基因枪法介导的辣椒花药遗传转化技术研究
|
摘要
为建立基因枪法介导的辣椒遗传转化体系,以小孢子胚胎发生率较高的辣椒品种L5的花药为受体,利用基因枪法将绿色荧光蛋白(GFP)基因导入L5花药小孢子中,并进行花药培养诱导小孢子胚胎发生,探讨基因枪法转化辣椒花药小孢子的适宜参数。结果表明,辣椒小孢子处于单核靠边期的花药在9℃下暗培养4 d时为最佳受体;CaMV35S启动子能启动GFP基因在辣椒花药培养的小孢子胚胎中表达;基因枪各轰击参数对小孢子胚胎发生有显著影响(P<0.05),随着基因枪轰击次数和轰击压力的增加以及轰击距离的减小,小孢子胚胎发生数量逐渐减少;最适的轰击参数为基因枪轰击2次、轰击压力1 350 psi和轰击距离9 cm,此时获得的转化胚胎数量最多;在最适的轰击参数下辣椒花药小孢子的平均转化效率为4.2%;体式荧光显微镜下共检测到14个转化胚胎。本研究结果为进一步建立基因枪介导的辣椒花药稳定遗传转化体系提供了一定的理论基础和技术支撑。
In order to establish the genetic transformation system of pepper( Capsicum annuumL.) using particle bombardment,the anthers of high embryogenesis pepper variety ‘L5 'were used as receptors,and the gene of green fluorescent protein( GFP) were transformed into the microspores of anthers. Then the microspore embryogenesis was induced by anther culture,the best parameters of particle bombardment for the genetic transformation of pepper have been preliminarily established. In the study,the best receptors for the particle bombardment were the anthers which were pre-cultured in the dark at 9℃ temperature for four days. The bombarding intensity had significant effect on the embryogenesis from anthers( P < 0. 05). The numbers of embryos were decreased with the increase of bombardment pressure and bombardment times,and decreased with the decrease of target distance. The best parameters of particle bombardment were shooting two times with 1350 psi rupture pressure and target distance of 9 cm. With which average transformation efciency was 4. 2%,and fourteen transformation embryos were identified using the Leica-microscope,but no transformation plants were obtained in this study. These results could be used in the stable genetic transformation of pepper using particle bombardment in the future.
In order to establish the genetic transformation system of pepper( Capsicum annuumL.) using particle bombardment,the anthers of high embryogenesis pepper variety ‘L5 'were used as receptors,and the gene of green fluorescent protein( GFP) were transformed into the microspores of anthers. Then the microspore embryogenesis was induced by anther culture,the best parameters of particle bombardment for the genetic transformation of pepper have been preliminarily established. In the study,the best receptors for the particle bombardment were the anthers which were pre-cultured in the dark at 9℃ temperature for four days. The bombarding intensity had significant effect on the embryogenesis from anthers( P < 0. 05). The numbers of embryos were decreased with the increase of bombardment pressure and bombardment times,and decreased with the decrease of target distance. The best parameters of particle bombardment were shooting two times with 1350 psi rupture pressure and target distance of 9 cm. With which average transformation efciency was 4. 2%,and fourteen transformation embryos were identified using the Leica-microscope,but no transformation plants were obtained in this study. These results could be used in the stable genetic transformation of pepper using particle bombardment in the future.
