Tissue-specific Expression of Acetolactate Synthase (ALS), Male Sterility-inducing Effect of Tribenuron-methyl and Its Effect on ALS Activity in Brassica napus L.
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摘要
[Objectives] This study was conducted to provide a basis for the rapid identification of the drug spraying effect in early stage and the molecular mechanism of chemical hybridizing in Brassica napus L. [Methods] Quantitative RT-PCR analysis showed that ALS was constitutively expressed in various tissues of 096030, including flower buds, four floral organs(calyxes, petals, stamens and pistils), roots, stems and leaves. ALS was prominently expressed in leaves and was expressed weakly in the petals and stamens. The male sterility-inducing effects of tribenuron-methyl on such two Brassica napus L. varieties as Ningyou18 and 096030 were investigated. [Results] Plants were twice sprayed with 0.2 μg/ml tribenuron-methyl on leaves. The results showed that 8-10 ml of tribenuron-methyl was applied per plant for the first time at bolting stage with 1-2 mm flower buds on 15-20 cm inflorescence, and the second spray was performed with 8-10 ml of tribenuron-methyl per plant 10 d later. The results showed that the percentage of the full sterile plants reached 100%, which lasted for the whole flowering period, and the relative seed setting rate was only about 4%. Thus, this method could fullfill the requirement of hybrid seed production in field. The in-vivo enzyme activity of acetolactate synthase(ALS) was assayed using 2 mm buds collected 3 d after spray. The results showed that 0.2 μg/ml tribenuron-methyl inhibited ALS activity. The ALS activity of Ningyou 18(CK) and Ningyou 18(0.2 μg/ml) was 3.20 and 1.30 μmol/(mg·h), respectively, and the ALS activity of 096030(CK) and 096030(0.2 μg/ml) was 3.37 and 1.25 μmol/(mg·h), respectively. The relative enzyme activity of ALS in Ningyou18 and 096030 was 40.63% and 37.23%, respectively, both of which decreased significantly. [Conclusions] These results showed that the change of ALS activity may be used as an index for quickly identifying and predicting the chemical hybridizing effect of tribenuron-methyl.
[Objectives] This study was conducted to provide a basis for the rapid identification of the drug spraying effect in early stage and the molecular mechanism of chemical hybridizing in Brassica napus L. [Methods] Quantitative RT-PCR analysis showed that ALS was constitutively expressed in various tissues of 096030, including flower buds, four floral organs(calyxes, petals, stamens and pistils), roots, stems and leaves. ALS was prominently expressed in leaves and was expressed weakly in the petals and stamens. The male sterility-inducing effects of tribenuron-methyl on such two Brassica napus L. varieties as Ningyou18 and 096030 were investigated. [Results] Plants were twice sprayed with 0.2 μg/ml tribenuron-methyl on leaves. The results showed that 8-10 ml of tribenuron-methyl was applied per plant for the first time at bolting stage with 1-2 mm flower buds on 15-20 cm inflorescence, and the second spray was performed with 8-10 ml of tribenuron-methyl per plant 10 d later. The results showed that the percentage of the full sterile plants reached 100%, which lasted for the whole flowering period, and the relative seed setting rate was only about 4%. Thus, this method could fullfill the requirement of hybrid seed production in field. The in-vivo enzyme activity of acetolactate synthase(ALS) was assayed using 2 mm buds collected 3 d after spray. The results showed that 0.2 μg/ml tribenuron-methyl inhibited ALS activity. The ALS activity of Ningyou 18(CK) and Ningyou 18(0.2 μg/ml) was 3.20 and 1.30 μmol/(mg·h), respectively, and the ALS activity of 096030(CK) and 096030(0.2 μg/ml) was 3.37 and 1.25 μmol/(mg·h), respectively. The relative enzyme activity of ALS in Ningyou18 and 096030 was 40.63% and 37.23%, respectively, both of which decreased significantly. [Conclusions] These results showed that the change of ALS activity may be used as an index for quickly identifying and predicting the chemical hybridizing effect of tribenuron-methyl.
[Objectives] This study was conducted to provide a basis for the rapid identification of the drug spraying effect in early stage and the molecular mechanism of chemical hybridizing in Brassica napus L. [Methods] Quantitative RT-PCR analysis showed that ALS was constitutively expressed in various tissues of 096030, including flower buds, four floral organs(calyxes, petals, stamens and pistils), roots, stems and leaves. ALS was prominently expressed in leaves and was expressed weakly in the petals and stamens. The male sterility-inducing effects of tribenuron-methyl on such two Brassica napus L. varieties as Ningyou18 and 096030 were investigated. [Results] Plants were twice sprayed with 0.2 μg/ml tribenuron-methyl on leaves. The results showed that 8-10 ml of tribenuron-methyl was applied per plant for the first time at bolting stage with 1-2 mm flower buds on 15-20 cm inflorescence, and the second spray was performed with 8-10 ml of tribenuron-methyl per plant 10 d later. The results showed that the percentage of the full sterile plants reached 100%, which lasted for the whole flowering period, and the relative seed setting rate was only about 4%. Thus, this method could fullfill the requirement of hybrid seed production in field. The in-vivo enzyme activity of acetolactate synthase(ALS) was assayed using 2 mm buds collected 3 d after spray. The results showed that 0.2 μg/ml tribenuron-methyl inhibited ALS activity. The ALS activity of Ningyou 18(CK) and Ningyou 18(0.2 μg/ml) was 3.20 and 1.30 μmol/(mg·h), respectively, and the ALS activity of 096030(CK) and 096030(0.2 μg/ml) was 3.37 and 1.25 μmol/(mg·h), respectively. The relative enzyme activity of ALS in Ningyou18 and 096030 was 40.63% and 37.23%, respectively, both of which decreased significantly. [Conclusions] These results showed that the change of ALS activity may be used as an index for quickly identifying and predicting the chemical hybridizing effect of tribenuron-methyl.
