芥菜型油菜黄籽候选基因BjuA.YSC表达载体构建和遗传转化
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摘要
研究表明,芥菜型油菜黄籽形成涉及类黄酮合成基因。初步研究表明芥菜型油菜种皮颜色黄黑籽差异的形成受到MYB类转录因子、bHLH类转录因子、WD40蛋白等协同调控,并且不断发现有新的调控基因影响原花色素的合成,如KAN4等转录因子。本研究以本实验室克隆的芥菜型油菜A9染色体上黄籽候选基因BjuA.YSC为研究对象,通过互补试验研究BjuA.YSC基因的功能,从而揭示油菜黄籽性状形成的分子机制,为甘蓝型黄籽油菜育种提供科学依据。
本研究构建了含黄籽候选基因的表达载体,以芥菜型油菜四川黄籽无菌苗的下胚轴为外植体,利用农杆菌介导法进行转化,抗性筛选和PCR分析鉴定目的基因已整合至受体植株中。试验结果如下：
1、通过酶切、连接等分子克隆技术,首次将BjuA.YSC整合入pBI121载体中,构建成表达载体pBI-BjuA.YSC。
2、建立了以下胚轴为外植体、农杆菌介导法的芥菜型油菜遗传转化体系。
3、从6400个芥菜型油菜四川黄籽下胚轴外植体中,共获得抗卡那霉素的再生苗24株,经PCR检测证实有外源基因整合的阳性植株为3株,转化效率为0.046%。3株转基因植株发育正常,能开花、结实,但T0代植株所结种子仍为黄色。
The yellow seed is showed to be associated with the expression of the genes for flavonoid synthesis in Brassicajuncea. It is suggested that the transcription factors MYB、 bHLH and WD40are the key factors responsible for difference in seed coat color. In this study, the gene BjuA.YSC our lab has cloned from Brassica juncea was used to study its function by complementation, which will provide the foundations for molecular mechanism of the yellow-seeded trait.
Expression vector of the gene BjuA. YSC was constructed and introduced into the Agrobacterium tumefaciens strain EHA105by the freeze-thaw method in this study. The hypocotyls from aseptic seedlings of B. juncea cv. Sichuan Yellow were used as explants for transformation. The main results are as follows:
1. The expression vector pBl-BjuA. YSC was constructed and transferred to competent E. coli cells for amplification.
2. The transformation system based on hypocotyl explants and Agrobacterium tumefaciens strain EHA105was established for the species Brassicajuncea.
3. The24kanamycin-resistant plantlets were regenerated from6400explants and three transgenic plants confirmed by PCR analysis. The transformation rate was estimated to be0.046%. The transgenic plants developed normally, flowered and set fruits and seeds. However, their seeds remained yellow like the untransformed parent.
本研究构建了含黄籽候选基因的表达载体,以芥菜型油菜四川黄籽无菌苗的下胚轴为外植体,利用农杆菌介导法进行转化,抗性筛选和PCR分析鉴定目的基因已整合至受体植株中。试验结果如下：
1、通过酶切、连接等分子克隆技术,首次将BjuA.YSC整合入pBI121载体中,构建成表达载体pBI-BjuA.YSC。
2、建立了以下胚轴为外植体、农杆菌介导法的芥菜型油菜遗传转化体系。
3、从6400个芥菜型油菜四川黄籽下胚轴外植体中,共获得抗卡那霉素的再生苗24株,经PCR检测证实有外源基因整合的阳性植株为3株,转化效率为0.046%。3株转基因植株发育正常,能开花、结实,但T0代植株所结种子仍为黄色。
The yellow seed is showed to be associated with the expression of the genes for flavonoid synthesis in Brassicajuncea. It is suggested that the transcription factors MYB、 bHLH and WD40are the key factors responsible for difference in seed coat color. In this study, the gene BjuA.YSC our lab has cloned from Brassica juncea was used to study its function by complementation, which will provide the foundations for molecular mechanism of the yellow-seeded trait.
Expression vector of the gene BjuA. YSC was constructed and introduced into the Agrobacterium tumefaciens strain EHA105by the freeze-thaw method in this study. The hypocotyls from aseptic seedlings of B. juncea cv. Sichuan Yellow were used as explants for transformation. The main results are as follows:
1. The expression vector pBl-BjuA. YSC was constructed and transferred to competent E. coli cells for amplification.
2. The transformation system based on hypocotyl explants and Agrobacterium tumefaciens strain EHA105was established for the species Brassicajuncea.
3. The24kanamycin-resistant plantlets were regenerated from6400explants and three transgenic plants confirmed by PCR analysis. The transformation rate was estimated to be0.046%. The transgenic plants developed normally, flowered and set fruits and seeds. However, their seeds remained yellow like the untransformed parent.
