【作者】 杨庆勇；

【导师】 周永明；

【作者基本信息】 华中农业大学 ， 作物生物技术， 2012， 博士

【摘要】 油菜是世界上最重要的油料作物之一。菜籽油中脂肪酸组成直接决定其食用品质和营养价值。高油酸、低亚麻酸菜籽油在加工、储运和煎炸时对氧化不敏感,具有很好的热稳定性和较长的货架期。高油酸菜籽油还可作为生产优质生物柴油、环保型润滑剂等工业产品的优质原料。因此,进一步提高菜籽油中油酸含量和降低亚麻酸含量是当前油菜脂肪酸改良的主要目标。脂肪酸脱氢酶基因FAD2和FAD3是控制植物种子中十八碳不饱和脂肪酸含量的主要基因,但目前对多倍体甘蓝型油菜中高油酸、低亚麻酸变异的遗传控制,以及FAD2和FAD3基因在甘蓝型油菜基因组中的拷贝数、分布、表达模式及各拷贝的功能还不完全清楚。本试验利用QTL定位、基因克隆和芸薹属基因组生物信息学分析,研究甘蓝型油菜中高油酸、低亚麻酸的遗传控制,以及其主效基因FAD2和FAD3在油菜中的拷贝数、分布、表达模式和基因功能,并开发出适用于选育高油酸、低亚麻酸油菜的稳定性好、适用范围较广的等位基因特异标记。取得的主要结果如下：1.通过生物信息学分析和基因克隆鉴定了白菜、甘蓝及甘蓝型油菜中所有的FAD2、FAD3基因拷贝。在白菜和甘蓝基因组中,FAD2各有两个拷贝；廿蓝型油菜存在4个FAD2基因拷贝,在A、C基因组各有两个。FAD3在甘蓝和白菜基因组中各有3个拷贝,甘蓝型油菜中共6个,在A、C基因组各有3个拷贝。序列分析表明,位于白菜A1染色体及甘蓝型油菜A1连锁群上的FAD2基因很可能是一个假基因。进化分析表明,白菜、甘蓝、甘蓝型油菜中FAD2和FAD3基因分别可分成2组和3组,同一组内的各拷贝在RNA水平和蛋白质水平上均高度相似；同一组内各个基因之间的相似性显著高于不同组的两个基因之间的相似性,而与其所处的基因组(A/C基因组)和物种无关；拟南芥、白菜、甘蓝和甘蓝型油菜中的FAD2基因在核酸和蛋白质水平上均高度保守,FAD3基因在mRNA水平和蛋白质水平上高度保守,而在基因组水平上差异很大,主要体现在内含子序列的长度和相似性上。2.QTL定位表明,控制本试验群体油酸含量的主效QTL位于甘蓝型油菜的A5连锁群上,该QTL解释了89%的表型变异。分子克隆发现,高油酸亲本SWHickory中BnaA.FAD2.a(LG A5)拷贝存在一个4bp的插入,该突变造成开放阅读框的移码,最终导致提前终止密码子。该突变与前人报道的完全不同,属于一种新的高油酸等位基因。在A4和C4连锁群上各检测到一个控制亚麻酸含量的主效QTL,分别解释了亚麻酸含量31%和60%的变异。低亚麻酸亲本SW Hickory中BnaA.FAD3.b (LG A4)和BnaC.FAD3.b (LG C4)基因编码区域分别存在2个和1个SNP位点,其中BnaA.FAD3.b位点上的第一个SNP位点为同义突变,另一个为错义突变；BnaC.FAD3.b上的单碱基突变造成基因内含子剪切失败,导致内含子通读,并造成开放阅读框的移码和提前终止密码子。同时,在A5、A6连锁群中各检测到1个效应较小的亚麻酸含量QTL,其贡献率分别为7%和2%。SW Hickory中BnaA.FAD3.a (LGA5)基因在第269位有一个T的缺失,该突变造成移码并最终导致提前终止密码子。3.利用RNA测序数据分析了甘蓝型油菜FAD2和FAD3基因各拷贝的时空表达模式。结果表明,本研究所鉴定的油酸含量功能拷贝BnaA. FAD2. a,以及三个亚麻酸含量功能拷贝(BnaA.FAD3.a, BnaA.FAD3.b和BnaC.FAD3.b),在油菜脂肪酸合成最为旺盛的种子发育中后期(14-25DAF)表达量急剧上升,为营养生长时期的7-20倍。各功能拷贝在14-25DAF种子中的表达量高低与其解释的油酸、亚麻酸含量遗传变异率一致。4.根据BnaA.FAD2.a, BnaA.FAD3.a, BnaA.FAD3.b和BnaC.FAD3.b在基因组序列水平上的差异,获得了油酸含量的等位基因特异共显性SCAR标记；利用引物3’-端错配技术设计了A、C基因组上亚麻酸含量的等位基因特异共显性SNP标记。上述标记在分子标记辅助选择中不受环境和遗传背景影响,与种子中油酸和亚麻酸含量高度一致,为甘蓝型油菜高油酸低亚麻酸育种提供了简单、快速、经济和有效的分子标记。更多还原

