【作者】 刘庆；

【导师】 邓秀新；

【作者基本信息】 华中农业大学 ， 果树学， 2008， 博士

【摘要】 芽变在柑橘中经常发生。长期以来,芽变选种是柑橘育种的主要方法,但是人们对于芽变性状形成的分子机理仍知之甚少。利用现代分子生物学技术对芽变性状形成的机理进行研究,将加深对芽变规律的认识:同时对提高芽变育种效率以及提高果实品质具有重要的指导作用。本研究以‘暗柳’甜橙(Citrus sinensis[L.]Osbeck)的红肉突变体‘红暗柳’为研究材料,以野生型作参照,分析了两者果实在发育过程中糖、酸、色素的变化规律和相关基因的表达,并采用SSH结合Microarray技术分析了两者基因组表达的差异。主要研究结果如下:1.完善了不同发育时期各类柑橘组织的RNA提取方法。该方法已成功从不同发育时期的柑橘有色层、白皮层、囊衣、汁胞、愈伤组织、茎、叶、花等组织中提取出高质量的RNA。而且,将整个RNA提取时间缩短为4个小时。2.利用HPLC测定了不同发育时期‘红暗柳’与‘暗柳’果实有色层、白皮层、囊衣、汁胞中类胡萝卜素的组成与含量变化。结果表明突变体果实中白皮层、囊衣和汁胞呈红色是番茄红素大量累积所致,其番茄红素的含量比野生型相应组织高上千倍。但两者叶片中类胡萝卜素的种类没有差异。3.利用Real-time PCR测定‘红暗柳’与‘暗柳’果实有色层、白皮层、囊衣、汁胞中类胡萝卜素代谢途径关键酶基因的表达。结果发现‘红暗柳’汁胞中的番茄红素积累是由于类胡萝卜素代谢途径中上游基因的上调表达和下游基因的下调共同作用导致的。然而,‘红暗柳’果实囊衣和白皮层中类胡萝卜素代谢途径上的基因表达量与‘暗柳’相比几乎没有变化,仅下游的Lcye基因似乎受到累积的番茄红素的诱导而上调表达。这表明调控‘红暗柳’果实白皮层和囊衣中番茄红素积累的机制与调控汁胞中番茄红素积累的模式不相同,即在‘红暗柳’果实中至少存在两种模式调控番茄红素的积累。结合其它实验结果,本文提出如下假说:番茄红素首先在汁胞中大量合成,然后被运输到囊衣和白皮层中从而导致了番茄红素也在这两个部位的积累。4.采用GC和Real-time PCR测定了‘红暗柳’与‘暗柳’果实有色层、白皮层、囊衣、汁胞中主要有机酸的含量以及代谢途径关键酶基因的表达。结果发现‘红暗柳’汁胞中的柠檬酸含量极低,仅为‘暗柳’的1/11到1/4之间。而且‘红暗柳’汁胞中柠檬酸代谢上游和下游关键酶基因的表达均要高于‘暗柳’,但下游基因升高的幅度要大于上游基因升高的幅度。通过测定两者汁胞中关键酶活性发现‘红暗柳’中柠檬酸上游和下游的酶活性均要高于‘暗柳’。似乎说明‘红暗柳’汁胞中柠檬酸大幅度减少与其下游基因的大量表达有联系。5.采用GC和Real-time PCR测定了‘红暗柳’与‘暗柳’果实色层、白皮层、囊衣、汁胞中主要可溶性糖的含量以及代谢途径关键酶基因的表达。结果发现‘红暗柳’汁胞中蔗糖的含量要显著高于‘暗柳’。合成蔗糖的蔗糖磷酸合成酶和分解蔗糖的蔗糖合成酶与转化酶在‘红暗柳’汁胞中的表达均要高于‘暗柳’。推测‘红暗柳’汁胞中积累蔗糖的原因可能是上游蔗糖磷酸合成酶的上调表达,当然也有可能是蔗糖在叶片中合成以后再运输至汁胞中。6.为了获得与突变体‘红暗柳’果实特异性状相关的基因,我们构建了‘红暗柳’与‘暗柳’果实的抑制性差减文库(SSH文库),然后采用Micrarray技术从文库中筛选在果实发育过程中差异表达的基因。在对差异表达的基因进行测序以及序列分析后,我们总共得到267条非重复性的基因,其中有182条与已知序列具有高同源性。很少有基因在果实发育过程中持续的上调或者下调表达,仅有一个基因(半胱氨酸蛋白酶前体)在‘红暗柳’果实中下调表达。经过SOTA聚类分析表明,95.1%的差异表达基因在170DAF时差异表达,而此时正是‘红暗柳’中大量积累番茄红素的时候。与细胞代谢、初生代谢、定位以及大分子代谢等相关的基因数目最多。这些基因所属的代谢途径包括:丙酮酸代谢途径、淀粉和蔗糖代谢途径、三羧酸代谢途径、糖酵解代谢途径已及类胡萝卜素合成途径等。而且,还发现13个与信号转导以及转录调控相关的基因,这些基因在170DAF时均差异表达。更多还原