引文
[1]严明理.芥菜型油菜黄籽形成的分子机理研究.长沙：湖南农业大学博士学位论文,2007.
[2]Nesi N, Debeaujona I, Jonda C, et al.The TRANSPARENT TESTA 16 Locus Encodes the ARABIDOPSIS BSISTER MADS Domain Protein and Is Required for Proper Development and Pigmentation of the Seed Coat. Plant Cell,2002,14:2463-2479.
[3]Broun P. Transcriptional control of flavonoid biosynthesis:a complex network of conserved regulators involved in multiple aspects of differentiation in Arabidopsis. Current Opinion in Plant Biology,2005,8:272-279.
[4]Gonzalez, A, Zhao M., Leavitt J.M, et al. Regulation of the anthocyaninbiosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant Journal,2008,53:814-827.
[5]Debeaujon I, Nesi N, Perez P, et al.Proanthocyanidin-accumulating cells in Arabidopsis testa:regulation of differentiation and role in seed development. Plant Cell,2003,15:2514-2531.
[6]Tohge T, Nishiyama Y, Hirai M Y, et al. Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor. The Plant Journal,2005,42:218-235.
[7]刘强,张贵友,陈受宜.植物转录因子的结构与调控作用.科学通报,2000,45：1465-1474.
[8]Sagasser M, Lu G H, Hahlbrock K, et al. A.thaliana TRANSPARENTTESTA 1 is involved in seed coat development and defines the WIP subfamily of plant zinc finger proteins. Genes Dev,2002,16:138-149.
[9]Walker A R, Davison P A, Bolognesi-Winfield A C, et al. The TRANSPARENT TESTAGLABRA 1 locus, which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis, encodes a WD40 repeat protein. Plant Cell,1999, 11:1337-1350.
[10]Johnson C S, Kolevski B, Smyth D R. TRANSPARENT TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor. Plant Cell,2002:14:1359-75.
[11]杨致荣,王兴春,杨西明,等.高等植物转录因子的研究进展,遗传,2004,26：403-408.
[12]黄泽军,黄荣峰,黄大景.植物转录因子功能分析法,2002,10：259-300.
[13]Zhang, J F,Lu, Y,Yuan, Y X, et al.Map-based cloning and characterization of a gene controlling hairiness and seed coat color traits in Brassica rapa. Plant Mol Biol, 2009,69:553-563.
[14]程振东,卫志明,许智宏.芸薹属作物的遗传转化.植物生理学通讯,1992,28：161-164.
[15]佘小玲,李栒.油菜下胚轴再生频率影响因素研究,湖南农业大学学报(自然版),2000,26：418-421.
[16]欧阳丽莹,唐章林,殷家明,等.甘蓝型黄籽油菜下胚轴再生体系的初步研究,西南农业大学学报(自然科学版),2004,26：525-528.
[17]王艳,曾幼玲,张富春,等.新疆甘蓝型油菜下胚轴的组织培养和植株再生研究,新疆农业科学,2005,42：24-28.
[18]刘晓庆,沈源,蔡小宁,等.甘蓝型油菜下胚轴离体培养再生植株研究,安徽农业科学,2008,36：13547-13548.
[19]Cardoza V, Stewart CN. Agrobacterium mediated transformation of canola,In Transgenic Crops of the world-Essential Protocols,2004:379-387.
[20]Cardoza V. Stewart CN. Canola (Brassica napus L.),Methods in molecular biology (Clifton, NJ),2006,343:257-266.
[21]杨柳,刘忠松.油菜农杆菌介导遗传转化体系研究进展,作物研究,2007,21：650-653.
[22]Maheshwari P, Selvaraj G, Kovalchuk Ⅰ. Optimization of Brassica napus (canola) explant regeneration for genetic transformation, New Biotechnology,2011,29:144-155.
[23]Ali, H. et al. In vitro regeneration of Brassica napus L, cultivars (star, cyclone, and westar) from hypocotyls and cotyledonary leaves, Pak. J. Bot.2007,39:1251-1256.
[24]Cardoza V, Stewart CN. Increased Agrobacterium mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants, Plant Cell Rep.2003,21:599-604.
[25]Khan MR, Rashid H, Quraishi A. High frequency shoot regeneration and Agrobacterium mediated DNA transfer in Canola (Brassica napus L.), Plant Cell Tissue Organ Cult.2003,75:223-231.
[26]Pandian A. Hurlstone C, Liu Q, et al. Agrobacterium-mediated transformation protocol to overcome necrosis in elite Australian Brassica juncea lines, Plant Molecular Biology Reporter.2006,24:103a-103i.
[27]王景雪,杜建中,荣二花,等.油菜下胚轴农杆菌介导法转化影响因素探讨,华北农学报,2005,20：12-15.
[28]张承妹,钱炳俊,许赞,等.利用高效农杆菌遗传转化系统将ACP反义基因片段导入油菜,上海农业学报,2003,19：5-8.
