摘要
油菜属于十字花科芸薹属,是产油效率最高的油料作物之一,它不仅是食用油的主要来源,也是解决全球能源短缺的重要原料作物。油菜种子发育过程中的油脂积累和脂肪酸代谢很大程度上存在变异,变异的原因除了控制油脂合成的关键基因以外,也包括应对环境和内源性刺激的大量基因。研究人员和育种家们花费了大量的精力鉴定参与油菜油脂形成的关键基因,但是基因与环境存在互作使鉴定过程非常复杂,至今尚无通过改变单个基因大幅提高含油量的报导。本论文的研究目的在于：利用拟南芥与油菜的比较基因组学,开发一系列油菜油脂功能基因分子标记；在DH系的分子标记连锁遗传图谱上进行含油量的基因定位并鉴定关键候选基因；以响应高温的含油量近等基因系为材料,探讨环境因子与QTL互作的分子机制。主要研究结果如下：
1.油菜油脂功能基因分子标记的开发：根据拟南芥油脂基因数据库(http://lipids.plantbiology.msu.edu/)中通过对拟南芥全基因组序列上与油脂相关基因的序列预测,并结合每个基因在各个器官中的表达序列标签(EST)及其分布的统计分析,从中选出75个在种子中高表达的基因通过与油菜数据库的搜索比对,利用油菜中的同源EST来设计引物,分析有关基因在油菜品种Tapidor和Ningyou 7之间的等位多态性,共在75个基因上设计了150对引物,平均每个基因包含两对引物。筛选标记发现,共51对引物具有多态性,多态性比率超过30%。
2.含油量QTL的定位分析：在甘蓝型油菜DH系中,将开发的分子标记连锁到华中农业大学已有的TN-DH图谱上并进行QTL扫描,结果连锁到遗传图谱上的标记共15个；共检测到5个标记在含油量QTL区间峰内,分别位于第1、3、10、11以及12连锁群上,因此我们推测这5个候选基因可能在油菜种中油脂合成过程中扮演着非常重要的角色。生物信息学分析表明,这5个候选基因的功能均与油脂代谢相关。
3.含油量QTL区间内关键候选基因的鉴定：为了进一步验证QTL置信区间内的候选基因的功能,我们利用其拟南芥同源基因的相关位点的T-DNA插入突变体,测定突变体成熟种子的含油量及脂肪酸组分,并与野生型进行比较分析,结果5个关键基因位置上的等位突变体中,有3个基因位点上的等位变化导致总含油量发生了显著变化,T-DNA插入导致突变体含油量变低；2个基因位点上的等位变化没有导致总含油量的显著变化,但却导致了亚麻酸和油酸的含量发生了显著变化,因此这些标记可以用于分子标记辅助育种选择。
4.温度对油菜近等基因系含油量和脂肪酸组分的影响：油菜近等基因系总含油量随着成熟期温度的升高而降低,亚油酸(C18：2)、亚麻酸(C18：3)以及芥酸(C22：1)含量也随着温度的升高而降低。在T1、T2、T3三种温度下,NIL-9的总含油量均高于NIL-1,但是差异度并不一致,最大的差异是在高温T3条件下,低温T1次之,差异最小的在中温T2条件下。
5.利用基因芯片研究了温度对近等基因系种子发育过程中基因表达的影响,结果表明：基因型、温度以及基因型与温度的互作在基因组水平分别引起4982个、19111个839个转录本的差异表达。两个近等基因系在T2条件下差异表达基因数目最少,基因组水平有251个,位于qOC.C2.2 QTL区间的有39个；2个近等基因系在T1条件下的差异表达基因数目,全基因组水平有2933个,位于qOC.C2.2 QTL区间的有460个；2个近等基因系在T3条件下差异表达基因数目最多,全基因组水平有3499个,qOC.C2.2 QTL区间有558个。这一研究结果与含油量的表型差异是一致的。
6.目标QTL qOC.C2.2区间内的差异表达基因功能分类：定位于QTL区间内的基因型差异表达基因可以被分成很多的大类,包括：调控胚发育、DNA的转录调控、生物非生物逆境响应(例如：氧化还原反应；植物盐胁迫生理；热激反应；植物受伤反应；镉胁迫反应)、光合作用相关(例如：光呼吸；光合系统化学调节；糖酵解作用；苹果酸代谢)等生理过程的基因家族。除此之外,还有一些差异表达基因的功能与蛋白质代谢相关,例如蛋白质的合成、蛋白质折叠以及蛋白质的胞间运输、转录起始、茉莉酸刺激响应等。
7.温度影响QTL区间内的差异表达基因功能分类：种子成熟期温度降至T1或者增至T3均可引起一系列基因的上调,这些基因涉及：DNA的转录调控、胚发育、生物非生物逆境响应(例如：抑菌反应；镉胁迫反应)、蛋白质的合成、氨基酸磷酸化、蛋白质折叠等。与T2条件相比,T3上调了一系列基因的表达,这些基因的功能涉及：热激反应以及泛素依赖的蛋白质降解过程,但是下调了控制脂肪酸的合成、响应红光信号、光合作用,赤霉素刺激响应以及转录延伸的有关基因。与T2条件相比,T1上调了一系列基因的表达,这些基因的功能包括：糖酵解、苹果酸代谢、脱落酸刺激响应、三羧酸循环以及脱水应激反应。
8.不同温度条件下油脂合成关键基因的表达模式：NIL-1和NIL-9之间的遗传差异对含油量合成相关基因BnLACS1, BnOLEO1, BnCLO1, BnFatA以及BnLHY有显著影响。与NIL-1相比,NIL-9的BnLACS1, BnCLO1以及BnLHY基因表达量高,但BnOLEO1以及BnFatA的表达水平低于NIL-1。温度升高上调了BnAB13, BnFUS3, BnTAG1, BnOLEO1,BnCLO}以及BnFaTA的表达量,但是下调了BnLEC1, BnWRI1, BnFAD2, BnFAD3,以及BnLPAT2的表达量。G×T互作显著影响了BnFAB2基因的表达,只有在T1条件下,NIL-9的表达量高于NIL-1。
