rolled leaves.Planta,2010,232(2):313-324.
[9]Zhao S S,Zhao L,Liu F X,Wu Y Z,Zhu Z F,Sun C Q,Tan L B.NARROW AND ROLLED LEAF 2 regulates leaf shape,male fertility,and seed size in rice.J Integr Plant Biol,2016,58(12):983-996.
[10]Liu X F,Li M,Liu K,Tang D,Sun M F,Li Y F,Shen Y,Du G J,Cheng Z K.Semi-Rolled Leaf2 modulates rice leaf rolling by regulating abaxial side cell differentiation.J Exp Bot,2016,67(8):2139-2150.
[11]Ma L,Sang X C,Zhang T,Yu Z Y,Li Y F,Zhao F M,Wang Z W,Wang Y T,Yu P,Wang N,Zhang C W,Ling YH,Yang Z L,He G H.ABNORMAL VASCULARBUNDLES regulates cell proliferation and procambium cell establishment during aerial organ development in rice.New Phytol,2017,213(1):275-286.
[12]Xu J,Wang L,Zhou M Y,Zeng D L,Hu J,Zhu L,Ren DY,Dong G J,Gao Z Y,Guo L B,Qian Q,Zhang W Z,Zhang G H.Narrow albino leaf 1 is allelic to CHR729,regulates leaf morphogenesis and development by affecting auxin metabolism in rice.Plant Growth Regul,2017,82(1):175-186.
[13]Ma X D,Ma J,Zhai H H,Xin P Y,Chu J F,Qiao Y L,Han L Z.CHR729 is a chd3 protein that controls seedling development in rice.PLoS One,2015,10(9):e0138934.
[14]Zhang G H,Li S Y,Wang L,Ye W J,Zeng D L,Rao Y C,Peng Y L,Hu J,Yang Y L,Xu J,Ren D Y,Gao Z Y,Zhu L,Dong G J,Hu X M,Yan M X,Guo L B,Li C Y,Qian Q.LSCHL4,from japonica cultivar,which is allelic to nal1,increases yield of indica super rice 93-11.Mol Plant,2014,7(8):1350-1364.
[15]桑贤春,林婷婷,何沛龙,王晓雯,廖红香,张孝波,马玲,何光华.水稻显性窄叶突变体Dnal1的鉴定与基因定位.中国农业科学,2014,47(9):1819-1827.Sang X C,Lin T T,He P L,Wang X W,Liao H S,Zhang X B,Ma L,He G H.Identification and gene mapping of a dominant narrow leaf mutant Dnal1 in rice(Oryza sativa).Sci Agric Sin,2014,47(9):1819-1827.(in Chinses with English abstract)
[16]方云霞,朱丽,潘江杰,余海平,薛大伟,饶玉春,王小虎,石珍源,张栋,祝阳舟,董国军,曾大力,张光恒,郭龙标,胡江,钱前.水稻窄叶突变体nal10的鉴定与基因精细定位.中国水稻科学,2015,29(6):587-594.Fang Y X,Zhu L,Pan J J,Yu H P,Xue D W,Rao Y C,Wang X H,Shi Z Y,Zhang D,Zhu Y Z,Dong G J,Zeng D L,Zhang G H,Guo L B,Hu J,Qian Q.Identification and fine mapping of a narrow leaf mutant nal10 in rice.Chin J Rice Sci,2015,29(6):587-594.(in Chinses with English abstract)
[17]Kim C M,Park S H,Je B I,Park S H,Park S J,Piao H L,Eun M Y,Dolan L,Han C D.OsCSLD1,a cellulose synthase-like D1 gene,is required for root hair morphogenesis in rice.Plant Physiol,2007,143(3):1220-1230.
[18]Takahisa Y,Kenji S,Naoki S,Akio M,Hirohiko H,Masahiko I,Taketa S.Root hairless 2(rth2)mutant represents a loss-of-function allele of the cellulose synthase-like gene OsCSLD1 in rice.Breeding Sci,2011,61(3):225-233.
[19]Vanneste S,Friml J.Auxin:A trigger for change in plant development.Cell,2009,136(6):1005-1016.
[20]Zhao Y.Auxin biosynthesis and its role in plant development.Annu Rev Plant Biol,2010,61:49-64.
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