[29]周小梅,君剑,军良,等.农杆菌介导抗虫基因转化芥菜型油菜的研究,山西大学学报(自然科学版),2005,28：192-195.
[30]高武军.根癌农杆菌介导的几丁质酶基因对油菜的遗传转化研究,山西农业大学,2000.
[31]Bhuiyan MSU, Min SR, Jeong WJ, et al. An improved method for Agrobacterium-mediated genetic transformation from cotyledon explants of Brassica juncea, Plant Biotechnology,2011,28:17-23.
[32]Zhang Y, Xu J, Han L, et al. Efficient shoot regeneration and Agrobacterium-mediated transformation of Brassica juncea,Plant Mol Biol Rep,2006,24:255 a-2 55i.
[33]Kamal G.B, lllich K.G, Asadollah A. Effects of genotype, explants type and nutrient medium components on canola(Brassica napus L.) shoot in vitro organogenesis, African J Biotechnol,2007,6:861-867.
[34]De Block M, De Bmuwer D,Tenning P. Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaeiens and the expression of the bar and neo gene sinthey transgenie plants, Plant Physiol,1989,91:694-701.
[35]徐光硕,饶勇强,陈雁等in planta方法转化甘蓝型油菜,作物学报,2004,30：1-5.
[36]付绍红,张汝全,牛应泽Floral dip转化法将PEPC基因ihpRNA干扰表达载体导入甘蓝型油菜,中国生物化学与分子生物学报,2009,24：764-769.
[37]Bechtlod N, Ellis Ⅰ, Pelletier G. In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants, Compt Rend Acad Sci Paris,1993, 316:1194-1199.
[38]Clough S I, Steven I, Bent A F. Floral dip:a simplifid method for Agrobacterium mediated transformation of Arabidopsis thaliana, Plant J,1998,16:735-743.
[39]付绍红,牛应泽,杨洪全等,表面活性齐ijSilwet L-77对floral-dip转化甘蓝型油菜效果的影响,分子植物育种,2004,2：661-666.
[40]王道杰,杨翠,陆鸣.真空渗透法转化油菜及转化种子的筛选,植物学报,2009,44：216-222.
[41]Sambrook J, Russell D分子克隆实验指南[M],科学出版社,2002.
[42]刘显军.芥菜型油菜种皮颜色相关基因克隆、分析及RNAi载体构建,长沙：湖南农业大学硕士学位论文,2008.
[2]Nesi N, Debeaujona I, Jonda C, et al.The TRANSPARENT TESTA 16 Locus Encodes the ARABIDOPSIS BSISTER MADS Domain Protein and Is Required for Proper Development and Pigmentation of the Seed Coat. Plant Cell,2002,14:2463-2479.
[3]Broun P. Transcriptional control of flavonoid biosynthesis:a complex network of conserved regulators involved in multiple aspects of differentiation in Arabidopsis. Current Opinion in Plant Biology,2005,8:272-279.
[4]Gonzalez, A, Zhao M., Leavitt J.M, et al. Regulation of the anthocyaninbiosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant Journal,2008,53:814-827.
[5]Debeaujon I, Nesi N, Perez P, et al.Proanthocyanidin-accumulating cells in Arabidopsis testa:regulation of differentiation and role in seed development. Plant Cell,2003,15:2514-2531.
[6]Tohge T, Nishiyama Y, Hirai M Y, et al. Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor. The Plant Journal,2005,42:218-235.
[7]刘强,张贵友,陈受宜.植物转录因子的结构与调控作用.科学通报,2000,45：1465-1474.
[8]Sagasser M, Lu G H, Hahlbrock K, et al. A.thaliana TRANSPARENTTESTA 1 is involved in seed coat development and defines the WIP subfamily of plant zinc finger proteins. Genes Dev,2002,16:138-149.
[9]Walker A R, Davison P A, Bolognesi-Winfield A C, et al. The TRANSPARENT TESTAGLABRA 1 locus, which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis, encodes a WD40 repeat protein. Plant Cell,1999, 11:1337-1350.
[10]Johnson C S, Kolevski B, Smyth D R. TRANSPARENT TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor. Plant Cell,2002:14:1359-75.
[11]杨致荣,王兴春,杨西明,等.高等植物转录因子的研究进展,遗传,2004,26：403-408.
[12]黄泽军,黄荣峰,黄大景.植物转录因子功能分析法,2002,10：259-300.
[13]Zhang, J F,Lu, Y,Yuan, Y X, et al.Map-based cloning and characterization of a gene controlling hairiness and seed coat color traits in Brassica rapa. Plant Mol Biol, 2009,69:553-563.
[14]程振东,卫志明,许智宏.芸薹属作物的遗传转化.植物生理学通讯,1992,28：161-164.
