草莓生长素合成限速酶FaYUC11基因的克隆和功能分析
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摘要
【目的】克隆草莓YUC家族新基因,探究其在草莓中的具体调控作用。【方法】以‘久香’草莓为材料,利用同源克隆技术克隆草莓果实大小调控相关的新基因,利用病毒诱导基因沉默(virus-induced gene silencing,VIGS)结合定量RT-PCR技术,分析新基因在草莓果实膨大调控中的作用。【结果】从‘久香’草莓果实中克隆到1个草莓果实大小调控相关的新基因,命名为FaYUC11(Gen Bank登录号:JX417083.1),用草莓幼果微注射法建立了病毒诱导FaYUC11基因沉默的转基因草莓体系,结果发现,FaYUC11基因沉默后果实的膨大和正常生长均受到影响,并且导致果实瘦果(种子)中游离生长素含量下降、果实纵横径增长率降低。【结论】FaYUC11基因是草莓生长素合成途径中的关键基因,该基因通过调控生长素的合成继而调控果实的膨大。
【Objective】Cloning and functional verification of a new crucial regulator gene in strawberry auxin biosynthesis.【Methods】The FaYUC11 for fruit enlargement was isolated from the‘Jiuxiang'cultivated strawberry(Fragaria×ananassa‘Jiuxiang') by using RT-PCR(reverse transcription PCR), and then the function of FaYUC11 in strawberry fruit was analyzed by VIGS(virus-induced gene silencing)technology to complete our investigation. The total RNA of the strawberry fruit was extracted by CTAB(Hexadecyl trimethyl ammonium Bromide). Specific primers were designed according to the homologous gene sequences of F. vesca for PCR amplification. After the PCR products were verified by sequencing, they were then compared with the YUC gene family members, and according to the comparison results, specific areas were selected close to the 5'end of FaYUC11 to design the interference primers, using the plasmid with FaYUC11 full-sequence as the template for amplification. Then the products were integrated into the virus RNA plasmid p TRV2, in order to obtain a p TRV2-d YUC11 virus induced gene silencing carrier. The infection vector p TRV2-d YUC11 was constructed and transformed into the Agrobacterium strain GV3101. The resultant Agrobacterium was microinjected into the strawberry fruit to induce gene silencing. 34 days after infection, an analysis was done of each index of the virus induced gene silencing strawberries. The expression of FaYUC11 was analyzed using q RT-PCR(Quantita-tive Real-time PCR), the content of the free IAA in the strawberry achenes(seeds) was detected by GCMS, and the phenotypic changes of the strawberry fruits infected by p TRV2-YUC11 were analyzed, and then compared with the percentage increase of the longitudinal and traverse diameters of the strawberry fruits.【Results】Compared with wild type strawberry fruits, the expression of fruits with the treatment of the p TRV2 empty vector was 1.13, while the expression of fruits with the p TRV2-YUC11 infection were 0.18 and 0.44. The results of q RT-PCR showed that, the FaYUC11 decreased in transcript accumulation levels with different degrees in the strawberry fruits infected with p TRV2-YUC11. The content of the free IAA in the strawberry achenes(seeds) was detected by GC-MS, the results showed that, compared with the group that were treated with the p TRV2 empty vector(515.2 ng· g-1), it significantly decreased in the fruits infected by p TRV2-YUC11(64.86 ng· g-1, 307.19 ng· g-1), which indicated that FaYUC11 is a significant regulator for IAA accumulation. Phenotypic change analysis of the strawberry fruits at 34 days after infection showed that there were mildly sunken at the top of the fruits that were treated with p TRV2 empty vector, but it did not affect fruit enlargement and its morphology, while the fruits infected by p TRV2-YUC11 were significantly suppressed, with some of the fruits enlargement of inhibition being minor(Ri YUC11-3), but part of the fruits were severely inhibited(Ri YUC11-2), with little change showing after 34 days growth, with some still being in the small green fruit stage. Furthermore, by analyzing the percentage increase of longitudinal and traverse diameters of the strawberry fruits found, compared with the wild type fruits, there was a significant decrease in the fruits infected by p TRV2-YUC11. This indicates that FaYUC11 regulates the increase of longitudinal and traverse diameters of strawberry fruits.【Conclusion】The lack of FaYUC11 led to free IAA content in strawberry achenes(seeds) and the percentage increase of the longitudinal and traverse diameters of strawberry fruits were reduced, and at the same time, this influenced the fruit enlargement and normal growth. It indicated that FaYUC11 plays a significantly important role in the regulation of strawberry fruit size. This provides good application prospects in the regulatory functions of strawberry fruit size and has great significance in the development of molecular markers for functional SNP and large strawberry fruit breeding.
