【作者】 曾文芳；

【导师】 邓秀新；

【作者基本信息】 华中农业大学 ， 果树学， 2013， 博士

【摘要】 柑橘果实因富含类胡萝卜素而具有丰富的色泽,并广受消费者的喜爱,因此,具有很重要的经济价值。同时由于不同柑橘品种间类胡萝卜素组成与含量的多样性而具有很高的科学研究价值。前人已针对柑橘类胡萝卜素做了大量的研究,然而类胡萝卜素代谢是许多基因参与并协调互作的过程,只研究主要的类胡萝卜素生物合成基因对研究类胡萝卜素代谢的分子机理是远远不够的。通过对编码类胡萝卜素生物合成限速酶(PSY)的基因启动子的研究可以探究它的作用机理,找到其调控模式,得到上游调控基因的信息。本研究主要包括两个部分内容：分离‘暗柳’甜橙类胡萝卜素关键合成酶CsPsy1a和CsPsy1b基因,并通过工程菌进行体外功能分析比较两个基因编码蛋白的酶活性,为柑橘色泽突变提供更多理论方面的依据；分离‘暗柳’甜橙类胡萝卜素代谢关键基因CsPsy1a和CsPsy1b的启动子序列,连接报告基因并转入模式植物拟南芥,通过检测报告基因的强弱来鉴定控制CsPsy1a和CsPsy1b的启动子的活性。主要研究结果如下：1甜橙八氢番茄红素合成酶基因的克隆与功能分析(1)利用RT-PCR从‘暗柳’甜橙中克隆到两条CsPsy1基因序列,分别命名为CsPsy1a和CsPsy1b,这两个基因的最大开放读码框分别为1311bp和1317bp,分别编码436和438个氨基酸。序列比对表明两个基因的核酸序列及其推导的氨基酸序列的相似性均在98%左右,序列之间存在16个碱基的差异,将推导的氨基酸序列进行比对后发现,共有9个氨基酸位点发生了改变,其中7个位点是单核苷酸多态性引起的,另2个氨基酸差异则来源于一个简单序列重复多态性(SSR)位点。(2)利用Rea1time RT-PCR对CsPsy1a和CsPsy1b在‘暗柳’甜橙果实不同发育时期的表达分析表明,CsPsyIb在果皮与果肉中的表达趋势一致,均在果实发育前期表达量逐渐增强,170DAFB时达到其表达高峰,而后趋于稳定,CsPsy1a在果皮和果肉中的表达量则随着果实的成熟持续上升。果皮中两个等位基因的表达差异较小,果肉中CsPsy1a的表达量显著高于CsPsy1b.(3)利用RT-PCR的方法从‘高班’柚克隆Psy1基因,经测序分析,从‘高班’柚中仅克隆到一条Psyl序列,且其编码区的核苷酸序列与‘暗柳’甜橙CsPsy1b完全相同。利用HPLC对‘高班’柚和‘暗柳’甜橙类胡萝卜含量的分析表明,两个品种果肉的颜色不同是由于类胡萝卜素含量有较大的差异。利用实时定量PCR的方法分析类胡萝卜素主要生物合成基因在‘高班’柚和‘暗柳’甜橙成熟时期果肉的表达,结果表明两个品种中类胡萝卜素的差异可能与类胡萝卜素主要合成基因的表达没有直接的联系。(4)利用RT-PCR的方法从‘马叙’葡萄柚中克隆Psyl基因,经测序分析,‘马叙’葡萄柚存在两条Psy1基因序列,且其中一个基因CpPsy1a序列与甜橙中的CsPsy1a序列一致,另一个基因CpPsy1b基因编码区核苷酸序列长度为1314bp,与CsPsy1a和CsPsy1b基因均相差3个碱基,分别编码437、436和438个氨基酸,且差异位点恰好出现在微卫星位点。这三个基因编码的氨基酸共有10个差异,其中8个是由单核苷酸多态性引起的差异,而另外2个由简单序列重复数目引起。(5)构建了CsPsy1a、CsPsy1b和CpPsy1b基因的原核表达载体pET-CsPSY1a, pET-CsPSY1b和pET-CpPSY1b,并转化含有pACCRT-E质粒的BL21菌株。工程菌功能分析表明,CsPsy1a、CsPsy1b和CpPsy1b基因均可编码功能蛋白,催化两分子GGPP缩合形成八氢番茄红素,然而表达CsPSY1a、CsPSY1b和CpPSY1b蛋白的工程菌株表现出不同程度的八氢番茄红素积累量,表明三个蛋白具有不同的催化效率。而这三个酶的催化效率恰好随着重复单元数目的增多活性逐步下降。(6)利用点突变实验定点突变原核表达载体pET-CsPSY1a,构建了四个在微卫星位点含不同数目重复单元突变体(pET-CsPSY1a-1、pET-CsPSY1a-2, pET-CsPSY1a-3(?)pET-CsPSY1a-4),并转化含有pACCRT-E质粒的BL21菌株。工程菌功能分析证实了微卫星重复单元的数目与PSY的酶活有着重要的联系。2甜橙八氢番茄红素合成酶基因启动子的克隆与功能分析(1)利用染色体步移法从‘暗柳’甜橙基因组中分离得到CsPsy1a启动子序列1476bp,在线分析软件表明其含有多种光调控元件,激素诱导响应元件和非生物逆境相关感应元件。通过5’-RACE技术定位CsPsy1a基因转录起始位点,结果发现CsPsy1a基因在‘暗柳’甜橙果肉中存在两个转录起始位点,一个是胞嘧啶(C),位于翻译起始位点上游186bp处,另一个是腺嘌呤(A),位于翻译起始位点上游518bp处。将后一个转录起始位点(A)设为‘+1’,经PLACE和PlantCARE预测,发现CsPsy1a基因启动子是一个典型的TATA盒型的启动子,TATA box和CAAT box分别位于-33bp和-83bp处。