【作者】 赵毅；

【导师】 李学军；

【作者基本信息】 西北农林科技大学 ， 作物学， 2019， 硕士

【摘要】 我国是世界上小麦生产与消费大国,小麦生产在国家粮食安全方面占据着举足轻重的地位。在当今环境下,提高小麦产量是保障我国粮食安全的一个重要途径。小麦株高是影响小麦产量的一个重要因素,株高相关基因的发掘与研究,对小麦育种有着重要的意义。本研究以中国春为材料,使用同源克隆方法,依据水稻P450蛋白家族基因Dwarf11序列,在小麦中克隆得到了TaDwarf11基因,并对其进行了时空表达、标记开发、基因定位及功能分析等研究得到了以下重要结论:1.依据基因数据库比对得到序列设计引物,在小麦中国春的gDNA与cDNA中进行了PCR片段扩增与测序验证,得到了完整的TaDwarf11序列,其cDNA全长为1428bp,gDNA全长为3607bp,含有9个长度分别为215bp、325bp、154bp、235bp、89bp、75bp、107bp、125bp和102bp的外显子,以及8个长度分别为365bp、241bp、157bp、91bp、281bp、189bp、470bp和386bp的内含子。所编码的蛋白质含有475个氨基酸,存在一个P450结构域。2.该基因在小麦根、茎、叶、幼穗中均有表达,但相对表达量在幼穗中最高,在叶片中次之,在根中的表达量较低,在茎中的相对表达量最低,推测这种表达差异可能是该基因表达与植物营养生长与生殖生长阶段功能差异性的体现。3.通过测序分析,在不同小麦品种间发现了TaDwarf11基因第一外显子138bp处的T-C碱基差异。根据此SNP开发出的分子标记,在小麦品种中区分出了TaDwarf11-A基因型(138bp处是T碱基)与TaDwarf11-B基因型(138bp处是C碱基)。利用中国春缺体-四体对该基因进行了染色体定位,该基因位于小麦2B染色体上。4.通过检测268份栽培种的基因型与其株高表型性状,进而进行关联分析,发现本研究中开发出的功能标记与小麦株高之间具有极显著相关性(P=3.32E-7)。5.本研究在检测268份栽培种与205份农家种的基因型后发现,小麦农家种中的高杆基因型(TaDwarf11-B)所占比例(66.3%)略高于其在栽培种中的比例(64.2%),推测该现象可能是育种过程中人工选择的结果。6.通过检测205份农家种的基因型与其种植地区,本研究发现TaDwarf11-A基因型(矮杆)在北方春麦区所占比例最高(58.33%),TaDwarf11-B基因型(高杆)在长江中下游冬麦区所占比例最高(75%)。相较南方而言,北方气候干旱,降水量少,气温较低,小麦营养生长较为缓慢,株高较低,故这种基因型的南北分布差异可能与南北方麦区种植环境差异有关。本研究发掘出了水稻Dwarf11基因在小麦中的同源基因TaDwarf11,并依据其第一外显子上的SNP开发出了该基因与小麦株高紧密相关的功能标记,证明了该基因与小麦株高性状的关联性。在该基因外显子区域开发的标记可以准确分辨高杆小麦品种与矮杆小麦品种,能够辅助小麦育种工作,为小麦株型改良提供了有效的选择工具。更多还原

【Abstract】 China is a big country of wheat production and consumption in the world,and wheat production plays an important role in national food security.In today’s environment,improving wheat yield is an important way to ensure food security in China.Plant height is an important factor affecting wheat yield.The discovery and research of plant height-related genes is of great significance to wheat breeding.In this study,TaDwarf11 gene was cloned from wheat using homologous cloning method based on the sequence of rice P450 protein family gene Dwarf11.The following important conclusions were obtained from the studies of temporal and spatial expression,marker development,gene localization and functional analysis of TaDwarf11 gene.1.Sequence design primers were obtained by gene database alignment.The full length of TaDwarf11 was 1428 BP and that of gDNA was 3607 bp.It contained 9 exons of 215 bp,325 bp,154 bp,235 bp,89 bp,75 bp,107 bp,125 BP and 102 bp,and 8 exons,respectively.The introns were 365 bp,241 bp,157 bp,91 bp,281 bp,189 bp,470 BP and 386 bp.The encoded protein contains 475 amino acids and has a P450 domain.2.This gene is expressed in roots,stems,leaves and young spikes of wheat,but its relative expression is highest in young spikes,second in leaves,lower in roots,and lowest in stems.It is speculated that this difference may be the reflection of the functional difference between the expression of this gene and vegetative and reproductive growth stages of plants.3.By sequencing analysis,the difference of T-C base at 138 bp of exon 1 of TaDwarf11 gene was found among different wheat varieties.Based on the molecular markers developed by this SNP,the TaDwarf11-A genotype(T base at 138bp)and TaDwarf11-B genotype(C base at 138bp)were identified in wheat varieties.The gene was located on chromosome 2B of Wheat by using Chinese spring deletion tetrasome.4.By detecting the genotype and plant height phenotypic traits of 268 cultivars,and then making correlation analysis,it was found that the functional markers developed in this study had a very significant correlation with plant height of wheat(P=3.32E-7).5.In this study,268 cultivars and 205 cultivars were tested for genotypes.It was found that the proportion of TaDwarf11-B in wheat cultivars was slightly higher than that in cultivars(66.3%).It was speculated that this phenomenon might be the result of artificial selection in breeding process.6.By detecting 205 genotypes of farm species and their growing areas,this study found that TaDwarf11-A genotype(dwarf pole)accounted for the highest proportion(58.33%)in the northern spring wheat region and TaDwarf11-B genotype(tall pole)accounted for the highest proportion(75%)in the middle and lower reaches of the Yangtze River winter wheat region.Compared with southern China,northern China has arid climate,less precipitation,lower temperature,slower vegetative growth and lower plant height.Therefore,the North-South distribution difference of this genotype may be related to the difference of planting environment between northern and southern wheat regions.In this study,the homologous gene TaDwarf11 of rice Dwarf11 in wheat was discovered.Based on SNP in its first exon,functional markers closely related to plant height of wheat were developed,which proved the correlation between the gene and plant height traits of wheat.The markers developed in the exon region of this gene can accurately distinguish high-stem wheat varieties from Low-stem wheat varieties,assist wheat breeding,and provide an effective selection tool for wheat plant type improvement.更多还原