引文
[1]李锡香,张宝玺.辣椒种质资源描述规范和数据标准[M].北京:中国农业出版社,2006:1-9
[2]HeidmannⅠ,Boutilier K.Pepper,sweet(Capsicum annuum)[J].Methods in Molecular Biology,2015,1223:321-334
[3]Aarrouf J,Castro-quezada P,Mallard S,Caromel B,Lizzi Y,LefebvreⅤ.Agrobacterium rhizogenes-dependent production of transformed roots from foliar explants of pepper(Capsicum annuum):a new and efficient tool for functional analysis of genes[J].Plant Cell Reports,2012,31(2):391-401
[4]Bulle M,Yarra R,Abbagani S.Enhanced salinity stress tolerance in transgenic chilli pepper(Capsicum annuum L.)plants overexpressing the wheat antiporter(Ta NHX2)gene[J].Molecular Breeding,2016,36(4):1-12
[5]Kim J,Park M,Jeong E S,Lee J M,Choi D.Harnessing anthocyanin-rich fruit:a visible reporter for tracing virus-induced gene silencing in pepper fruit[J].Plant Methods,2017,13(3):1-10
[6]Kumar RⅤ,SharmaⅤK,Chattopadhyay B,Chakraborty S.An improved plant regeneration and agrobacterium-mediated transformation of red pepper(Capsicum annuum L.)[J].Physiology and Molecular Biology of Plants,2012,18(4):357-364
[7]Jung M,Shin S H,Park J M,Lee S N,Lee M Y,Ryu K H,Paek K Y,Harn C H.Detection of transgene in early developmental stage by GFP monitoring enhances the efficiency of genetic transformation of pepper[J].Plant Biotechnology Reports,2011,5(2):157-167
[8]Kim Y S,Kuk YⅠ,Kim K M.Inheritance and expression of transgenes through anther culture of transgenic hot pepper[J].Zeitschrift Fur Naturforschung C,2007,62(9/10):743-746
[9]Kim D S,Choi H W,Hwang B K.Pepper mildew resistance locus O interacts with pepper calmodulin and suppresses xanthomonas Avr Bs T-triggered cell death and defense responses[J].Planta,2014,240(4):827-39
[10]Ji J,Huang W,Li D,Yin Y,Chai W,Gong Z.A CMS-related gene,[Psi]atp6-2,causes increased ATP hydrolysis activity of the mitochondrial F1F0-ATP synthase and induces male sterility in pepper(Capsicum annuum L.)[J].Plant Molecular Biology Reporter,2014,32(4):888-899
[11]Arthikala M K,Reddy K N,Manjulatha M,Arellano E S,Sreevathsa R,Ganeshan G.A rapid novel and high-throughput identification of putative bell pepper transformants generated through in planta transformation approach[J].Scientia Horticulturae,2011,129(4):898-903
[12]Pozueta-Romero J,HoulnéG,Ca1as L,Schantz R,Chamarro J.Enhanced regeneration of tomato and pepper seedling explants for Agrobacterium-mediated transformation[J].Plant Cell Tissue and Organ Culture,2001,67(2):173-180
[13]李秋庆,陈国菊,曹必好,陈长明,雷建军.农杆菌介导PSAG12-IPT基因转化辣椒的研究[J].辣椒杂志,2015,13(2):23-29
[14]Nie S,Tan C,Huang L,Pan C,Li H,Gong M,Shi J,He W,Huang H,Zhang Z.University Sichuan agricultural files Chinese patent application for high-efficiency genetic transformation and plantlet regeneration method of hot peppers:China,CN106106149(A)[P].2017-12-05
[15]Han Y,Blechl A,Wang D.The distribution of cotransformed transgenes in particle bombardment-mediated transformed wheat[J].Transgenic Research,2015,24(6):1055-1063
[16]Hamada H,Linghu Q,Nagira Y,Miki R,Taoka N,Imai R.An in planta biolistic method for stable wheat transformation[J].Scientific Reports(Nature Publisher Group),2017,7(1):1-8