【Objective】Cloning and functional verification of a new crucial regulator gene in strawberry auxin biosynthesis.【Methods】The FaYUC11 for fruit enlargement was isolated from the‘Jiuxiang'cultivated strawberry(Fragaria×ananassa‘Jiuxiang') by using RT-PCR(reverse transcription PCR), and then the function of FaYUC11 in strawberry fruit was analyzed by VIGS(virus-induced gene silencing)technology to complete our investigation. The total RNA of the strawberry fruit was extracted by CTAB(Hexadecyl trimethyl ammonium Bromide). Specific primers were designed according to the homologous gene sequences of F. vesca for PCR amplification. After the PCR products were verified by sequencing, they were then compared with the YUC gene family members, and according to the comparison results, specific areas were selected close to the 5'end of FaYUC11 to design the interference primers, using the plasmid with FaYUC11 full-sequence as the template for amplification. Then the products were integrated into the virus RNA plasmid p TRV2, in order to obtain a p TRV2-d YUC11 virus induced gene silencing carrier. The infection vector p TRV2-d YUC11 was constructed and transformed into the Agrobacterium strain GV3101. The resultant Agrobacterium was microinjected into the strawberry fruit to induce gene silencing. 34 days after infection, an analysis was done of each index of the virus induced gene silencing strawberries. The expression of FaYUC11 was analyzed using q RT-PCR(Quantita-tive Real-time PCR), the content of the free IAA in the strawberry achenes(seeds) was detected by GCMS, and the phenotypic changes of the strawberry fruits infected by p TRV2-YUC11 were analyzed, and then compared with the percentage increase of the longitudinal and traverse diameters of the strawberry fruits.【Results】Compared with wild type strawberry fruits, the expression of fruits with the treatment of the p TRV2 empty vector was 1.13, while the expression of fruits with the p TRV2-YUC11 infection were 0.18 and 0.44. The results of q RT-PCR showed that, the FaYUC11 decreased in transcript accumulation levels with different degrees in the strawberry fruits infected with p TRV2-YUC11. The content of the free IAA in the strawberry achenes(seeds) was detected by GC-MS, the results showed that, compared with the group that were treated with the p TRV2 empty vector(515.2 ng· g-1), it significantly decreased in the fruits infected by p TRV2-YUC11(64.86 ng· g-1, 307.19 ng· g-1), which indicated that FaYUC11 is a significant regulator for IAA accumulation. Phenotypic change analysis of the strawberry fruits at 34 days after infection showed that there were mildly sunken at the top of the fruits that were treated with p TRV2 empty vector, but it did not affect fruit enlargement and its morphology, while the fruits infected by p TRV2-YUC11 were significantly suppressed, with some of the fruits enlargement of inhibition being minor(Ri YUC11-3), but part of the fruits were severely inhibited(Ri YUC11-2), with little change showing after 34 days growth, with some still being in the small green fruit stage. Furthermore, by analyzing the percentage increase of longitudinal and traverse diameters of the strawberry fruits found, compared with the wild type fruits, there was a significant decrease in the fruits infected by p TRV2-YUC11. This indicates that FaYUC11 regulates the increase of longitudinal and traverse diameters of strawberry fruits.【Conclusion】The lack of FaYUC11 led to free IAA content in strawberry achenes(seeds) and the percentage increase of the longitudinal and traverse diameters of strawberry fruits were reduced, and at the same time, this influenced the fruit enlargement and normal growth. It indicated that FaYUC11 plays a significantly important role in the regulation of strawberry fruit size. This provides good application prospects in the regulatory functions of strawberry fruit size and has great significance in the development of molecular markers for functional SNP and large strawberry fruit breeding.