【Abstract】 Bud mutations arise often in citrus. The selection of bud mutants is one of the most important breeding channels in citrus. However, the molecular basis of bud mutation has rarely been studied.In this study we describe a novel, pleiotropic sweet orange （Citrus sinensis [L.] Osbeck） mutant, ’Hong Anliu’. This mutation causes carotenoid accumulation, high sugar and low acid in the fruits. Gas chromatography analysis revealed that high sugar and low acid in the fruit were caused by the accumulation of sucrose and the deficiency of citric acid. The dominant carotenoid accumulated in albedo, segment membranes and juice sacs is lycopene, which can reach levels that are a thousand-fold higher than those in comparable wild-type fruits. This mutation does not affect carotenoid composition of leaves. Carotenoid concentration and biosynthetic gene expression of albedo, segment membranes and juice sacs were dramatically altered by the mutation. Lycopene accumulation in the juice sacs was regulated by coordinate expression of carotenoid biosynthetic genes. However, in albedo and segment membranes, the expression of downstream carotenogenic genes seems to be feedback induced by lycopene accumulation. This implies that there must be at least two modes regulating lycopene accumulation in ’Hong Anliu’ fruit. Taken together, these results suggest that massive lycopene might be synthesized in the juice sacs and then transported to the segment membrane and the albedo, which leads to lycopene accumulation there.Organic acid concentration and biosynthetic gene expression of albedo, segment membranes and juice sacs were dramatically altered by the mutation. In juice sacs of ’Hong anliu’, the concerntration of citric acid was among 1/4 and 1/11 of ’Anliu’. The expression of both upstream （CS） and downstream （Acon, IDH, MDH） genes of ’Hong Anliu’in the citric acid metabolism were higher than those of ’Anliu’. Moreover, the enzymes activities of downstream genes in citric acid metabolism were higher in ’Hong anliu’than those of ’Anliu’. Thus, we speculate that, the citric acid deficiency in the juice sacs of ’Hong anliu’ was caused by the up-regulation of the downstream genes.Sugar concentration and metablic gene expression of albedo, segment membranes and juice sacs were dramatically altered by the mutation. The concerntration of sucrose in the juice sacs of ’Hong anliu’ was significangly higher than that of ’Anliu’. The expression of sucrose cleavage genes （Ivr and SPS） and sucrose synthetic gene （SuS） in the juice sacs of ’Hong anliu’ were higher than those of ’Anliu’. We speculate that the sucrose accumulation in the juice of ’Hong anliu’ may be caused by the up-regulation of SuS. However, the sucrose accumulation in the juice of ’Hong anliu’ could also be synthesized in the leaves and transported to the juice sacs.To identify potential important or novel genes involved in a spontaneous sweet orange （C. sinensis [L.] Osbeck） bud mutation causing lycopene accumulation, low citric acid, and high sucrose in fruit, suppression subtractive hybridization （SSH） and microarray were performed to decipher this bud mutation during fruit development.After sequencing of the differentially expressed clones, a total of 267 non-redundant transcripts were obtained and 182 （68.2%） of them shared homology (E-value≤1×10^-10) with known gene products. Few genes were consecutively up- or down-regulated （fold change≥2） in the bud mutation during fruit development. SOTA Algorithm analysis results showed that 95.1% of the differentially expressed genes were extensively coordinated with the initiation of lycopene accumulation. Cellular metabolic-, primary metabolic-, localization-, macromolecular metabolic-related transcripts were among the most regulated genes. These genes were involved in many metabolic pathways such as pyruvate metabolism, starch and sucrose metabolism, citrate cycle, glycolysis, and carotenoid biosynthesis pathways. Moreover, 13 genes which were differentially regulated at 170DAF shared homology with previously described signal transduction or transcription factors.This is the first global approach addressing a simultaneous evaluation of transcription changes to bud mutation, and these differentially expressed genes constitute relevant candidates for bud mutation in citrus fruits.更多还原