[17]Belide S,Vanhercke T,Petrie J R,Singh S P.Robust genetic transformation of sorghum(Sorghum bicolor L.)using differentiating embryogenic callus induced from immature embryos[J].Plant Methods,2017,13:109
[18]Visarada K B R S,Prasad G S,Royer M.Genetic transformation and evaluation of two sweet sorghum genotypes for resistance to spotted stemborer Chilo partellus(Swinhoe)[J].Plant Biotechnology Reports,2016,10(5):277-289
[19]Himuro Y,Ishiyama K,Mori F,Gondo T,Takahashi F,Shinozaki K,Kobayashi M,Akashi R.Arabidopsis galactinol synthase At Gol S2 improves drought tolerance in the monocot model Brachypodium distachyon[J].Journal of Plant Physiology,2014,171(13):1127-1131
[20]Chaithra N,Gowda R P H,Guleria N.Transformation of tomato with Cry2ax1 by biolistic gun method for fruit borer resistance[J].International Journal of Agriculture Environment and Biotechnology,2015,8(4):795-803
[21]Sidhu M K,Dhatt A S,Sandhu J S,Gosal S S.Biolistic transformation of cry 1Ac gene in eggplant(Solanum melongena L.)[J].International Journal of Agriculture Environment and Biotechnology,2014,7(4):679-687
[22]NianiouⅠ,Karavangeli M,Zambounis A,Mooibroek H.Development of pepper transgenic plants via Agrobacterium and biolistic transformation[J].Acta Horticulturae,2002,579(2):83-88
[23]刘凡,赵泓,陈斌,张月云.辣椒游离小孢子细胞团培养的胚状体形成[J].分子细胞生物学报,2007(6):371-375
[24]Sánchez-Díaz R A,Castillo A M,Vallés M P.Microspore embryogenesis in wheat:new marker genes for early,middle and late stages of embryo development[J].Plant Reproduction,2013,26(3):287
[25]Soriano M,Li H,Boutilier K.Microspore embryogenesis:establishment of embryo identity and pattern in culture[J].Plant Reproduction,2013,26(3):181-196
[26]Nguyen T D,Moon S,NguyenⅤN,Gho Y,Chandran A K,Soh M S,Song J T,An G,Oh S A,Park S K,Jung K H.Genome-wide identification and analysis of rice genes preferentially expressed in pollen at an early developmental stage[J].Plant Molecular Biology,2016,92(1/2):71-88
[27]杜志钊,高润红,陈志伟,何婷,李梁,郭桂梅,陆瑞菊,黄剑华.利用基因枪轰击花药将GUS基因导入大麦小孢子[J].麦类作物学报,2011,31(6):1025-1029
[28]李菲,张淑江,章时蕃,武剑,王晓武,张慧,孙日飞,张振贤.基因枪法介导大白菜小孢子转基因技术研究初报[J].园艺学报,2017,44(1):62-68
[29]Abdollahi M R,Moieni A,Mousavi A,Salmanian A H.High frequency production of rapeseed transgenic plants via combination of microprojectile bombardment and secondary embryogenesis of microspore-derived embryos[J].Molecular Biology Reports,2011,38(2):711-719
[30]Rustgi S,Ankrah N O,Brew-Appiah R,Sun Y,Liu W,von Wettstein D.Doubled haploid transgenic wheat lines by microspore transformation[M]//Bhalla P L,Singh M B.Weat Biotechnology.New York:Springer,2017:213-234
[31]薛建平,张爱民.不同基因型小麦花药培养过程中药壁变化的研究[J].植物学通报,2002(3):354-358
[32]Verónica PⅤ,Seguí-Simarro J M.Anther culture in pepper(Capsicum annuum L.)[J].Acta Physiologiae Plantarum,2016,1359(5):467
[33]Xia B,Dong C,Zhang W,Lu Y,Chen J,Shi J.Highly efficient uptake of ultrafine mesoporous silica nanoparticles with excellent biocompatibility by liriodendron hybrid suspension cells[J].Science China Life Sciences,2013,56(1):82-89
[34]Jez M,Bas T,Veber M,Kosir A,Dominko T,Page R,Rozman P.The hazards of DAPI photoconversion:effects of dye,mounting media and fixative,and how to minimize the problem[J].Histochemistry and Cell Biology,2013,139(1):195-204