(2)将-479nt-+447nt的启动子片段连接GUS报告基因后,通过农杆菌介导的花序侵染法转化拟南芥植株,全发育时期的组织化学染色结果表明,不论是茎生叶还是莲座叶,叶脉的GUS活性最强。充分展开的花朵中,在雄蕊、雌蕊、花托、萼片、花柄以及花萼等组织中均有表达,且集中在叶脉组织,而在花药和花瓣中未检测到GUS的表达。在花和果荚中,GUS活性的表达随着器官的成熟逐渐增强。这些结果表明CsPsyla启动子在光能合成和非光能合成器官中均有活性,并受到发育的调控。(3)构建5个CsPsy1ap5’端缺失启动子体,命名为V1-V5。转化拟南芥植株后,组织化学染色表明：除V1外,其他缺失启动子都能有效地驱动GUS表达,表明控制该启动子活性关键位点位于-46nt～+21nt;另外,在-479nt--306nt和-306nt--93nt区域分别存在增强子元件和沉默子元件。转基因拟南芥植株在去黄化过程中经不同光质处理后,携带V5的转基因植株经红光和白光处理后GUS活性相对黑暗处理显著性增强,而经远红光和蓝光处理后GUS活性减弱。缺失分析表明,在-479nt到-306nt区域存在着光调控元件。转基因拟南芥植株经激素和非生物逆境处理后,结果表明：在不同激素和非生物逆境的处理下,仅蔗糖处理后的GUS活性有显著性增强,表明CsPsy1a基因启动子受到高浓度蔗糖的诱导。缺失分析表明,在-479nt到-306nt区域同样存在着蔗糖响应元件。(4)利用染色体步移法从‘暗柳’甜橙基因组中分离得到CsPsy1b启动子序列1754bp,在线软件分析元件,并与CsPsy1a启动子鉴定出的顺式元件相比较,发现两个启动子中均含有大量光调控元件,激素诱导响应元件和非生物逆境相关感应元件。尽管两个启动子序列中都存在多种光调控的元件,但类型完全不同，如在CsPsy1b启动子序列中发现多个与光能合成相关的普遍存在的G-Box元件,而CsPsy1a启动子序列中却无该类型光调控元件。这些结果表明,CsPsy1b与CsPsy1a一样,主要受光信号调控,同时受到诸多非生物逆境的诱导。(5)将CsPsy1b启动子连接GUS报告基因后,通过农杆菌介导的花序侵染法转化拟南芥植株,全发育时期的组织化学染色结果表明,和CsPsy1a启动子一样,CsPsy1b启动子在光能合成和非光能合成器官中均有活性,并受到发育的调控。(6)将携带CsPsy1ap::GUS和CsPsy1bp::GUS的转基因植株经不同光照条件处理,结果表明：在去黄化过程中不同光质的照射下,携带CsPsy1ap::GUS的转基因植株GUS活性相对黑暗处理均显著性上升。在红光处理时GUS活性最强,其次是远红光和蓝光,最后是白光；而携带CsPsy1bp::GUS的转基因植株在远红光处理下GUS活性最强,其次是红光,白光,最后是蓝光。表明两个启动子在去黄化过程中均受到不同光质的调控,但由于启动子序列中光调控元件的数目及类型不一致,因此,两个基因启动子在不同的光质处理下表现不尽相同；在不同强度光照的照射下,携带CsPsy1ap::GUS的转基因植株响应不同强度光照的调控,表现在弱光下启动子活性更强,而携带CsPsy1bp::GUS的转基因植株可能不响应；在不同光周期的处理下,长日照下CsPsy1a启动子活性较强,而CsPsy1b启动子不响应。更多还原

【Abstract】 Due to the accumulation of specific carotenoids, citrus fruits display a wide range of colorations and are appreciated by consumers with significant economic value. Citrus fruits are also of high scientific value owing to the diversity of carotenoid composition and content among varieties. So far, citrus carotenoids have been well characterized, however, carotenoid metabolism is a complex process with many genes participating in and coordinating interaction. Research on the molecular mechanism of the major carotenoid biosynthetic genes is far from knowing enough, further investigations to understand the regulatory mechanism of carotenoid metabolism is needed. The mechanism has been conducted in this research through the research on the promoter of the gene (Psy) which encoding the rate-limiting enzyme of carotenoid biosynthesis pathway, to find the