【Abstract】 Oilseed rape(Brassica napus L.) is one of the most important oil crops worldwide. The edible and processing quality of rapeseed oil is mainly determined by the fatty acid composition of the triglycerol lipid in its seeds. High C18:1and low C18:3content in rapeseed oil is not sensitive to the oxidation when processing, storage and frying, therefore desirable for its good thermal stability and long shelf life. In addition, high oleic acid oil is also a renewable raw material for biodiesel and environmentally friendly lubricants production. Therefore, one of the major goals for quality breeding after removal of erucic acid in oilseed rape is to increase the C18:1content and reduce the C18:3content in the seeds of oilseed rape cultivars. The fatty acid desaturase genes FAD2and FAD3have been shown as the major genes for the control of C18:1and C18:3contents. However, the copy number, locus distribution, genes structure, gene expression and gene function of the two gene families in amphidiploid Brassica napus are still not completely understood to date. Based on QTL mapping, molecular cloning and bioinforamtic analysis of the transcriptome data from Brassica napus, we studied the copy number, locus distribution, genes structure, gene expression and gene function of the FAD2gene and FAD3gene families of amphidiploid Brassica napus in this study. Furthermore, based on the results of genetic mapping and identified sequences, allele-specific markers of FAD2and FAD3genes were developed for breeding of high oleic, low linolenic acid rapeseed. The main results are as follows:1. In the present study, all copies of FAD2and FAD3genes in the A-and C-genome of B. napus and its two diploid progenitor species, B.rapa and B.oleracea, were identified through bioinformatic analysis and extensive molecular cloning. Two FAD2genes exist in B.rapa and B.oleracea, and four copies of FAD2genes exist in B.napus. Three and six copies of FAD3genes were identified in diploid species and amphidiploid species, respectively. Through data mining and molecular cloning of FAD2, FAD3genes in Brassica species, we showed that:1) there is a naturally mutated ORF for one of the FAD2locus in A-genome (Al) of both B.rapa and B.napus. The locus contained five insertions or deletions, which resulted in a frame shift starting and premature termination codon (PTC), likely representing a pseudogene of FAD2in A-genome.2) Based on the protein sequences, the FAD2and FAD3genes from B.napus and their corresponding counterparts in two diploid species could be grouped into two, three clusters, respectively. And members belonging to the same category were more closely related to each other regardless of their genome and species origins.3) The sequences of the FAD2gene in Arabidopsis and three examined Brassica species were highly similar among the loci. On the other hand, the genomic sequences of the FAD3gene are rather divergent, mainly reflected in the length and similarity of the introns sequences. Interestingly, at protein and cDNA levels, both FAD2and FAD3members among the loci share high similarities.2. The genetic control of high C18:1and low C18:3contents in a double haploid population was investigated through mapping of the quantitative trait loci (QTL) for the traits and the molecular cloning of the underlying genes. One major QTL of BnaA.FAD2.a located on A5chromosome was responsible for the high C18:1content, which accounting for89%of the trait variation. A4bp insertion mutation in the BnaA.FAD2.a locus was uncovered, which resulted in a frame shift starting and PTC. And that represented a previously unidentified allele for the high oleic variation in Brassica napus species. Two major QTLs on A4and C4chromosomes were found to be responsible for the low C18:3content in the DH population as well as in SW Hickory, which explained about31%,60%of the variation of C18:3content, respectively. Two single nucleotide mutations in the parents SW Hickory were identified in the locus of BnaA.FAD3.b (LG A4). The first one was a T to C substitution in exon2leading to a synonymous mutation, and the second a C to T transition in exon7, which resulted in an amino acid substitution from arginine to cysteine. BnaC.FAD3.b (LG C4) was a G to A substitution in the5’-end of intron6in SW Hickory, which resulted in an abnormal splicing with the entire sixth intron being retained in the mature BnaC.FAD3.b transcript, eventually resulted in frameshift and the PTC. Meanwhile, LNAA5and LNAA6were detected in three and one years, which explained about7%,2%of the variation of C18:3content, respectively. BnaA.FAD3.a (LG A5) was a T deletion mutation in269th base of the genome and cDNA sequences in SW Hickory, which resulted in frameshift and eventually led to the PTC.3. With the RNA sequencing data of Brassica napus, gene expression in FAD2and FAD3genes has been compared in different tissues and developmental stages. The results showed that, in the mid-late stages of seed development (14-25DAF), which is the most vigorous in fatty acid synthesis in rapeseed, the expressions of function locus(BnaA.FAD2.a) of oleic acid content, and three functional loci (BnaA.FAD3.a, BnaA.FAD3.b and BnaC.FAD3.b) of linolenic acid content exhibited a sharp rise, as much as7-20time higher than in other developmental stages. The gene expression pattern of each locus is highly consistent with their contributions to the variation of oleic acid or linolenic acid content.4. Based on the polymorphism information of BnaA.FAD2.a, BnaA.FAD3.a, BnaA.FAD3.b and BnaC.FAD3.b genes sequence, allele-specific codominant SCAR marker were developed for oleic acid. And single-nucleotide amplified polymorphisms (SNAP) codominant markers for FADS genes by utilizing the SNAPER program. The allele-specific codominant markers aided selection can greatly enhance the efficiency and effectiveness of plant breeding relative to conventional methods, thereby overcoming the shortcomings of phenotypic selection in the process of the HOLL breeding of Brassica napus. Genotyping can be reliably performed at an early stage of plant growth, avoiding the need to conduct large scale of field trials. The allele-specific marker is demonstrated to be time-and cost-effective in developing HOLL varieties.更多还原