引文
[1]THIMANN K V,KOEPFLI J B.Identity of the growth-promoting and root-forming substances of plants[J].Nature,1935,135(4):101-102.
[2]伍涛.丰水、鸭梨果实发育过程中糖积累特性及其与果实大小关系研究[D].南京:南京农业大学,2011.WU Tao.The characteristic of sugar accumulation and its relationship with fruit size during the fruit development in‘Hosui’and‘Yali’pear.[D].Nanjing:Nanjing Agricultural University,2011.
[3]NITSCH J P.Free auxins and free tryptophane in the strawberry[J].Plant Physiology,1955,30(1):33-39.
[4]LIU H,XIE W F,ZHANG L,VALPUESTA V,YE Z W,GAO Q H,DUAN K.Auxin biosynthesis by the YUCCA6 flavin monooxygenase gene in woodland strawberry(Fragaria vesca)[J].Journal of Integrative Plant Biology,2014,56(4):350-363.
[5]QUITTENDEN L J,DAVIES N W,SMITH J A,MOLESWORTH P P,TIVENDALE N D,ROSS J J.Auxin biosynthesis in pea:characterization of the tryptamine pathway[J].Plant Physiology,2009,151(3):1130-1138.
[6]WON C,SHEN X L,MASHIGUCHI K,ZHENG Z Y,DAI X H,CHENG Y F,KASAHARA H,KAMIYA Y,CHORY J,ZHAO Y D.Conversion of tryptophan to indole-3-acetic acid by TRYPTOPHAN AMINOTRANSFERASES OF ARABIDOPSIS and YUCCAs in Arabidopsis[J].Proceedings of the National Academy of Sciences,2011,108(45):18518-18523.
[7]ZHAO Y D,HULL A K,GUPTA N R,GOSS K A,ALONSO J,ECKER J R,NORMANLY J,CHORY J,CELENZA J L.Trp-dependent auxin biosynthesis in Arabidopsis:involvement of cytochrome P450s CYP79B2 and CYP79B3[J].Genes&Development,2002,16(23):3100-3112.
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[9]SUGAWARA S,HISHIYAMA S,JIKUMURA Y,HANADA A,NISHIMURA T,KOSHIBA T,KASAHARA H.Biochemical analyses of indole-3-acetaldoxime-dependent auxin biosynthesis in Arabidopsis[J].Proceedings of the National Academy of Sciences,2009,106(13):5430-5435.
[10]COHEN J D,SLOVIN J P,HENDRICKSON A M.Two genetically discrete pathways convert tryptophan to auxin:more redundancy in auxin biosynthesis[J].Trends in Plant Science,2003,8(5):197-199.
[11]KHASIN M,CAHOON R R,NICKERSON K W,RIEKHOF W R.Molecular machinery of auxin synthesis,secretion,and perception in the unicellular chlorophyte alga Chlorella sorokiniana UTEX 1230[J].Bio Rxiv,2017,8:172833.
[12]EKLUND D M,ISHIZAKI K,FLORES-SANDOVAL E,KIKUCHI S,TAKEBAYASHI Y,TSUKAMOTO S,BHALERAO R P.Auxin produced by the indole-3-pyruvic acid pathway regulates development and gemmae dormancy in the liverwort Marchantia polymorpha[J].The Plant Cell,2015,27(6):1650-1669.
[13]MASHIGUCHI K,TANAKA K,SAKAI T,SUGAWARA S,KAWAIDEH,NATSUME M,MCSTEEN P.The main auxin biosynthesis pathway in Arabidopsis[J].Proceedings of the National Academy of Sciences,2011,108(45):18512-18517.
