油菜突变体库构建与激素反应基因克隆分析
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摘要
激活标记技术(activation tagging)是构建功能获得突变体为研究对象,在植物功能基因组学的研究中具有重要的作用。该技术在模式植物拟南芥中已经得到广泛的应用,但是在油菜中还未见报道。
我们以优质高产的油菜品种1244W6为材料,采用浸花蕾方法,将含有激活标记和Basta筛选标记质粒pSKI015的农杆菌进行转化,成功构建了油菜激活标记突变体库。通过Basta筛选,结果共获得约30000个转基因株系(T1代)。转化效率约为1%。对突变体库的T1代转基因植株表型进行观察,共发现39个株系有明显的表型变化,约占转化植株总数的千分之五,包括叶色、分枝数、植株的育性以及开花期等方面的变异。这将为开展油菜分子遗传学研究提供丰富的资源。
将突变体T2代种子播种在含不同浓度植物激素的营养液中,结果筛选出激素反应突变体63个。这些激素反应突变体的获得,为揭示油菜激素代谢调控及激素调控油菜生长发育、产量和含油量的分子机理提供了材料。
采用TAIL-PCR技术,克隆了W1016赤霉素反应突变体T-DNA插入位点基因组旁邻序列。通过序列比对,发现该序列与拟南芥基因组中的细胞色素P450单加氧酶基因(At1g01190)序列相似。接着我们便以这旁邻序列作为出发点,采用Genome Walker的方法进一步克隆到了油菜中P450基因的全长序列,这为深入研究该基因在油菜激素代谢调控中的功能奠定了基础,同时也建立了克隆油菜突变体相关基因的新方法。
Activation tagging is an important strategy in plant genomics by generating gain-of-function mutants. This method in arabidopsis has been widely applied, but not reported in napus.
An activation tagging mutant library of Brassica napus 1244w6 was constructed by inflorescence dipping method, and pSKI015 plasmid with Basta screening marker was transformed by Agrobacterial tumefaciens containing 30000 independent T1 transformants were obtained through basta screening. The efficiency of transformation is about 1%. Library of mutant T1 generation of transgenic plants have observed phenotype, a total of 39 lines have significant phenotypic change about 5%o of transformed plants, changes in leaf color, branch number, plant fertility as well as the variation in flowering periods.This would be provided abundant resources for the development of molecular genetics of Brassica napus.
About 63 phytohormone response mutants were obtained in the seeds of T2 transformants screened, which was sowed in nutrient solution with different phytohormone. So, these mutants could provide some materials to reveal and clarify the molecular mechanism of the phytohormone metabolism in Brassica napus growth and development, and regulation in yield and oil content of Brassica napus.
The T-DNA flanking sequence of gibberellin response mutant W1016 was cloned by TAIL-PCR. which is quite similar to the cytochrome P450 monooxygenase gene (At1g01190) in arabidopsis thaliana genome. Then we take this known flanking sequence, to clone the full-length sequence of Brassica napus P450 genes by using Genome Walker. This research lays the foundation of further study on this gene in oilseed rape in the functional regulation of hormone metabolism, and also established a new approach of clone rape-related genes from mutant.
我们以优质高产的油菜品种1244W6为材料,采用浸花蕾方法,将含有激活标记和Basta筛选标记质粒pSKI015的农杆菌进行转化,成功构建了油菜激活标记突变体库。通过Basta筛选,结果共获得约30000个转基因株系(T1代)。转化效率约为1%。对突变体库的T1代转基因植株表型进行观察,共发现39个株系有明显的表型变化,约占转化植株总数的千分之五,包括叶色、分枝数、植株的育性以及开花期等方面的变异。这将为开展油菜分子遗传学研究提供丰富的资源。
将突变体T2代种子播种在含不同浓度植物激素的营养液中,结果筛选出激素反应突变体63个。这些激素反应突变体的获得,为揭示油菜激素代谢调控及激素调控油菜生长发育、产量和含油量的分子机理提供了材料。
采用TAIL-PCR技术,克隆了W1016赤霉素反应突变体T-DNA插入位点基因组旁邻序列。通过序列比对,发现该序列与拟南芥基因组中的细胞色素P450单加氧酶基因(At1g01190)序列相似。接着我们便以这旁邻序列作为出发点,采用Genome Walker的方法进一步克隆到了油菜中P450基因的全长序列,这为深入研究该基因在油菜激素代谢调控中的功能奠定了基础,同时也建立了克隆油菜突变体相关基因的新方法。
Activation tagging is an important strategy in plant genomics by generating gain-of-function mutants. This method in arabidopsis has been widely applied, but not reported in napus.
An activation tagging mutant library of Brassica napus 1244w6 was constructed by inflorescence dipping method, and pSKI015 plasmid with Basta screening marker was transformed by Agrobacterial tumefaciens containing 30000 independent T1 transformants were obtained through basta screening. The efficiency of transformation is about 1%. Library of mutant T1 generation of transgenic plants have observed phenotype, a total of 39 lines have significant phenotypic change about 5%o of transformed plants, changes in leaf color, branch number, plant fertility as well as the variation in flowering periods.This would be provided abundant resources for the development of molecular genetics of Brassica napus.
