本文選題：甘藍型油菜 + 隱性核不育��； 參考：《西南大學》2017年碩士論文

【摘要】：甘藍型油菜是世界上重要的油料作物,利用隱性雄性不育授粉系統(tǒng)制備強優(yōu)勢雜交種是甘藍型油菜育種的重要內(nèi)容。本研究通過石蠟切片和掃描電鏡對甘藍型油菜隱性雜合兩型系SLA和SLB花藥發(fā)育的特點進行細胞學分析,初步確定不育系SLA發(fā)生敗育的類型和時期。利用NGS技術對兩型系SLA和SLB進行了轉(zhuǎn)錄組和基因組重測序分析,在基因組水平上分析SLA和SLB之間的遺傳變異;用轉(zhuǎn)錄組和qRT-PCR方法篩選在SLA和SLB中的差異表達基因;應用在線分析軟件Venny比較分析轉(zhuǎn)錄組差異表達基因和重測序發(fā)生變異的基因,確定與花藥敗育相關的候選基因。其主要結果如下:掃描電鏡分析結果顯示:不育系SLA的花藥顯著小于可育系SLB,整個發(fā)育過程花藥無明顯的伸長生長。當花蕾大小為2mm時,不育系SLA和可育系SLB花藥表層細胞無顯著差異;花蕾大小為4mm時,不育系SLA花藥表層細胞開始塌陷萎縮,背面細胞排列緊密;當花蕾開放后,不育系SLA花藥細胞完全萎縮、退化,花粉囊不能正常開裂釋放花粉。不同發(fā)育期花蕾石蠟切片甲苯胺藍染色的細胞學結果顯示:在四分體時期,不育系SLA和可育系SLB的花藥存在顯著差異,SLA的絨氈層細胞高度液泡化,不能皺縮、轉(zhuǎn)化為分泌型;隨后在小孢子發(fā)育過程中逐漸降解消失,不能正常發(fā)育為成熟小孢子。因此,不育系SLA發(fā)生敗育主要原因可能是在四分體時期或四分體之前絨氈層細胞的發(fā)育異常而導致。通過對兩型系SLA和SLB花蕾轉(zhuǎn)錄組數(shù)據(jù)比較分析,共獲得101個在不育材料SLA中顯著上調(diào)表達的差異基因。采用GO功能分類將三個類別中已注釋基因劃分為25個功能類別。101個差異表達基因中,在分子功能方面富集最多,有55個基因發(fā)生富集,主要在離子結合、電子載體活性等能量代謝相關代謝過程;在生物過程方面有44個基因發(fā)生富集,主要在碳水化合物代謝、蛋白水解、抗氧化反應等條目中;在細胞成分方面富集到2個基因,主要在胞外部分的條目中,說明甘藍型油菜育性調(diào)控是一個復雜的生物過程。KEGG代謝途徑分析發(fā)現(xiàn),差異表達基因僅在苯丙烷代謝途徑中顯著富集,共有4個顯著差異表達基因,包括1個ACOS5基因(BnaC09g10950D)和3個過氧化物酶家族基因(BnaA01g01280D、BnaA07g01880D、BnaC01g02300D),說明苯丙烷代謝途徑在不育系SLA發(fā)生敗育過程中起重要作用。通過對SLA和SLB全基因組重測序數(shù)據(jù)比較分析,在整個基因組中檢測到119,863個SNP位點和18,370個Indel變異位點,這些變異位點大部分發(fā)生在基因間或內(nèi)含子等非編碼區(qū)域,在各條染色體上的分布存在差異性。根據(jù)變異位點在參考基因組上對應的基因物理位置,共注釋到影響基因表達或者功能的錯義突變、移碼突變等變異的基因14,705個;同時針對這些變異基因進行GO功能富集分析,發(fā)現(xiàn)這些基因主要在核苷結合、碳水化合物衍生物結合、有機物代謝過程通路等條目發(fā)生富集。綜合轉(zhuǎn)錄組和全基因組重測序結果,共檢測到11個育性關鍵差異表達基因,其中3個基因(BnaA03Rf-4、BnaA10Rf-7、BnaC05Rf-10)存在2種以上位點變異類型。另外,在內(nèi)含子區(qū)域發(fā)生變異的基因有4個(BnaA03Rf-3、BnaA04Rf-5、BnaA10Rf-6、BnaAnnRf-8),發(fā)生同義突變基因2個(BnaA03Rf-4、BnaC03Rf-9),在上游或下游區(qū)域發(fā)生變異的有6個基因(BnaA01Rf-1、BnaA02Rf-2、BnaA03Rf-4、BnaA10Rf-7、BnaC05Rf-10、BnaC07Rf-11)。qRT-PCR結果顯示11個基因中有8個基因在SLA的Bu1(1 mm花蕾)中均顯著上調(diào)表達,qRT-PCR驗證的這8個基因可作為關鍵育性候選基因。
[Abstract]:Brassica napus is an important oil crop in the world. Using the Recessive Male Sterile pollination system to prepare strong heterosis is an important content of Brassica napus breeding. In this study, the characteristics of the anther development of SLA and SLB in the recessive heterozygous line of Brassica napus were analyzed by paraffin section and scanning electron microscope. The type and period of abortion in line SLA, the transcriptional and genomic resequencing analysis of SLA and SLB in type two lines was carried out by NGS technology, the genetic variation between SLA and SLB was analyzed at the genomic level, and the differential expression genes in SLA and SLB were screened by the transcriptional group and qRT-PCR method, and the transcriptional analysis was compared with the online analysis software Venny. The results of the scanning electron microscope analysis showed that the anther of SLA was significantly smaller than that of the fertile line SLB, and the anther was not elongated in the whole development process. When the bud size was 2mm, the sterile line SLA and the fertile line SLB were found. When the size of buds was 4mm, the surface cells of the sterile line SLA anther began to collapse and atrophy and the back cells were closely arranged. When the buds were open, the SLA anther cells of the sterile line were completely atrophied and degenerated. The pollen sac could not normally crack and release pollen. The cytological junction of the paraffin section of the bud in different development stages was stained with toluidine blue. The results showed that there were significant differences in anthers