[35]Pechan M P.Successful co-cultivation of Brassica napus microspores and proembryos with Agrobacterium[J].Plant Cell Reports,1989(8):387-390
[36]Folling L,Olesen A.Transformation of wheat(Triticum aestivum L.)microspore-derived callus and microspores by particle bombardment[J].Plant Cell Reports,2002,20:1098-1105
[37]Liu W,Parrott W A,Hildebrand D F,Collins G B,Williams E G.Agrobacterium induced gall formation in bell pepper(Capsicum annuum L.)and formation of shoot-like structures expressing introduced genes[J].Plant Cell Reports,1990,9(7):360-364
[38]Supena E D,Suharsono S,Jacobsen E,Custers J B.Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper(Capsicum annuum L.)[J].Plant Cell Reports,2006,25:1-10
[39]王仲慧,刘金兵,刁卫平,王述彬,潘宝贵,戈伟.辣椒花药培养胚状体发生途径影响因子研究[J].核农学报,2015,29(8):1471-1478
[40]Popova T,Grozeva S,TodorovaⅤ,Stankova G,Anachkov N,RodevaⅤ.Effects of low temperature,genotype and culture media on in vitro androgenic answer of pepper(Capsicum annuum L.)[J].Acta Physiologiae Plantarum,2016,38(11):1-11
[41]Abdollahi M R,Moieni A,Mousavi A,Salmanian A H,Jalali Javaran M,Majdi M.Effect of integrated bombardment and Agrobacterium transformation system on transient GUS expression in hypocotyls of rapeseed(Brassica napus L.cv.PF704)microsporederived embryos[J].Pakistan Journal of Biological Sciences,2007,10(18):3141-3145
[42]杨国顺,谢丙炎,杨宇红,王晓武,卢向阳,蒋芳玲.应用绿色荧光蛋白报告基因优化辣椒的遗传转化体系[J].园艺学报,2004(6):737-742
[43]Kumar A M,Reddy K N,Sreevathsa R,Ganeshan G,Udayakumar M.Towards crop improvement in bell pepper(Capsicum annuum L.):Transgenics(uid A:hptⅡ)by a tissue-culture-independent Agrobacterium-mediated in planta approach[J].Scientia Horticulturae,2009,119(4):362-370
[2]HeidmannⅠ,Boutilier K.Pepper,sweet(Capsicum annuum)[J].Methods in Molecular Biology,2015,1223:321-334
[3]Aarrouf J,Castro-quezada P,Mallard S,Caromel B,Lizzi Y,LefebvreⅤ.Agrobacterium rhizogenes-dependent production of transformed roots from foliar explants of pepper(Capsicum annuum):a new and efficient tool for functional analysis of genes[J].Plant Cell Reports,2012,31(2):391-401
[4]Bulle M,Yarra R,Abbagani S.Enhanced salinity stress tolerance in transgenic chilli pepper(Capsicum annuum L.)plants overexpressing the wheat antiporter(Ta NHX2)gene[J].Molecular Breeding,2016,36(4):1-12
[5]Kim J,Park M,Jeong E S,Lee J M,Choi D.Harnessing anthocyanin-rich fruit:a visible reporter for tracing virus-induced gene silencing in pepper fruit[J].Plant Methods,2017,13(3):1-10
[6]Kumar RⅤ,SharmaⅤK,Chattopadhyay B,Chakraborty S.An improved plant regeneration and agrobacterium-mediated transformation of red pepper(Capsicum annuum L.)[J].Physiology and Molecular Biology of Plants,2012,18(4):357-364
[7]Jung M,Shin S H,Park J M,Lee S N,Lee M Y,Ryu K H,Paek K Y,Harn C H.Detection of transgene in early developmental stage by GFP monitoring enhances the efficiency of genetic transformation of pepper[J].Plant Biotechnology Reports,2011,5(2):157-167
[8]Kim Y S,Kuk YⅠ,Kim K M.Inheritance and expression of transgenes through anther culture of transgenic hot pepper[J].Zeitschrift Fur Naturforschung C,2007,62(9/10):743-746
[9]Kim D S,Choi H W,Hwang B K.Pepper mildew resistance locus O interacts with pepper calmodulin and suppresses xanthomonas Avr Bs T-triggered cell death and defense responses[J].Planta,2014,240(4):827-39