[14]ABU-ZAITOON Y M,BENNETT K,NORMANLY J,NONHEBEL H M.A large increase in IAA during development of rice grains correlates with the expression of tryptophan aminotransferase Os TAR1 and a grain-specific YUCCA[J].Physiologia Plantarum,2012,146(4):487-499.
[15]STEPANOVA A N,YUN J,ROBLES L M,NOVAK O,HE W,GUO H,LJUNG K,ALONSO J M.The Arabidopsis YUCCA1flavin monooxygenase functions in the indole-3-pyruvic acid branch of auxin biosynthesis[J].The Plant Cell,2011,23(11):3961-3973.
[16]ZHAO Y D,CHRISTENSEN S K,FANKHAUSER C,CASHMAN J R,COHEN J D,WEIGEL D,CHORY J.A role for flavin monooxygenase-like enzymes in auxin biosynthesis[J].Science,2001,291(5502):306-309.
[17]YAMAMOTO Y,KAMIYA N,MORINAKA Y,MATSUOKA M,SAZUKA T.Auxin biosynthesis by the YUCCA genes in rice[J].Plant Physiology,2007,143(3):1362-1371.
[18]EXPóSITO R M,BOREGS A A,BORGES P A,PéREZ J A.Gene structure and spatiotemporal expression profile of tomato genes encoding YUCCA-like flavin monooxygenases:The To FZY gene family[J].Plant Physiology and Biochemistry,2011,49(7):782-791.
[19]GALLAVOTTI A,BARAZESH S,MALCOMBER S,HALL D,JACKSON D,SCHMIDT R J,MCSTEEN P.Sparse in fl orescence1 encodes a monocot-specific YUCCA-like gene required for vegetative and reproductive development in maize[J].Proceedings of the National Academy of Sciences,2008,105:15196-15201.
[20]YE X,KANG B,OSBURN L D,LI Y,MAX C Z M.Identification of the flavin-dependent monooxygenase encoding YUCCA gene family in Populus trichocarpa and their expression in vegetative tissues and in response to hormone and environmental stresses[J].Plant Cell,Tissue and Organ Culture,2009,97(3):271-283.
[21]JIA H F,CHAI Y M,LI C L,LU D,LUO J J,QIN L,SHEN Y Y.Abscisic acid plays an important role in the regulation of strawberry fruit ripening[J].Plant Physiology,2011,157(1):188-199.
[22]BEYENE G,CHAUHAN R D,TAYLOR N J.A rapid virus-induced gene silencing(VIGS)method for assessing resistance and susceptibility to cassava mosaic disease[J].Virology Journal,2017,14(1):47.
[23]LANGE M,YELLINA A L,ORASHAKOVA S,BECKER A.Virus-induced gene silencing(VIGS)in plants:an overview of target species and the virus-derived vector systems[J].Methods in Molecular Biology,2013,975(5):1-14.
[24]PURKAYASTHA A,DASGUPTA I.Virus-induced gene silencing:a versatile tool for discovery of gene functions in plants[J].Plant Physiology and Biochemistry,2009,47(11):967-976.
[25]LIU Y,SCHIFF M,DINESH-KUMAR S P.Virus-induced gene silencing in tomato[J].The Plant Journal,2002,31(6):777-786.
[26]JIA H F,CHAI Y M,LI C L,LU D,LUO J J,QIN L,SHEN Y Y.Abscisic acid plays an important role in the regulation of strawberry fruit ripening[J].Plant Physiology,2011,157(1):188-199.
[27]EDLUND A,EKLOF S,SUNDBERG B,MORITZ T,SANDBERG G.A microscale technique for gas chromatography-mass spectrometry measurements of picogram amounts of indole-3-acetic acid in plant tissues[J].Plant Physiology,1995,108(3):1043-1047.
[28]KOCH M,VORWERK S,MASUR C,SHARIFI-SIRCHI G,OLIVIERI N,SCHLAICH N L.A role for a flavin-containing monooxygenase in resistance against microbial pathogens in Arabidopsis[J].The Plant Journal,2006,47(4):629-639.
