【作者】 李霞；

【导师】 涂金星；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2013， 博士

【摘要】 油菜是我国重要的油料作物,菜籽油是人们生活用油的主要来源。随着国民经济的发展,生活水平的不断提高,对食用油品质的要求也在日益提高。因此,油菜品质育种逐渐成为现代油菜育种的重要组成部分。相同遗传背景下,黄籽油菜较黑籽油菜具有种皮薄、纤维素含量低、含油量高、饼粕蛋白质含量高等诸多优点。同时其榨取的菜籽油杂质少、清澈透明,易受消费者青睐,业已成为油菜品质育种中优良的种质资源。但大面积种植的甘蓝型油菜中并不存在天然的黄籽材料,使得这一优良性状在实际生产中的推广应用成为难题。目前很多科学家利用油菜的近缘种来创造甘蓝型油菜黄籽种质。白菜型油菜作为栽培油菜基本种,蕴含着丰富的黄籽资源,是创建甘蓝型油菜黄籽种质的重要物种之一。本研究通过白菜型黄籽油菜种皮色泽基因的克隆与功能分析,探讨了白菜型黄籽油菜形成的生物学基础。主要内容如下：1.初步定位显示,白菜型油菜种皮色泽基因位于A9连锁群SSR标记Lsr126与bsrl之间。在此基础上,利用http://brassicadb.org/brad/网站提供的白菜Scaffold序列信息,开发与目标基因共分离的SSR标记,最终锁定目标基因区段。该区段与拟南芥基因组同源线性比对分析显示,共包含at4g09820到at4g10620共计22个拟南芥同源基因。2.依据目标基因区段与拟南芥线性比对结果及白菜型油菜IP引物群体验证,选定BrTT8基因为候选基因。利用Tail技术和白菜基因组序列信息,设计特异引物TuLl/Rl,对白菜型油菜BrTT8基因进行全长扩增。3.等位基因序列分析显示,在近等基因系黄籽材料中BrTT8基因内存在大片段序列插入。插入序列分析表明,该序列具有新型转座子Helitron的结构特征：插入序列位于保守碱基AT之间,5’端起始于TC,3’末端结束于CTAG,在3’端上游6bp处有一段17bp长的回文序列。4. BrTT8基因在白菜型油菜近等基因系黑、黄籽材料中表达差异检测显示,黄籽材料开花后10天、20天、30天的种子中均未检测到BrTT8基因转录产物,而在黑籽材料中BrTT8基因正常转录。5.BrTT8基因全序列提交http://www.expasy.org/tools/protparam/网站进行蛋白结构预测,结果显示BrTT8蛋白包含520个氨基酸,分子量为59.5kD,其编码一个bHLH蛋白,蛋白羧基端附近有一个典型的bHLH结构域。6.利用农杆菌介导的转化方法,将BrTT8基因转入拟南芥tt8-1突变体,最终获得25个表型恢复的T1代拟南芥转化株。同时通过香草醛染色分析,转基因植株表型恢复源于原花色素在种皮的积累。由此说明,BrTT8基因有着与拟南芥TT8基因相似的功能,都参与了原花色素合成途径。7.白菜型油菜近等基因系黑、黄籽材料间,类黄酮途径结构基因Real-Time PCR分析显示,在黄籽材料中,晚期结构基因BrDFR、BrBAN表达量明显下降。但早期结构基因BrTT6和BrTT7的表达量在黑、黄籽材料间没有显著差异。以上说明在白菜型油菜中,BrTT8同样参与了类黄酮途径晚期结构基因的表达调控。8.对白菜型油菜近等基因系黑、黄籽材料,发育中的种子切片观察发现,在白菜型油菜黑籽材料中原花色素经过染色处理后,可以形成红色小颗粒沉积于内种皮细胞层中,而在黄籽材料种皮细胞中,则没有原花色素的沉积。而且,原花色素积累的细胞层在黄籽材料中已不复存在,取而代之的是细胞残片类似物,散落在内种皮细胞层间。但在拟南芥tt8-1突变体与野生型拟南芥的种皮结构显微观察发现,突变体tt8-1内种皮与野生型相比,只存在色素积累的差别,并没有细胞层结构的变化。推测白菜型油菜BrTT8基因与拟南芥TT8基因在功能上虽然相似,但并非完全相同。9.白菜型油菜黄籽沙逊是甘蓝型黄籽油菜No.2127-17重要亲本之一,但在以No.2127-17为亲本的近等基因系群体间BrTT8并未表现出多态性,说明甘蓝型油菜No.2127-17种皮色泽性状相关的N9连锁群上的主效QTL位点并非是BrTT8基因位点。综上所述,通过白菜型油菜种皮色泽相关基因BrTT8的克隆及功能分析,可初步将白菜型油菜黄籽沙逊与黑籽材料3H129所构建的近等基因系群体中,种皮色泽分离的分子机制概括如下：在该群体中,黄籽材料BrTT8基因由于转座子的大片段插入,引起了BrTT8基因序列的突变,从而导致这一重要转录因子功能的紊乱,使得类黄酮代谢途径中晚期结构基因不能正常转录,原花色素合成受阻,继而不能在黄籽材料内种皮中积累色素,最终种皮呈现无色透明,种子发育为黄籽。更多还原