[15]佘小玲,李栒.油菜下胚轴再生频率影响因素研究,湖南农业大学学报(自然版),2000,26：418-421.
[16]欧阳丽莹,唐章林,殷家明,等.甘蓝型黄籽油菜下胚轴再生体系的初步研究,西南农业大学学报(自然科学版),2004,26：525-528.
[17]王艳,曾幼玲,张富春,等.新疆甘蓝型油菜下胚轴的组织培养和植株再生研究,新疆农业科学,2005,42：24-28.
[18]刘晓庆,沈源,蔡小宁,等.甘蓝型油菜下胚轴离体培养再生植株研究,安徽农业科学,2008,36：13547-13548.
[19]Cardoza V, Stewart CN. Agrobacterium mediated transformation of canola,In Transgenic Crops of the world-Essential Protocols,2004:379-387.
[20]Cardoza V. Stewart CN. Canola (Brassica napus L.),Methods in molecular biology (Clifton, NJ),2006,343:257-266.
[21]杨柳,刘忠松.油菜农杆菌介导遗传转化体系研究进展,作物研究,2007,21：650-653.
[22]Maheshwari P, Selvaraj G, Kovalchuk Ⅰ. Optimization of Brassica napus (canola) explant regeneration for genetic transformation, New Biotechnology,2011,29:144-155.
[23]Ali, H. et al. In vitro regeneration of Brassica napus L, cultivars (star, cyclone, and westar) from hypocotyls and cotyledonary leaves, Pak. J. Bot.2007,39:1251-1256.
[24]Cardoza V, Stewart CN. Increased Agrobacterium mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants, Plant Cell Rep.2003,21:599-604.
[25]Khan MR, Rashid H, Quraishi A. High frequency shoot regeneration and Agrobacterium mediated DNA transfer in Canola (Brassica napus L.), Plant Cell Tissue Organ Cult.2003,75:223-231.
[26]Pandian A. Hurlstone C, Liu Q, et al. Agrobacterium-mediated transformation protocol to overcome necrosis in elite Australian Brassica juncea lines, Plant Molecular Biology Reporter.2006,24:103a-103i.
[27]王景雪,杜建中,荣二花,等.油菜下胚轴农杆菌介导法转化影响因素探讨,华北农学报,2005,20：12-15.
[28]张承妹,钱炳俊,许赞,等.利用高效农杆菌遗传转化系统将ACP反义基因片段导入油菜,上海农业学报,2003,19：5-8.
[29]周小梅,君剑,军良,等.农杆菌介导抗虫基因转化芥菜型油菜的研究,山西大学学报(自然科学版),2005,28：192-195.
[30]高武军.根癌农杆菌介导的几丁质酶基因对油菜的遗传转化研究,山西农业大学,2000.
[31]Bhuiyan MSU, Min SR, Jeong WJ, et al. An improved method for Agrobacterium-mediated genetic transformation from cotyledon explants of Brassica juncea, Plant Biotechnology,2011,28:17-23.
[32]Zhang Y, Xu J, Han L, et al. Efficient shoot regeneration and Agrobacterium-mediated transformation of Brassica juncea,Plant Mol Biol Rep,2006,24:255 a-2 55i.
[33]Kamal G.B, lllich K.G, Asadollah A. Effects of genotype, explants type and nutrient medium components on canola(Brassica napus L.) shoot in vitro organogenesis, African J Biotechnol,2007,6:861-867.
[34]De Block M, De Bmuwer D,Tenning P. Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaeiens and the expression of the bar and neo gene sinthey transgenie plants, Plant Physiol,1989,91:694-701.
[35]徐光硕,饶勇强,陈雁等in planta方法转化甘蓝型油菜,作物学报,2004,30：1-5.
[36]付绍红,张汝全,牛应泽Floral dip转化法将PEPC基因ihpRNA干扰表达载体导入甘蓝型油菜,中国生物化学与分子生物学报,2009,24：764-769.
[37]Bechtlod N, Ellis Ⅰ, Pelletier G. In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants, Compt Rend Acad Sci Paris,1993, 316:1194-1199.
[38]Clough S I, Steven I, Bent A F. Floral dip:a simplifid method for Agrobacterium mediated transformation of Arabidopsis thaliana, Plant J,1998,16:735-743.
[39]付绍红,牛应泽,杨洪全等,表面活性齐ijSilwet L-77对floral-dip转化甘蓝型油菜效果的影响,分子植物育种,2004,2：661-666.
[40]王道杰,杨翠,陆鸣.真空渗透法转化油菜及转化种子的筛选,植物学报,2009,44：216-222.
[41]Sambrook J, Russell D分子克隆实验指南[M],科学出版社,2002.
[42]刘显军.芥菜型油菜种皮颜色相关基因克隆、分析及RNAi载体构建,长沙：湖南农业大学硕士学位论文,2008.