[29]CHENG Y,DAI X,ZHAO Y.Auxin synthesized by the YUCCA flavin monooxygenases is essential for embryogenesis and leaf formation in Arabidopsis[J].The Plant Cell,2007,19(8):2430-2439.
[2]伍涛.丰水、鸭梨果实发育过程中糖积累特性及其与果实大小关系研究[D].南京:南京农业大学,2011.WU Tao.The characteristic of sugar accumulation and its relationship with fruit size during the fruit development in‘Hosui’and‘Yali’pear.[D].Nanjing:Nanjing Agricultural University,2011.
[3]NITSCH J P.Free auxins and free tryptophane in the strawberry[J].Plant Physiology,1955,30(1):33-39.
[4]LIU H,XIE W F,ZHANG L,VALPUESTA V,YE Z W,GAO Q H,DUAN K.Auxin biosynthesis by the YUCCA6 flavin monooxygenase gene in woodland strawberry(Fragaria vesca)[J].Journal of Integrative Plant Biology,2014,56(4):350-363.
[5]QUITTENDEN L J,DAVIES N W,SMITH J A,MOLESWORTH P P,TIVENDALE N D,ROSS J J.Auxin biosynthesis in pea:characterization of the tryptamine pathway[J].Plant Physiology,2009,151(3):1130-1138.
[6]WON C,SHEN X L,MASHIGUCHI K,ZHENG Z Y,DAI X H,CHENG Y F,KASAHARA H,KAMIYA Y,CHORY J,ZHAO Y D.Conversion of tryptophan to indole-3-acetic acid by TRYPTOPHAN AMINOTRANSFERASES OF ARABIDOPSIS and YUCCAs in Arabidopsis[J].Proceedings of the National Academy of Sciences,2011,108(45):18518-18523.
[7]ZHAO Y D,HULL A K,GUPTA N R,GOSS K A,ALONSO J,ECKER J R,NORMANLY J,CHORY J,CELENZA J L.Trp-dependent auxin biosynthesis in Arabidopsis:involvement of cytochrome P450s CYP79B2 and CYP79B3[J].Genes&Development,2002,16(23):3100-3112.
[8]TROMAS A,PERROT-RECHENMANN C.Recent progress in auxin biology[J].Comptes Rendus Biologies,2010,333(4):297-306.
[9]SUGAWARA S,HISHIYAMA S,JIKUMURA Y,HANADA A,NISHIMURA T,KOSHIBA T,KASAHARA H.Biochemical analyses of indole-3-acetaldoxime-dependent auxin biosynthesis in Arabidopsis[J].Proceedings of the National Academy of Sciences,2009,106(13):5430-5435.
[10]COHEN J D,SLOVIN J P,HENDRICKSON A M.Two genetically discrete pathways convert tryptophan to auxin:more redundancy in auxin biosynthesis[J].Trends in Plant Science,2003,8(5):197-199.
[11]KHASIN M,CAHOON R R,NICKERSON K W,RIEKHOF W R.Molecular machinery of auxin synthesis,secretion,and perception in the unicellular chlorophyte alga Chlorella sorokiniana UTEX 1230[J].Bio Rxiv,2017,8:172833.
[12]EKLUND D M,ISHIZAKI K,FLORES-SANDOVAL E,KIKUCHI S,TAKEBAYASHI Y,TSUKAMOTO S,BHALERAO R P.Auxin produced by the indole-3-pyruvic acid pathway regulates development and gemmae dormancy in the liverwort Marchantia polymorpha[J].The Plant Cell,2015,27(6):1650-1669.
[13]MASHIGUCHI K,TANAKA K,SAKAI T,SUGAWARA S,KAWAIDEH,NATSUME M,MCSTEEN P.The main auxin biosynthesis pathway in Arabidopsis[J].Proceedings of the National Academy of Sciences,2011,108(45):18512-18517.
[14]ABU-ZAITOON Y M,BENNETT K,NORMANLY J,NONHEBEL H M.A large increase in IAA during development of rice grains correlates with the expression of tryptophan aminotransferase Os TAR1 and a grain-specific YUCCA[J].Physiologia Plantarum,2012,146(4):487-499.
