小麦独脚金内酯合成相关基因TaDWARF27的分离与功能分析
发布时间：2019-02-24 19:35
【摘要】：小麦是我国重要的粮食作物。小麦的产量由亩穗数、穗粒数和千粒重决定,分蘖数在一定程度上决定了最终的亩穗数。目前对小麦分蘖的研究大多集中在生理方面,对其发生的分子机理及调控方面的研究国内外报道很少。已有研究表明独角金内酯(SLs)作为一种植物激素可以抑制水稻和拟南芥侧枝的生长。DWARF27(D27)基因编码一种含铁的β类胡萝卜素异构酶,参与SLs的合成。d27突变体表现为侧枝增多,株高下降,株型紧凑。为理解SLs调控小麦分蘖的分子机理,本研究分离了TaDWARF27基因,并对其表达模式及功能进行了初步研究,具体结果如下:TaDWARF27基因具有三对等位基因,定位于7号染色体上。三对等位基因的序列同源性高达97.71%。cDNAs全长分别为837bp(TaD27-7A)、840bp(TaD27-7B)和840bp(TaD27-7D),分别编码279、280、280个氨基酸残基组成的蛋白质。该基因gDNA具有7个外显子和6个内含子,全长分别为:4675bp(TaD27-7A)、4896bp(TaD27-7B)和4703bp(TaD27-7D)。与OsD27相似,TaD27蛋白具有一个保守的结构域DUF4033。系统进化树分析表明TaD27蛋白与粗山羊草D27蛋白亲缘关系最近,其次为大麦和二穗短柄草的D27蛋白。亚细胞定位分析显示TaD27蛋白定位于叶绿体上。定量PCR分析结果显示TaD27的转录物主要在叶片中表达。原位杂交显示TaD27主要在叶片、叶腋和幼穗原基表达。TaD27-7B的过表达可以恢复拟南芥d27突变体的表型,这说明在拟南芥和小麦中TaD27的功能具有一定的保守性。为进一步探究TaD27的功能,我们构建了RNA interference载体和过表达载体,以栽培品种科农199(KN199)的幼胚为受体,进行农杆菌浸染介导的小麦遗传转化,共得到了TaD27 RNAi转基因植株295株和过表达转基因植株255株。除草剂抗性筛选和PCR分子鉴定得到16株RNAi阳性苗和10株过表达阳性苗。定量PCR分析结果显示RNAi阳性苗叶片中TaD27的表达量明显下调。我们从中选出13个RNAi株系和2个过表达株系进行了加代,目前正进行植株鉴定及表型观察。综上所述,TaD27很可能在小麦分蘖调控过程中起重要作用,该实验结果可以为提高小麦产量提供分子理论依据。
[Abstract]:Wheat is an important food crop in China. The yield of wheat is determined by the number of ears per mu, the number of grains per ear and the weight of 1000 grains, and the number of tillers determines the number of ears per mu to a certain extent. At present, the studies on wheat tillers are mostly focused on physiology, and few reports on molecular mechanism and regulation of wheat tillers are reported at home and abroad. It has been shown that (SLs), as a phytohormone, can inhibit the growth of lateral branches of rice and Arabidopsis thaliana. DWARF27 (D27) gene encodes an iron-containing 尾 -carotene isomerase. D 27 mutants were characterized by increased lateral branches, decreased plant height and compact plant type. In order to understand the molecular mechanism of SLs regulating wheat tiller, TaDWARF27 gene was isolated, and its expression pattern and function were studied. The results are as follows: TaDWARF27 gene has three alleles located on chromosome 7. The sequence homology of the three alleles is as high as 837bp (TaD27-7A), 840bp (TaD27-7B) and 840bp (TaD27-7D), which encode 279280280 amino acid residues. There are 7 exons and 6 introns in gDNA, which are 4675bp (TaD27-7A), 4896bp (TaD27-7B) and 4703bp (TaD27-7D). Similar to OsD27, TaD27 protein has a conserved domain DUF4033. Phylogenetic tree analysis showed that TaD27 protein was most closely related to D27 protein of Leymus chinensis, followed by D27 protein from barley and short stalks. Subcellular localization analysis showed that TaD27 protein was located on chloroplast. Quantitative PCR analysis showed that TaD27 transcripts were mainly expressed in leaves. In situ hybridization showed that TaD27 was mainly expressed in leaf, axil and young panicle primordia. Overexpression of TaD27-7B could restore the phenotype of Arabidopsis d27 mutant, which indicated that the function of TaD27 was conserved in Arabidopsis and wheat. In order to further explore the function of TaD27, we constructed RNA interference vector and overexpression vector. The immature embryos of the cultivar KN199 were used as the receptor for Agrobacterium tumefaciens soaking mediated wheat genetic transformation. A total of 295 TaD27 RNAi transgenic plants and 255 overexpressed transgenic plants were obtained. 16 RNAi positive seedlings and 10 overexpression positive seedlings were obtained by herbicide resistance screening and PCR molecular identification. Quantitative PCR analysis showed that the expression of TaD27 in the leaves of RNAi positive seedlings was significantly down-regulated. 13 RNAi lines and 2 overexpression lines were selected for culture. Plant identification and phenotypic observation were carried out. In conclusion, TaD27 may play an important role in the regulation of wheat tillering, and the results can provide molecular theoretical basis for improving wheat yield.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S512.1;Q943.2
