利用BSA-seq图位克隆两个水稻重要基因

发布时间：2018-04-17 12:05

本文选题：利用 + BSA-seq　；参考：《福建农林大学》2016年博士论文

【摘要】：水稻分蘖是营养生长的重要特性,其分蘖数决定了有效穗数,进而决定了水稻产量。穗是水稻的营养器官,由若干小花和小穗组成。小花和小穗在不同水平上的发育决定了水稻穗部性状和产量。因此分蘖和穗部发育是决定营养生长和生殖生长的两个重要农艺性状。本论文分离了在水稻籼稻品种明恢86组织培养过程中发现的两个突变体srt1和pshl-1/2,通过将BSA和全基因组测序方法定位和克隆了这两个基因,并对这两个基因的功能进行了初步分析。其结果如下：1. Sterile and Reduced Tillering 1:现单杆和雌性不育的突变体。(1)srtl表现为很少或不分蘖。与对照明恢86(分蘖数为9.0±3.6)相比,突变体srtl的分蘖明显少于对照,只有1-4个分蘖(0.5±0.7)。(2)srtl的茎比野生型粗壮。从茎的横切片结果来看,srtl的茎微管束发育比野生型多,能提供足够的养分给整个植株；纵切结果显示其细胞排列比野生型更有规律。(3)srtl的叶片显得比野生型长且宽。野生型与突变体srt1叶片的长宽比分别为：52.2±3.34：59.5±2.1和1.55±0.2：2.05±0.1。(4)srtl表现为完全不育。花药碘染实验显示突变体的花药表现正常。正反交实验结果显示,用srtl作为父本,野生型作为母本结实率为9.9土6.8%,然而反过来srt1作为母本时颗粒无收,说明srtl表现为雌性不育。(5)通过BSA-seq方法比较突变DNA池和野生DNA池间SNP频率分布,计算CAAFD值,发现在水稻4号染色体长臂末端约(29-34M)处CAAFD有一个明显的峰。在该期间开发INDEL标记,将目标基因定位在231Kb范围内。(6)在231kb区间内共有37个ORFs。我们发现其中LOC_Os04g56780与野生型相比有21个碱基缺失。LOC_Os04g56780编码水稻中与拟南芥Wuschel (AtWUS)的同源基因OsWUS。(7)突变体中OsWUS的21bp发生在第一个外显子上,使OsWUS的一个高度保守结构域Homeobox中缺失了7个氨基酸。该缺失可能导致OsWUS功能丧失,说明Homeobox结构域在OsWUS的功能上起重要作用。2. Pepper-Shaped husk 1：一个小花形状异常的突变体。(1)突变体pshl-1表现为穗变短且密,其穗长仅有野生型明恢86的20%。pshl-1的小花呈现辣椒状,其外稃正常,外稃明显比野生型小。(2)pshl-1的结实率仅有5.8%±7.2,远远小于野生型的结实率(85.2%±4.9)。开花时突变体的表型不正常,仅有一小部分突变体6.55%±5.3的花开启后能正常关闭,而野生型种大部分(84.65%±6.7)的花都能正常开花及关闭。(3)psh1-1的种子比野生型要小。突变体的粒长(8.7±0.48)和粒宽(1.975±0.07)明显小于野生型。pshl-1的粒形表现为辣椒状,其千粒重(28.67±0.65)明显低于野生型(15.0±1.18)。(4)通过F2群体的遗传分析显示,突变体pshl-1表现为辣椒颖和低结实率的性状受一对隐性等位基因控制。(5)应用BSA-seq方法定位PSH1,通过计算CAAFD值发现在水稻4号染色体断臂末端0-3Mb处有一个高峰。经连锁分析将该基因定位在218Kb的区间内。(6)该218Kb区间内有31个ORFs。通过不同基因池中序列比对发现,LOC_Os04g01590基因内部的一个单碱基替换(G→T)导致终止密码TAA的产生,是蛋白质提前终止。(7) LOC_Os04g01590编码精氨酸酶。它有6个外显子,编码340个氨基酸。突变体pshl-1中的第三个外显子上的G到T的替换,及pshl-2中的第二个外显子上的G到T的替换都能产生终止密码TAG,使蛋白质提前终止。这些结果显示突变体表现出的突变性状是由基因LOC_Os04g01590突变造成的。(8)氧化应激分析显示突变体pshl和野生型无明显差异。
[Abstract]:Rice tillering is an important characteristic of vegetative growth, tiller number determines the effective panicle number, and then determine the yield of rice. Spike is rice vegetative organs, composed of several flowers and spikelets. Flowers and spikelet development at different levels determines the panicle traits and yield of rice. So the tiller and panicle is two the important agronomic traits of vegetative growth and reproductive growth and development. This paper decided to separate the training process of the two mutants of SRT1 and pshl-1/2 in indica rice cultivar Minghui 86, by BSA and genome sequencing method for mapping and cloning of the two genes analyzed and the function of the two genes the results are as follows: 1.. The Sterile and Reduced Tillering 1: is now a single rod and the female sterile mutants. (1) srtl showed little or no tillers. Compared with Minghui 86 (the tiller number was 9 + 3.6) compared with mutant SR TL was significantly less than the control, only 1-4 tiller (0.5 + 0.7). (2) srtl stem than the wild type. From the cross section of the stout stem, srtl stem vascular bundles development than wild type, can provide enough support to the whole plant; the results showed that the longitudinal arrangement of cells is more than law of the wild type. (3) srtl than the wild type leaves look long and wide. The length and width of the wild type and