[15]STEPANOVA A N,YUN J,ROBLES L M,NOVAK O,HE W,GUO H,LJUNG K,ALONSO J M.The Arabidopsis YUCCA1flavin monooxygenase functions in the indole-3-pyruvic acid branch of auxin biosynthesis[J].The Plant Cell,2011,23(11):3961-3973.
[16]ZHAO Y D,CHRISTENSEN S K,FANKHAUSER C,CASHMAN J R,COHEN J D,WEIGEL D,CHORY J.A role for flavin monooxygenase-like enzymes in auxin biosynthesis[J].Science,2001,291(5502):306-309.
[17]YAMAMOTO Y,KAMIYA N,MORINAKA Y,MATSUOKA M,SAZUKA T.Auxin biosynthesis by the YUCCA genes in rice[J].Plant Physiology,2007,143(3):1362-1371.
[18]EXPóSITO R M,BOREGS A A,BORGES P A,PéREZ J A.Gene structure and spatiotemporal expression profile of tomato genes encoding YUCCA-like flavin monooxygenases:The To FZY gene family[J].Plant Physiology and Biochemistry,2011,49(7):782-791.
[19]GALLAVOTTI A,BARAZESH S,MALCOMBER S,HALL D,JACKSON D,SCHMIDT R J,MCSTEEN P.Sparse in fl orescence1 encodes a monocot-specific YUCCA-like gene required for vegetative and reproductive development in maize[J].Proceedings of the National Academy of Sciences,2008,105:15196-15201.
[20]YE X,KANG B,OSBURN L D,LI Y,MAX C Z M.Identification of the flavin-dependent monooxygenase encoding YUCCA gene family in Populus trichocarpa and their expression in vegetative tissues and in response to hormone and environmental stresses[J].Plant Cell,Tissue and Organ Culture,2009,97(3):271-283.
[21]JIA H F,CHAI Y M,LI C L,LU D,LUO J J,QIN L,SHEN Y Y.Abscisic acid plays an important role in the regulation of strawberry fruit ripening[J].Plant Physiology,2011,157(1):188-199.
[22]BEYENE G,CHAUHAN R D,TAYLOR N J.A rapid virus-induced gene silencing(VIGS)method for assessing resistance and susceptibility to cassava mosaic disease[J].Virology Journal,2017,14(1):47.
[23]LANGE M,YELLINA A L,ORASHAKOVA S,BECKER A.Virus-induced gene silencing(VIGS)in plants:an overview of target species and the virus-derived vector systems[J].Methods in Molecular Biology,2013,975(5):1-14.
[24]PURKAYASTHA A,DASGUPTA I.Virus-induced gene silencing:a versatile tool for discovery of gene functions in plants[J].Plant Physiology and Biochemistry,2009,47(11):967-976.
[25]LIU Y,SCHIFF M,DINESH-KUMAR S P.Virus-induced gene silencing in tomato[J].The Plant Journal,2002,31(6):777-786.
[26]JIA H F,CHAI Y M,LI C L,LU D,LUO J J,QIN L,SHEN Y Y.Abscisic acid plays an important role in the regulation of strawberry fruit ripening[J].Plant Physiology,2011,157(1):188-199.
[27]EDLUND A,EKLOF S,SUNDBERG B,MORITZ T,SANDBERG G.A microscale technique for gas chromatography-mass spectrometry measurements of picogram amounts of indole-3-acetic acid in plant tissues[J].Plant Physiology,1995,108(3):1043-1047.
[28]KOCH M,VORWERK S,MASUR C,SHARIFI-SIRCHI G,OLIVIERI N,SCHLAICH N L.A role for a flavin-containing monooxygenase in resistance against microbial pathogens in Arabidopsis[J].The Plant Journal,2006,47(4):629-639.
[29]CHENG Y,DAI X,ZHAO Y.Auxin synthesized by the YUCCA flavin monooxygenases is essential for embryogenesis and leaf formation in Arabidopsis[J].The Plant Cell,2007,19(8):2430-2439.