Oilseed rape(Brassica napus L.) is one of the most efficient oil crops throughout the world, it is not only a major source of edible oil but also can be used as biodiesel to solve the shortage of petrolic oil. Oil accumulation and fatty acid metabolism during seed development exist in large variations, including a large number of genes response to environmental and endogenous stimuli. Researchers and breeders have spent a lot of efforts to identify key genes related to oil formation, but so far not suffcient knowledge reflecting the genetic control of seed oil has been acuqired. This study is aim to:(1) develop a series of molecular markers of functional genes involved in oil accumulation based on the comparation of the Arabidopsis and Brassica genomes; (2) map molecular markers involved in oil accumulation on the genetic linkage map using DH lines and to identify key candidate genes contributing to certain QTL effct; (3) investigate the molecular mechanisms of the interaction between QTL and environmental factors using NILs whose oil content was in response to growth temperature during seed development. The main results are as follows:
1. The genome of Arabidopsis has been searched for sequences of genes involved in acyl lipid metablism(http://lipids.plantbiology.msu.edu/). According to the sequence information of Arabidopsis lipid gene database and the analysis of the distribution of ESTs in organs,sequences of 75 seed-specific lipid genes were selected to do BLAST for homologous Brassica ESTs, a search for intron polymorphisms within the EST database was conducted. A set of 150 PCR primer pairs was designed according to Brassica ESTs. For each gene we designed 2 primer pairs, of which 51 showed polymorphisms between Tapidor and Ningyou 7. The polymorphsim rate was over 30%.
2. A total of 15 markers on the seed expressing function genes were linked to the TN-DH map, which was made up by a total of 19 linkage groups and 700 markers that span over a 2060 cM distance with an average interval of 3.3 cM. Of these,5 unique markers were detected in the QTLs region contributing to oil seed oil cntent. The genes located on the 1,3,10,11,12 linkage group. Bioinformatic analysis suggested that these candidate genes involved in oil formation.