[10]Ji J,Huang W,Li D,Yin Y,Chai W,Gong Z.A CMS-related gene,[Psi]atp6-2,causes increased ATP hydrolysis activity of the mitochondrial F1F0-ATP synthase and induces male sterility in pepper(Capsicum annuum L.)[J].Plant Molecular Biology Reporter,2014,32(4):888-899
[11]Arthikala M K,Reddy K N,Manjulatha M,Arellano E S,Sreevathsa R,Ganeshan G.A rapid novel and high-throughput identification of putative bell pepper transformants generated through in planta transformation approach[J].Scientia Horticulturae,2011,129(4):898-903
[12]Pozueta-Romero J,HoulnéG,Ca1as L,Schantz R,Chamarro J.Enhanced regeneration of tomato and pepper seedling explants for Agrobacterium-mediated transformation[J].Plant Cell Tissue and Organ Culture,2001,67(2):173-180
[13]李秋庆,陈国菊,曹必好,陈长明,雷建军.农杆菌介导PSAG12-IPT基因转化辣椒的研究[J].辣椒杂志,2015,13(2):23-29
[14]Nie S,Tan C,Huang L,Pan C,Li H,Gong M,Shi J,He W,Huang H,Zhang Z.University Sichuan agricultural files Chinese patent application for high-efficiency genetic transformation and plantlet regeneration method of hot peppers:China,CN106106149(A)[P].2017-12-05
[15]Han Y,Blechl A,Wang D.The distribution of cotransformed transgenes in particle bombardment-mediated transformed wheat[J].Transgenic Research,2015,24(6):1055-1063
[16]Hamada H,Linghu Q,Nagira Y,Miki R,Taoka N,Imai R.An in planta biolistic method for stable wheat transformation[J].Scientific Reports(Nature Publisher Group),2017,7(1):1-8
[17]Belide S,Vanhercke T,Petrie J R,Singh S P.Robust genetic transformation of sorghum(Sorghum bicolor L.)using differentiating embryogenic callus induced from immature embryos[J].Plant Methods,2017,13:109
[18]Visarada K B R S,Prasad G S,Royer M.Genetic transformation and evaluation of two sweet sorghum genotypes for resistance to spotted stemborer Chilo partellus(Swinhoe)[J].Plant Biotechnology Reports,2016,10(5):277-289
[19]Himuro Y,Ishiyama K,Mori F,Gondo T,Takahashi F,Shinozaki K,Kobayashi M,Akashi R.Arabidopsis galactinol synthase At Gol S2 improves drought tolerance in the monocot model Brachypodium distachyon[J].Journal of Plant Physiology,2014,171(13):1127-1131
[20]Chaithra N,Gowda R P H,Guleria N.Transformation of tomato with Cry2ax1 by biolistic gun method for fruit borer resistance[J].International Journal of Agriculture Environment and Biotechnology,2015,8(4):795-803
[21]Sidhu M K,Dhatt A S,Sandhu J S,Gosal S S.Biolistic transformation of cry 1Ac gene in eggplant(Solanum melongena L.)[J].International Journal of Agriculture Environment and Biotechnology,2014,7(4):679-687
[22]NianiouⅠ,Karavangeli M,Zambounis A,Mooibroek H.Development of pepper transgenic plants via Agrobacterium and biolistic transformation[J].Acta Horticulturae,2002,579(2):83-88
[23]刘凡,赵泓,陈斌,张月云.辣椒游离小孢子细胞团培养的胚状体形成[J].分子细胞生物学报,2007(6):371-375
[24]Sánchez-Díaz R A,Castillo A M,Vallés M P.Microspore embryogenesis in wheat:new marker genes for early,middle and late stages of embryo development[J].Plant Reproduction,2013,26(3):287
[25]Soriano M,Li H,Boutilier K.Microspore embryogenesis:establishment of embryo identity and pattern in culture[J].Plant Reproduction,2013,26(3):181-196