between the sterile line SLA and the fertile line SLB during the four division. The tapetum cells in SLA were highly vacuolated, and they could not be crinkled and converted to secretory type. Then, the degradation of the microspore was gradually disappearing and could not normally develop into mature microspore. Therefore, the main cause of abortion of the sterile line SLA may be in the process of abortion. The development of tapetum cells was abnormal before or before the four division or four division. Through comparison and analysis of the data of the SLA and SLB buds transcriptional group of the two type system, 101 differentially expressed genes were significantly up-regulated in the sterile material SLA. The GO functional classification was used to divide the annotated genes in the three categories into 25 functional categories.101 difference tables In the gene, the most abundant gene is enriched in the molecular function, and 55 genes are enriched, mainly in the energy metabolism related metabolic processes, such as ion binding and the activity of electron carrier. In the biological process, 44 genes are enriched, mainly in carbohydrate metabolism, protein hydrolysis, antioxidant reaction and so on. The enrichment of 2 in the cell composition is 2. A gene, mainly in the entry of the extracellular part, shows that the fertility regulation of Brassica napus is a complex biological process.KEGG metabolic pathway analysis. The differentially expressed genes are enriched only in the phenylpropane metabolic pathway, and there are 4 significant differentially expressed genes, including 1 ACOS5 genes (BnaC09g10950D) and 3 peroxidase families. Gene (BnaA01g01280D, BnaA07g01880D, BnaC01g02300D), indicating that the phenylpropane metabolic pathway plays an important role in the abortion process of the sterile line SLA. Through the comparative analysis of the whole genome resequencing data of SLA and SLB, 119863 SNP loci and 18370 Indel mutation sites are detected in the whole genome. Most of these mutation sites occur. In non coding regions such as the INTERGENE or intron, there are differences in the distribution of the chromosomes. According to the location of the genes corresponding to the reference genome, 14705 mutations, such as the missense mutation and the shift mutation, which affect the gene expression or function, are also annotated, and the GO function for these variants is performed at the same time. Enrichment analysis showed that these genes were mainly enriched in nucleoside binding, carbohydrate derivative binding, and organic matter metabolic pathway pathway, and 11 key differentially expressed genes were detected in the comprehensive transcriptional group and total genome resequencing, of which 3 genes (BnaA03Rf-4, BnaA10Rf-7, BnaC05Rf-10) had 2 kinds of epistasis In addition, 4 genes (BnaA03Rf-3, BnaA04Rf-5, BnaA10Rf-6, BnaAnnRf-8) were mutated in the intron region, and 2 (BnaA03Rf-4, BnaC03Rf-9) of the synonymous mutation (BnaA03Rf-4, BnaC03Rf-9) were occurring, and 6 genes (BnaA01Rf-1, BnaA02Rf-2, BnaA03Rf-4, BnaA10Rf-7, BnaC05Rf-10, BnaC07Rf-11) occurred in the upstream or downstream regions. The results showed that 8 of the 11 genes were significantly up-regulated in Bu1 (1 mm buds) of SLA, and these 8 genes verified by qRT-PCR could be used as key fertility candidate genes.
【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S565.4

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本文編號：1995562