3. In order to confirm the function of the candidate genes putatively contributing to the QTLs, we analysized the allelic variation of the respective loci among Arabidopsis T-DNA insertional mutant. The results indicated that the T-DNA insertion on three of the five loci resulted in signifcant change of seed oil content, whereas, the T-DNA insertion on the rest loci did not give rise to the change of total seed oil content, but instead to the change of specific fatty acid ratio.
4. Temperature had a clear effect on seed oil content as well as fatty acid composition. Overall, the NIL plants responded to increasing temperature with decreasing levels of total seed oil, linoleic acid (C18:2), linolenic acid (C18:3), and erucic acid (C22:1). NIL-9 had higher seed oil content than did NIL-1 in all three growth chambers. However, the degree of surplus varied. The highest surplus was found in Chamber T3 and the lowest in Chamber T2.
5. Statistical calculations indicated that the effect of genotype, temperature, and the interaction between genotype and temperature on the expression of the genes in 25 DAF seeds were all significant at a 0.01% false discovery rate, generated 19 111,4 982 and 839 DEGs (Diffenentially expressed genes, DEGs) respectively. The smallest expression differences were found under T2, under which NIL-1 differed from NIL-9 with the expression of 251 genes globally and 39 genes at the qOC.C2.2 region. More genetic divergences were observed under T1, under which the NILs differed from each other with 2 933 DEGs globally and 460 DEGs at the qOC.C2.2 region. The greatest difference was caused by T3, under which the NILs differed from each other with 3 499 DEGs globally and 558 DEGs at the qOC.C2.2 region. This results were consistant with the oil content phenotype.
6. The 246 DEGs resulting from genotype can be grouped into various GO categories. Of these, a high proportion of GO slims were specifically related to embryo development, DNA-dependant transcription regulations, stress responses (such as oxidation reduction; salt stress response; heat response; wound response; cadmium stress response), and photosynthesis (photorespiration; photosystem stoichiometry adjustment; glycolysis; malate metabolism). These were over represented on the list of DEGs arising from genotype. A number of DEGs also belonging to the GO categories are involved in more general biological processes. Of these, some are associated with protein biosynthesis, protein folding and protein intercellular transport, translational initials, jasmonic acid response.
7. Either increasing the temperature to T3 or decreasing the temperature to T1 resulted in the up-regulation of the genes related to DNA-dependent transcription regulations, embryo development, stress response (bacterium defending response; cadmium stress response), protein biosynthesis, amino acid phosphorylation, and protein folding. Increasing the temperature to T3 caused the up-regulation of the genes involved in heat response and ubiquitin-dependent protein catabolism, but the down-regulation of several genes associated with fatty acid biosynthesis, red light response, photosynthesis, gibberellic acid stimulus response, and translational elongations. Decreasing the temperature to T1 led to the down-regulation of some genes regulating glycolysis, malate metabolism, abscisic acid stimulus response, the tricarboxylic acid cycle, and water deprivation response. Either increasing temperature to T3 or decreasing temperature to T1 resulted in the down-regulation of the genes of the cold response category and the up- and down-regulation of the genes of the oxidation reduction category.
8. The genetic difference between NIL-9 and NIL-1 had a significant effect on the expression of the genes BnLACS1, BnOLEO1, BnCLO1, BnFatA, and BnLHY. Relative to NIL-1, NIL-9 had a higher expression level of BnLACS1, BnCLO1, and BnLHY, but a lower expression level of BnOLEO1 and BnFatA. Increasing temperature led to a higher expression of BnAB13, BnFUS3, BnTAG1, BnOLEO1, BnCLO1, and BnFaTA, but a lower expression of BnLEC1, BnWRI1, BnFAD2, BnFAD3, and BnLPAT2. The G×T interaction significantly affected the expression of BnFAB2. Treated only under T1, NIL-9 had a higher expression of BnLPAT2 than NIL-1.
引文
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