水稻小粒突变体sg1的分离和基因克隆
时间:2021-12-10 21:48 来源:毕业论文 作者:毕业论文 点击:次
摘要水稻产量的提高一直是科学家研究的课题,那么水稻的产量与什么因素相关呢?众所周知,水稻之所以重要是因为它是人类主要的食物。人类一天活动主要依靠的能量就是来自于水稻的淀粉。研究水稻产量,主要是从三个方面探究。一是水稻的穗数,二是水稻每条穗的颗粒数,三是每个颗粒的饱满程度,也就是水稻的重量。本次论文课题就是以小粒水稻为研究对象,定位克隆造成水稻小粒的基因。75377 水稻籽粒大小对水稻颗粒重量的影响较大,因此水稻的籽粒大小调控分子机理研究对提升水稻产量具有重大意义。为了研究籽粒大小调控的分子机理,我们用EMS诱变野生型水稻ZH11获得了系列粒型改变突变体,其中一个突变体被命名为sg1,主要表现为种子粒形小且圆,种子饱满程度低,发芽率低。为探究该突变是否为隐形突变,我将sg1与野生型水稻9311进行杂交,得到的F1代全为野生型,将F1代进行自交,F2代出现性状分离,野生型9311个体与突变体sg1个体比例为3:1,根据孟德尔定律可知该突变为隐性突变。为进行基因定位,将获得的突变体sg1与野生型9311回交,回交6-7次后,我们获得了两千多株突变体表型的F2单株。水稻基因组序列测定后,可自由设计引物。本次实验采用的引物共110对,均匀分布于水稻12条染色体。 实验时首先从F2群体中选取20个突变表型植株,提取DNA,进行PCR扩增,凝胶电泳,根据双亲多态性不同进行筛选,多态性不同的引物即为候选引物,此时候选引物范围较大。接着选取200株突变体单株,利用候选引物进行初定位。初定位后扩大模板数量,设计引物,进行精细定位。最终SG1基因被限定在第5条染色体上PM446和YP320两对引物之间,物理距离约为33kb。该区间包括7个ORF,其中第四个ORF的第1212个碱基上,由胞嘧啶(C)突变为胸腺嘧啶(T),该密码子由CAG变为TAG,从而造成蛋白翻译的提前终止。 Abstract The improvement of rice yield has been the subject of scientists for a long time。 So what are the factors related to rice yield? As is known to all, rice is important because it is the main food of human beings。 The energy that is mainly depended on the day of human beings is the starch from rice。 The research on rice yield is mainly from three aspects。 One is the number of rice panicle, two is the number of grains per spike of rice, the three is the full extent of each particle, which is the weight of rice。 This paper is to small rice as the research object, mapping and cloning the rice grain gene 。 The effect of grain size on rice grain weight was larger。 Therefore, research on the grain size control mechanism of rice is of great significance to improve the yield of rice。 In order to study the molecular mechanism of the grain size regulation, we made mutagenesis of the wild rice with EMS, and obtained a series of mutant altered in grain shape。 One of the mutants showed a small and circle grain shape (refer to sg1), which, also presented additional phenotypes, like reduced seed yield and low germination rate。 In order to explore the mutation is recessive mutant, I made sg1 and 9311 hybridization, F1 generation full for the wild type, F2 generation character separation and wild-type 9311 inpidual and mutant sg1 inpidual proportion 3:1。 According to Mendel's law , the mutation is recessive mutations。 For gene mapping, we made the mutant sg1 and wild-type 9311 backcross, backcross 6-7 times, we obtain the more than 2000 mutant phenotype of the F2 plants。 After the rice genome sequence was determined, the primers were designed。 The 110 pairs of primers were used in this experiment, which were evenly distributed on 12 chromosomes of rice。 First selecting 20 mutant phenotype from the F2 population, DNA extraction, PCR amplification, gel electrophoresis, according to the different parents polymorphism screening, different polymorphic primers is candidate primers。 At this time, the candidate primers a wide range。 Then select the 200 plant mutants, using the candidate primer for initial positioning。 After the initial positioning of the template to expand the number of primers designed to fine positioning。 The SG1 gene was finally delimited to a single BAC by marker PM446 and YP320 with a physical distance of 33Kb。 In this region, there were 7 open reading frame (ORFs), and the fourth ORF, had a site mutation (Cytosine (C) in wild type) and thymine (T) ) in mutant) in 1212th base after start codon ATG, which caused the premature stop code (CAG to TAG)。 (责任编辑:qin) |