About 63 phytohormone response mutants were obtained in the seeds of T2 transformants screened, which was sowed in nutrient solution with different phytohormone. So, these mutants could provide some materials to reveal and clarify the molecular mechanism of the phytohormone metabolism in Brassica napus growth and development, and regulation in yield and oil content of Brassica napus.
The T-DNA flanking sequence of gibberellin response mutant W1016 was cloned by TAIL-PCR. which is quite similar to the cytochrome P450 monooxygenase gene (At1g01190) in arabidopsis thaliana genome. Then we take this known flanking sequence, to clone the full-length sequence of Brassica napus P450 genes by using Genome Walker. This research lays the foundation of further study on this gene in oilseed rape in the functional regulation of hormone metabolism, and also established a new approach of clone rape-related genes from mutant.
引文
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[2]张建模,邹小云,宋来强.杂交油菜主要产量性状与品质性状的关系研究.江西农业学报,2006,18(6)：16～20.
[3]邓伟,肖华贵.杂交油菜产量与相关农艺性状的灰色关联度分析.安徽农业科学,2009,37(5)：1961～1962.
[4]李加纳.甘蓝型油菜主要农艺性状的遗传模型和基因效应分析.遗传学报,1992,19(2)：162～168.
[5]戚存扣,盖钧镒,傅寿仲.甘蓝型油菜(Brassica napus L.)千粒重性状遗传体系分析.作物学报,2004,30(12)：1274-1277.
[6]张书芬,马朝芝,朱家成,等.甘蓝型油菜含油量的主基因+多基因遗传效应分析.遗传学报,2006,33(2)：171～180.
[7]付三雄,戚存扣.甘蓝型油菜含油量的主基因+多基因遗传分析.江苏农业学报,2009,25(4)：731～736.
[8]张洁夫,戚存扣,浦惠明,等.甘蓝型油菜主要脂肪酸的主基因+多基因遗传分析.中国油料作物学报,2007,29(4)：359-364.
[9]牟同敏,刘后利.甘蓝型油菜种子中硫代葡萄糖甙总量的遗传分析.作物学报,1990,16(2)：97～105.
[10]李云昌,李英德.高产双低杂交油菜新品种中油杂2号的选育.中国油料作物学报,2002,24(2)：73～75.
[11]P B E M, Scarth R, W G D F, et al. Brassica seed quality breeding at the University of Manitoba, sustainable development in cruciferous oilseed crops production. Proceedings of 12th International Rapeseed Congress,2007,1:2～4.
[12]Wang H, Li Y, Hu Q, et al. Current status of double-low rapeseed variety development in OCRI-CAAS.2006, (8):29～36.
[13]傅廷栋.中国油菜生产和品种改良的现状与前景.安徽农学通报,2000,6(1)：2-9.
[14]Federspiel N. Deciphering a weed. Genomic sequencing of Arabidopsis. Plant Physiol,2000,124(4):1456～1459.
[15]Chory J, Ecker J R, Briggs S, et al. National Science Foundation-Sponsored Workshop Report:"The 2010 Project" functional genomics and the virtual plant. A blueprint for understanding how plants are built and how to improve them. Plant Physiol,2000,123(2):423～426.
[16]Parkin I A, Gμlden S M, Sharpe A G, et al. Segmental structure of the Brassica napus genome based on comparative analysis with Arabidopsis thaliana. Genetics, 2005,171(2):765～781.
[17]Zhao J Y, Becker H C, Ding H D, et al. QTL of three agronomically important traits and their interactions with environment in a European x Chinese rapeseed population. Yi Chuan Xue Bao,2005,32(9):969～978.
[18]刘仁虎,孟金陵.RFLP和AFLP分析白菜型油菜和甘蓝型油菜遗传多样性及其在油菜改良中的应用价值.遗传学报,2006,33(9)：814～823.
[19]刘春林,阮颖.油菜分子标记图谱构建及抗菌核病性状的QTL定位.遗传学报,2000,27(10)：918～924.
[20]涂金星,傅廷栋.甘蓝型油菜隐性核不育遗传标记的初步研究：Ⅱ.P6—9紫茎基因与可育.作物学报,1999,25(6)：669-673.
[21]Jourdren C, Barret P, Horvais R. Identification of RAPD markers linked to the loci controlling erucic level in rapeseed. Molecular Breeding,1996, (2):61～67.
[22]Horsch R B, Fry J E, Hoffman N. A simple and general method for transferring genes into plants. Science,1985,227:1229～1231.
[23]林良斌,官春云.转基因抗虫油菜中Bt杀虫蛋白基因稳定遗传和高效表达及抗虫性研究.作物学报,2002,28(2)：175-178.
[24]Knutzon D S, Thompson G A, Radke S E, et al. Modification of Brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene. Proc Natl Acad Sci U S A,1992,89(7):2624～2628.
[25]陈锦清,郎春秀.反义PEP基因调控油菜籽粒蛋白质／油脂含量比率的研究.农业生物技术学报,1999,7(4)：316-320.
[26]甘莉,王新.油菜种子贮藏蛋白的遗传多样性分析.华中农业大学学报,1999,18(6)：528～532.
[27]熊志勇,夏伏建.优质油菜籽粕蛋白质分类研究.武汉植物学研究,2001,19(3)：259～261.
[28]郭龙彪,储成才,钱前.水稻突变体与功能基因组学.植物学通报,2006,12(1)：1～13.
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