SRT1 mutant leaves are as below: 52.2 + 3.34:59.5 + 2.1 and 1.55 + 0.2:2.05 + 0.1. (4) srtl were completely sterile anther. Iodine staining experiments showed that the mutant anther showed normal reciprocal. The experimental results show that using srtl as male parent and wild type as the female parent setting rate is 9.9 6.8% of the earth, but in turn SRT1 as the female parent when erected, srtl showed that the female sterility. (5) by the method of BSA-seq mutation in DNA pool and DNA pool between wild SNP frequency distribution calculation CAAFD The value found in rice chromosome 4 (29-34M) at the end of about CAAFD has an obvious peak. During the development of INDEL markers, the target gene in the 231Kb range. (6) were in the range of 231kb 37 ORFs. we found the wild type and LOC_Os04g56780 21 base deletion.LOC_Os04g56780 encoding in rice and Arabidopsis Wuschel (AtWUS) compared with the homologous gene of OsWUS. (7) OsWUS mutant 21bp in the first exon, a highly conserved Homeobox domain to OsWUS deletion of 7 amino acids. The deletion may lead to the loss of OsWUS function, Homeobox domain in OsWUS the function plays an important role in.2. Pepper-Shaped husk 1: a flower shape abnormal mutant. (1) mutant pshl-1 showed shorter panicle and dense panicle length, the only wild type Minghui 86 20%.pshl-1 flower show like chili, besides Lemma normal lemma significantly smaller than those of the wild type. (2) pshl-1 seed rate of only 5.8% + 7.2, far less than the seed setting rate of wild type (85.2% + 4.9). Flowering mutant phenotype is not normal, can shut down only a small part of the mutant 6.55% + 5.3 start, and for most of the wild type (84.65% + 6.7) flowers can be normal flowering and closed. (3) psh1-1 seeds is smaller than the wild type. The mutant grain length (8.7 + 0.48) and grain width (1.975 + 0.07) was significantly less than that of wild type.Pshl-1 grain shape for pepper shape, its grain weight (28.67 + 0.65) significantly than the wild type (15 + 1.18). (4) through the genetic analysis of F2 groups showed that mutant pshl-1 showed pepper Ying and low seed setting rate was controlled by a pair of recessive alleles. (5) the application of BSA-seq method to locate the PSH1, found that there is a peak in rice chromosome 4 arm end 0-3Mb by calculating the value of CAAFD. By linkage analysis of this gene in the range of 218Kb. (6) the 218Kb range of 31 ORFs. by sequence alignment of different gene pools found that a single nucleotide substitution within LOC_Os04g01590 gene (G, T) to stop codon TAA, the protein is terminated (7) LOC_Os04g01590 encoding. Arginase. It has 6 exons, encoding 340 amino acids. The third exons of the G to replace T in pshl-1 mutant, second exons of the G to replace T can generate a stop codon TAG and pshl-2, the early termination of these protein. The results showed that the mutation characteristics showed is caused by a mutation of the LOC_Os04g01590 gene. (8) oxidative stress analysis showed no significant difference PSHL mutant and wild type.

【学位授予单位】：福建农林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S511

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