本文编号：2429863
[Abstract]:Wheat is an important food crop in China. The yield of wheat is determined by the number of ears per mu, the number of grains per ear and the weight of 1000 grains, and the number of tillers determines the number of ears per mu to a certain extent. At present, the studies on wheat tillers are mostly focused on physiology, and few reports on molecular mechanism and regulation of wheat tillers are reported at home and abroad. It has been shown that (SLs), as a phytohormone, can inhibit the growth of lateral branches of rice and Arabidopsis thaliana. DWARF27 (D27) gene encodes an iron-containing 尾 -carotene isomerase. D 27 mutants were characterized by increased lateral branches, decreased plant height and compact plant type. In order to understand the molecular mechanism of SLs regulating wheat tiller, TaDWARF27 gene was isolated, and its expression pattern and function were studied. The results are as follows: TaDWARF27 gene has three alleles located on chromosome 7. The sequence homology of the three alleles is as high as 837bp (TaD27-7A), 840bp (TaD27-7B) and 840bp (TaD27-7D), which encode 279280280 amino acid residues. There are 7 exons and 6 introns in gDNA, which are 4675bp (TaD27-7A), 4896bp (TaD27-7B) and 4703bp (TaD27-7D). Similar to OsD27, TaD27 protein has a conserved domain DUF4033. Phylogenetic tree analysis showed that TaD27 protein was most closely related to D27 protein of Leymus chinensis, followed by D27 protein from barley and short stalks. Subcellular localization analysis showed that TaD27 protein was located on chloroplast. Quantitative PCR analysis showed that TaD27 transcripts were mainly expressed in leaves. In situ hybridization showed that TaD27 was mainly expressed in leaf, axil and young panicle primordia. Overexpression of TaD27-7B could restore the phenotype of Arabidopsis d27 mutant, which indicated that the function of TaD27 was conserved in Arabidopsis and wheat. In order to further explore the function of TaD27, we constructed RNA interference vector and overexpression vector. The immature embryos of the cultivar KN199 were used as the receptor for Agrobacterium tumefaciens soaking mediated wheat genetic transformation. A total of 295 TaD27 RNAi transgenic plants and 255 overexpressed transgenic plants were obtained. 16 RNAi positive seedlings and 10 overexpression positive seedlings were obtained by herbicide resistance screening and PCR molecular identification. Quantitative PCR analysis showed that the expression of TaD27 in the leaves of RNAi positive seedlings was significantly down-regulated. 13 RNAi lines and 2 overexpression lines were selected for culture. Plant identification and phenotypic observation were carried out. In conclusion, TaD27 may play an important role in the regulation of wheat tillering, and the results can provide molecular theoretical basis for improving wheat yield.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S512.1;Q943.2
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1 武亭亭;小麦独脚金内酯合成相关基因TaDWARF27的分离与功能分析[D];山东农业大学;2016年
，本文编号：2429863
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