發(fā)布時(shí)間：2018-04-17 12:05

  本文選題：利用 + BSA-seq��； 參考：《福建農(nóng)林大學(xué)》2016年博士論文

【摘要】：水稻分蘗是營(yíng)養(yǎng)生長(zhǎng)的重要特性,其分蘗數(shù)決定了有效穗數(shù),進(jìn)而決定了水稻產(chǎn)量。穗是水稻的營(yíng)養(yǎng)器官,由若干小花和小穗組成。小花和小穗在不同水平上的發(fā)育決定了水稻穗部性狀和產(chǎn)量。因此分蘗和穗部發(fā)育是決定營(yíng)養(yǎng)生長(zhǎng)和生殖生長(zhǎng)的兩個(gè)重要農(nóng)藝性狀。本論文分離了在水稻秈稻品種明恢86組織培養(yǎng)過(guò)程中發(fā)現(xiàn)的兩個(gè)突變體srt1和pshl-1/2,通過(guò)將BSA和全基因組測(cè)序方法定位和克隆了這兩個(gè)基因,并對(duì)這兩個(gè)基因的功能進(jìn)行了初步分析。其結(jié)果如下：1. Sterile and Reduced Tillering 1:現(xiàn)單桿和雌性不育的突變體。(1)srtl表現(xiàn)為很少或不分蘗。與對(duì)照明恢86(分蘗數(shù)為9.0±3.6)相比,突變體srtl的分蘗明顯少于對(duì)照,只有1-4個(gè)分蘗(0.5±0.7)。(2)srtl的莖比野生型粗壯。從莖的橫切片結(jié)果來(lái)看,srtl的莖微管束發(fā)育比野生型多,能提供足夠的養(yǎng)分給整個(gè)植株；縱切結(jié)果顯示其細(xì)胞排列比野生型更有規(guī)律。(3)srtl的葉片顯得比野生型長(zhǎng)且寬。野生型與突變體srt1葉片的長(zhǎng)寬比分別為：52.2±3.34：59.5±2.1和1.55±0.2：2.05±0.1。(4)srtl表現(xiàn)為完全不育�；ㄋ幍馊緦�(shí)驗(yàn)顯示突變體的花藥表現(xiàn)正常。正反交實(shí)驗(yàn)結(jié)果顯示,用srtl作為父本,野生型作為母本結(jié)實(shí)率為9.9土6.8%,然而反過(guò)來(lái)srt1作為母本時(shí)顆粒無(wú)收,說(shuō)明srtl表現(xiàn)為雌性不育。(5)通過(guò)BSA-seq方法比較突變DNA池和野生DNA池間SNP頻率分布,計(jì)算CAAFD值,發(fā)現(xiàn)在水稻4號(hào)染色體長(zhǎng)臂末端約(29-34M)處CAAFD有一個(gè)明顯的峰。在該期間開發(fā)INDEL標(biāo)記,將目標(biāo)基因定位在231Kb范圍內(nèi)。(6)在231kb區(qū)間內(nèi)共有37個(gè)ORFs。我們發(fā)現(xiàn)其中LOC_Os04g56780與野生型相比有21個(gè)堿基缺失。LOC_Os04g56780編碼水稻中與擬南芥Wuschel (AtWUS)的同源基因OsWUS。(7)突變體中OsWUS的21bp發(fā)生在第一個(gè)外顯子上,使OsWUS的一個(gè)高度保守結(jié)構(gòu)域Homeobox中缺失了7個(gè)氨基酸。該缺失可能導(dǎo)致OsWUS功能喪失,說(shuō)明Homeobox結(jié)構(gòu)域在OsWUS的功能上起重要作用。2. Pepper-Shaped husk 1：一個(gè)小花形狀異常的突變體。(1)突變體pshl-1表現(xiàn)為穗變短且密,其穗長(zhǎng)僅有野生型明恢86的20%。pshl-1的小花呈現(xiàn)辣椒狀,其外稃正常,外稃明顯比野生型小。(2)pshl-1的結(jié)實(shí)率僅有5.8%±7.2,遠(yuǎn)遠(yuǎn)小于野生型的結(jié)實(shí)率(85.2%±4.9)。開花時(shí)突變體的表型不正常,僅有一小部分突變體6.55%±5.3的花開啟后能正常關(guān)閉,而野生型種大部分(84.65%±6.7)的花都能正常開花及關(guān)閉。(3)psh1-1的種子比野生型要小。突變體的粒長(zhǎng)(8.7±0.48)和粒寬(1.975±0.07)明顯小于野生型。pshl-1的粒形表現(xiàn)為辣椒狀,其千粒重(28.67±0.65)明顯低于野生型(15.0±1.18)。(4)通過(guò)F2群體的遺傳分析顯示,突變體pshl-1表現(xiàn)為辣椒穎和低結(jié)實(shí)率的性狀受一對(duì)隱性等位基因控制。(5)應(yīng)用BSA-seq方法定位PSH1,通過(guò)計(jì)算CAAFD值發(fā)現(xiàn)在水稻4號(hào)染色體斷臂末端0-3Mb處有一個(gè)高峰。經(jīng)連鎖分析將該基因定位在218Kb的區(qū)間內(nèi)。(6)該218Kb區(qū)間內(nèi)有31個(gè)ORFs。通過(guò)不同基因池中序列比對(duì)發(fā)現(xiàn),LOC_Os04g01590基因內(nèi)部的一個(gè)單堿基替換(G→T)導(dǎo)致終止密碼TAA的產(chǎn)生,是蛋白質(zhì)提前終止。(7) LOC_Os04g01590編碼精氨酸酶。它有6個(gè)外顯子,編碼340個(gè)氨基酸。突變體pshl-1中的第三個(gè)外顯子上的G到T的替換,及pshl-2中的第二個(gè)外顯子上的G到T的替換都能產(chǎn)生終止密碼TAG,使蛋白質(zhì)提前終止。這些結(jié)果顯示突變體表現(xiàn)出的突變性狀是由基因LOC_Os04g01590突變?cè)斐傻摹?8)氧化應(yīng)激分析顯示突變體pshl和野生型無(wú)明顯差異。
[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.

【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S511

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1 Mjomba Fredrick Mwamburi;利用BSA-seq圖位克隆兩個(gè)水稻重要基因[D];福建農(nóng)林大學(xué);2016年

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本文編號(hào)：1763533