【Abstract】 With the sustainable development of national economy, more and more breeders pay attention to rapeseed quality. Yellow-seeded rapeseed is characterized with thinner seed coat and lower hull percentage, which in turn is correlated with higher oil and protein content compared to black-seeded rapeseed under the same genetic background. In addition, the rough oil color is shallow and clear of yellow-seeded rapeseed, which can reduce the manufacturing cost and easy to popular with consumers. So they have been the excellent germplasm resources for rapeseed quality breeding. But there were no natural yellow-seeded material in Brassica napus. This situation brings difficult problem in yellow seeded traits application. As the basic species of rapeseed, B.rapa has abuntant yellow-seeded germplasm, which used to create several artificial synthetic yellow-seeded varieties of B. napus. Through the method of cytology and molecular biology, the seed coat color gene has been cloned in B.rapa. The main points in this study as follow:1. The seed coat color gene was located on linkage group9corresponding with the two molecular markers Isr126and bsrl. In addition, we developed co-separation SSR molecular markers that linked to the seed coat color gene from the Scaffold sequence on A9from http://brassicadb.org/brao/.Blast analysis with the Arabidopsis genome showed that the sequence was similar to a region of chromosome4. There are22Arabidopsis genes in the region including at4g09820to at4g10620.2. BrTT8gene was seleced as candidate gene, we used tail-PCR technology and B.rapa genome sequences as reference for designing specific primer pair Tu1L and Tu1R to clone the full-length TT8ortholog gene by PCR amplification.3. Sequence analysis of two alleles revealed a large insertion of a new class of transposable elements, Helitron in yellow sarson. The sequence analysis showed that the inserted fragment contained the structural characteristics of a recently discovered class of transposable elements in eukaryotes, termed Helitron. The insertion was4320bp, starting with5’TC, ending with3’CTAG, and containing two short palindromic sequences that were possibly formed by the17-bp hairpin that was located near its3’terminus.4. We determined the level of mRNA expression in the seeds of the yellow-seeded line in comparison with that of the black-seeded line, to detect the expression changes of the Brassica TT8orthologue gene due to the insertion. There was no mRNA expression detected in the immature seeds of yellow-seeded line. 5. The BrTT8gene encodes a putative bHLH protein consisting of520amino acids with a predicted molecular weight of59.5kD, and a pI of5.45(http://www.expasy.org/tools/protparam/). The BrTT8protein sequence contains a typical bHLH signature near the C terminus that corresponds with a putative binding domain (http://www.expasy.ch/tools/scanprosite/).6. Through the method of agrobacterium-mediated transformation, BrTT8gene was transported into Arabidopsis mutant tt8-1for function complementary experiments.The T2seed progeny originating from25independent T1kanamycin-resistant transformants exhibited phenotypic reversions in seed color compared with the wild type. Furthermore, analysis of PAs deposition during seed development was performed by staining PAs with vanillin in the wild type and T2. These data indicated BrTT8was also involved in the PA synthetize pathway.7. The expression level of five flavonoid genes, from two group EBGs and LBGs, were analyzed during the development of seeds in yellow-seed line and black-seed line, respectively. Quantitative Real-time PCR (QRT-PCR) analysis revealed that BrTT8was necessary for the expression of flavonoid LBGs, such as LDOX and BAN in the developing seeds, similar to TT8in Arabidopsis.8. Histological analysis showed that the black color of seeds was also due to the PAs accumulation in the plant endothelium layers of immature seeds. However, there was no pigment present in the seed coats of the yellow-seeded plants. Compared with the black-seeded plants, there was no accumulation of red granules in the endothelium layers of yellow seed coat after Safranine O and Fast Green treatments. But compare the seed coat structure of mutants tt8-1with wild-type in Arabidopsis, we found that only the pigment accumulation was different from the wild type, and no change was detected in structure of the seed coat cell Iyer in the tt8-1mutant. Which means BrTT8gene is similar, not completely the same, on the function with TT8gene that participate in the flavonoids synthesis. The BrTT8gene seems to have effect on the structure of seed coat.9. Yellow sarson is a major yellow-seeded germplasm, and used to create artificial synthetic yellow-seeded varieties of B. napus No.2127-17. BrTT8gene doesn’t show polymorphism in the nearly isogenic line population of No.2127-17. These data showed that the seed coat color main QTL locus of B. napus on N9linkage group is not derived from the BrTT8gene. In conclusion, we reported the isolation and functional characterization of the BrTT8in this study. The molecular mechanism of seed coat colore formation between the black and yellow seeded line in nearly isogenic line population as forllow:the alleles in the yellow seeded line showed that a transposable element could disturbed the BrTT8expression in B.rapa. BrTT8encodes bHLH proteins, which is important transcription factor in plant. Additional experiments also demonstrated that BrTT8modulated the expression of flavonoid "LBGs", and the BrBAN could not be normal transcripted, and the proanthocyanidin synthesis biological was blocked. So there was no pigment present in the seed coats of the yellow-seeded plants, and the seed coat showed the colorless, transparent. Therefore, the seed colore also became the yellow.更多还原