引文
[1]FU TD.Oil research and application of heterosis in rapeseed[J].Chinese Journal of Oil Crop Sciences,2008,30(z1):1-5.(in Chinese)
[2]ZHANG ZQ,WANG GH,GUAN CY,et al.Research Advances in Chemical Emasculation of Rape[J].Hunan Agricultural Sciences,2011,(5):19-22.(in Chinese)
[3]ZHANG BJ,ZHAO HX,HU SW.Male sterile-inducing ability of tribenuron-methyl to rapeseed cultivar Zhongshuang 9[J].Chinese Journal of Oil Crop Sciences,2010,32(4):467-471.(in Chinese)
[4]HUANG C,HU HB,PU XB,et al.Effects of male sterilization of Brassica napus L.induced by tribenuron-methyl in Chengdu region[J].Southwest China Journal of Agricultural Sciences,2015,28(3):1097-1104.(in Chinese)
[5]PANG SS,DUGGLEBY RG,GUDDAT LW.Crystal structure of yeast acetohydroxyacid synthase:A target for herbicidal inhibitors[J].J Mol Biol,2002,317:249-262.
[6]YU CY.Characterization and work mechanism of chemical hybridizing agents for plants[D].Yangling:Northwest Agriculture&Forestry University,2009.(in Chinese)
[7]MCCOURT JA,DUGGLEBY RG.Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids[J].Amino Acids,2006,31:173-210.
[8]CHEN SY,GUAN CY,WANG GH,et al.Breeding of a new double-low hybrid rapeseed cultivar Xiangzayou 6[J].Chinese Journal of Oil Crop Sciences,2005,27(2):37-39.(in Chinese)()(Continued from page 4)
[9]CHEN L,LI GN,TANG ZL,et al.Breeding of"Yuhuang 1"---Anew yellow-seeded hybrid cultivar of rape(Brassica napus L.)[J].Journal of Southwest Agricultural University,2002,24(1):45-47.(in Chinese)
[10]WANG AN,WANG H,ZHAO YJ,et al.Purity identification of chemical hybriding male varieties of hybrid rape Qinyou 33 using SSRmarkers[J].Chinese journal of agricultural biotechnology,2011,19(6):1011-1018.(in Chinese)
[11]FU SX,QI CK,GU H,et al.Breeding of high-quality two-line hybrid rape variety Ningza 1818 through chemical emasculation[J].Acta Agriculturae Jiangxi,2015,27(7):11-14.(in Chinese)
[12]LIU ZQ,LIU QQ,YU CY,et al.Evaluation of inhibiting effects of 5herbicides on acetolactate synthase activities and gametocidal effect in rapeseed[J].Chinese Journal of Oil Crop Sciences,2016,38(6):742-749.(in Chinese)
[13]SINGH BK,STIDHAM MA,SHANER DL.Assay of acetohydroxyacid synthase[J].Ann Biochem,1988,171:173-179.
[14]CROSS RB,MCCARTY LB,THARAYIL N,et al.Detecting annual bluegrass(Poa annua)resistance to ALS-inhibiring herbicides using a rapid diagnostic assay[J].Weed Sci,2013,61(3):384-389.
[15]VEGA T,BRECCIA G,GIL M,et al.Acetohydroxyacid synthase(AHAS)in vivo assay for screening imidazolinone-resistance in sun-:flower(Helianthus annuus L.)[J].Plant Physiol Biochem,2012,61:103-107.
[16]LI YD,DONG X,HE S,et al.Effects of pyroxsulam on acetolactate aynthetase activity of main wheat varieties in Xinjiang[J].Acta Agriculturae Boreali-occidentalis Sinica,2017,26(4):560-567.(in Chinese)
[17]LIU W,WANG JX,YANG GL,et al.Difference of tolerance and mechanism of various wheat varieties to tribenuron-methyl[J].Journal of Plant Protection,2005,32(3):300-304.(in Chinese)
[18]YAO X,WANG YQ YUE XP,et al.Generation of tribenuron-methyl herbicide-resistant Os CYP81A6-expressing rapeseed(Brassica napus L.)plants for hybrid seed production using chemical-induced male sterility[J].Plant Breeding,2016,135(3):349-354.
[19]LI T,LIU B,CHEN CY,et al.TALEN-mediated homologous recombination produces site-directed DNA base change and herbicide-resistant rice[J].J Genet Genom,2016,43(5):297-305.