[26]Nguyen T D,Moon S,NguyenⅤN,Gho Y,Chandran A K,Soh M S,Song J T,An G,Oh S A,Park S K,Jung K H.Genome-wide identification and analysis of rice genes preferentially expressed in pollen at an early developmental stage[J].Plant Molecular Biology,2016,92(1/2):71-88
[27]杜志钊,高润红,陈志伟,何婷,李梁,郭桂梅,陆瑞菊,黄剑华.利用基因枪轰击花药将GUS基因导入大麦小孢子[J].麦类作物学报,2011,31(6):1025-1029
[28]李菲,张淑江,章时蕃,武剑,王晓武,张慧,孙日飞,张振贤.基因枪法介导大白菜小孢子转基因技术研究初报[J].园艺学报,2017,44(1):62-68
[29]Abdollahi M R,Moieni A,Mousavi A,Salmanian A H.High frequency production of rapeseed transgenic plants via combination of microprojectile bombardment and secondary embryogenesis of microspore-derived embryos[J].Molecular Biology Reports,2011,38(2):711-719
[30]Rustgi S,Ankrah N O,Brew-Appiah R,Sun Y,Liu W,von Wettstein D.Doubled haploid transgenic wheat lines by microspore transformation[M]//Bhalla P L,Singh M B.Weat Biotechnology.New York:Springer,2017:213-234
[31]薛建平,张爱民.不同基因型小麦花药培养过程中药壁变化的研究[J].植物学通报,2002(3):354-358
[32]Verónica PⅤ,Seguí-Simarro J M.Anther culture in pepper(Capsicum annuum L.)[J].Acta Physiologiae Plantarum,2016,1359(5):467
[33]Xia B,Dong C,Zhang W,Lu Y,Chen J,Shi J.Highly efficient uptake of ultrafine mesoporous silica nanoparticles with excellent biocompatibility by liriodendron hybrid suspension cells[J].Science China Life Sciences,2013,56(1):82-89
[34]Jez M,Bas T,Veber M,Kosir A,Dominko T,Page R,Rozman P.The hazards of DAPI photoconversion:effects of dye,mounting media and fixative,and how to minimize the problem[J].Histochemistry and Cell Biology,2013,139(1):195-204
[35]Pechan M P.Successful co-cultivation of Brassica napus microspores and proembryos with Agrobacterium[J].Plant Cell Reports,1989(8):387-390
[36]Folling L,Olesen A.Transformation of wheat(Triticum aestivum L.)microspore-derived callus and microspores by particle bombardment[J].Plant Cell Reports,2002,20:1098-1105
[37]Liu W,Parrott W A,Hildebrand D F,Collins G B,Williams E G.Agrobacterium induced gall formation in bell pepper(Capsicum annuum L.)and formation of shoot-like structures expressing introduced genes[J].Plant Cell Reports,1990,9(7):360-364
[38]Supena E D,Suharsono S,Jacobsen E,Custers J B.Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper(Capsicum annuum L.)[J].Plant Cell Reports,2006,25:1-10
[39]王仲慧,刘金兵,刁卫平,王述彬,潘宝贵,戈伟.辣椒花药培养胚状体发生途径影响因子研究[J].核农学报,2015,29(8):1471-1478
[40]Popova T,Grozeva S,TodorovaⅤ,Stankova G,Anachkov N,RodevaⅤ.Effects of low temperature,genotype and culture media on in vitro androgenic answer of pepper(Capsicum annuum L.)[J].Acta Physiologiae Plantarum,2016,38(11):1-11
[41]Abdollahi M R,Moieni A,Mousavi A,Salmanian A H,Jalali Javaran M,Majdi M.Effect of integrated bombardment and Agrobacterium transformation system on transient GUS expression in hypocotyls of rapeseed(Brassica napus L.cv.PF704)microsporederived embryos[J].Pakistan Journal of Biological Sciences,2007,10(18):3141-3145
[42]杨国顺,谢丙炎,杨宇红,王晓武,卢向阳,蒋芳玲.应用绿色荧光蛋白报告基因优化辣椒的遗传转化体系[J].园艺学报,2004(6):737-742
[43]Kumar A M,Reddy K N,Sreevathsa R,Ganeshan G,Udayakumar M.Towards crop improvement in bell pepper(Capsicum annuum L.):Transgenics(uid A:hptⅡ)by a tissue-culture-independent Agrobacterium-mediated in planta approach[J].Scientia Horticulturae,2009,119(4):362-370