[20]BRECCIA G,VEGA T,FELITTI SA,et al.Differential expression of acetohydroxyacid synthase genes in sunflower plantlets and its response to imazapyr herbicide[J].Plant Sci,2013,208:28-33.
[21]FAN ZJ,CHEN JP,DANG HB,et al.Effect of monosulfuron on the activity of its target enzyme acetolactate synthase[J].Modern Agrochemicals,2003,2(2):16-17.(in Chinese)
[2]ZHANG ZQ,WANG GH,GUAN CY,et al.Research Advances in Chemical Emasculation of Rape[J].Hunan Agricultural Sciences,2011,(5):19-22.(in Chinese)
[3]ZHANG BJ,ZHAO HX,HU SW.Male sterile-inducing ability of tribenuron-methyl to rapeseed cultivar Zhongshuang 9[J].Chinese Journal of Oil Crop Sciences,2010,32(4):467-471.(in Chinese)
[4]HUANG C,HU HB,PU XB,et al.Effects of male sterilization of Brassica napus L.induced by tribenuron-methyl in Chengdu region[J].Southwest China Journal of Agricultural Sciences,2015,28(3):1097-1104.(in Chinese)
[5]PANG SS,DUGGLEBY RG,GUDDAT LW.Crystal structure of yeast acetohydroxyacid synthase:A target for herbicidal inhibitors[J].J Mol Biol,2002,317:249-262.
[6]YU CY.Characterization and work mechanism of chemical hybridizing agents for plants[D].Yangling:Northwest Agriculture&Forestry University,2009.(in Chinese)
[7]MCCOURT JA,DUGGLEBY RG.Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids[J].Amino Acids,2006,31:173-210.
[8]CHEN SY,GUAN CY,WANG GH,et al.Breeding of a new double-low hybrid rapeseed cultivar Xiangzayou 6[J].Chinese Journal of Oil Crop Sciences,2005,27(2):37-39.(in Chinese)()(Continued from page 4)
[9]CHEN L,LI GN,TANG ZL,et al.Breeding of"Yuhuang 1"---Anew yellow-seeded hybrid cultivar of rape(Brassica napus L.)[J].Journal of Southwest Agricultural University,2002,24(1):45-47.(in Chinese)
[10]WANG AN,WANG H,ZHAO YJ,et al.Purity identification of chemical hybriding male varieties of hybrid rape Qinyou 33 using SSRmarkers[J].Chinese journal of agricultural biotechnology,2011,19(6):1011-1018.(in Chinese)
[11]FU SX,QI CK,GU H,et al.Breeding of high-quality two-line hybrid rape variety Ningza 1818 through chemical emasculation[J].Acta Agriculturae Jiangxi,2015,27(7):11-14.(in Chinese)
[12]LIU ZQ,LIU QQ,YU CY,et al.Evaluation of inhibiting effects of 5herbicides on acetolactate synthase activities and gametocidal effect in rapeseed[J].Chinese Journal of Oil Crop Sciences,2016,38(6):742-749.(in Chinese)
[13]SINGH BK,STIDHAM MA,SHANER DL.Assay of acetohydroxyacid synthase[J].Ann Biochem,1988,171:173-179.
[14]CROSS RB,MCCARTY LB,THARAYIL N,et al.Detecting annual bluegrass(Poa annua)resistance to ALS-inhibiring herbicides using a rapid diagnostic assay[J].Weed Sci,2013,61(3):384-389.
[15]VEGA T,BRECCIA G,GIL M,et al.Acetohydroxyacid synthase(AHAS)in vivo assay for screening imidazolinone-resistance in sun-:flower(Helianthus annuus L.)[J].Plant Physiol Biochem,2012,61:103-107.
[16]LI YD,DONG X,HE S,et al.Effects of pyroxsulam on acetolactate aynthetase activity of main wheat varieties in Xinjiang[J].Acta Agriculturae Boreali-occidentalis Sinica,2017,26(4):560-567.(in Chinese)
[17]LIU W,WANG JX,YANG GL,et al.Difference of tolerance and mechanism of various wheat varieties to tribenuron-methyl[J].Journal of Plant Protection,2005,32(3):300-304.(in Chinese)
[18]YAO X,WANG YQ YUE XP,et al.Generation of tribenuron-methyl herbicide-resistant Os CYP81A6-expressing rapeseed(Brassica napus L.)plants for hybrid seed production using chemical-induced male sterility[J].Plant Breeding,2016,135(3):349-354.
[19]LI T,LIU B,CHEN CY,et al.TALEN-mediated homologous recombination produces site-directed DNA base change and herbicide-resistant rice[J].J Genet Genom,2016,43(5):297-305.
[20]BRECCIA G,VEGA T,FELITTI SA,et al.Differential expression of acetohydroxyacid synthase genes in sunflower plantlets and its response to imazapyr herbicide[J].Plant Sci,2013,208:28-33.
[21]FAN ZJ,CHEN JP,DANG HB,et al.Effect of monosulfuron on the activity of its target enzyme acetolactate synthase[J].Modern Agrochemicals,2003,2(2):16-17.(in Chinese)