regulatory pattern and to acquire the information about the upstream regulatory gene. The result was presented as two parts:Two similar but non-identical sequences (CsPsyla and CsPsy1b) were isolated from sweet orange. A functional analysis based on heterologous expression in E. coli indicated that CsPsyla was a more efficient converter of geranylgeranyl diphosphate to phytoene, to understand the mechanism of citrus color trait; The promoters of the sweet orange genes (CsPsy1a and CsPsy1b) were isolated, then fused to the β-glucuronidase (GUS) reporter gene and introduced as a transgene into Arabidopsis thaliana, to investigate the promoter activity based on GUS activity. The main results were as follows:1Isolation and functional characterization of phytoene synthase genes from sweet orange(1) Two CsPsyl sequences were isolated from sweet orange cv. Anliu, and named as CsPsy1a and CsPsy1b. The two sequences were98.6%identical to one another, one being of length1,311bp and the other1,317bp. They encoded436and438amino acids, respectively. The sequences differed from one another at18nucleotide positions, resulting in nine predicted peptide differences. Seven of which were generated by SNP and the other two by different number of AAT repeat units within a microsatellite.(2) Real time RT-PCR was used to study the expression of the two genes during fruit development. Transcription profiling showed that CsPsyla abundance increased throughout fruit development in both the peel and pulp, while that of CsPsy1b increased early in fruit development and then stabilized. The abundance of the two transcripts was similar in the peel, but that of CsPsy1a present was higher in the pulp.(3) A full length Psy cDNA was isolated from pummelo cv. Gaophuang. Its sequence proved to be identical to that of CsPsy1b. HPLC was used to compare the composition and content of carotenoids from these two cultivars, the result displayed a pronounced difference in their carotenoid content. The transcription in cv. Gaophuang of main carotenoid biosynthetic genes (except for Hyb) was higher than in cv. Anliu (yellow fleshed fruit), these results showed that the transcription of other major carotenoid synthesis genes could not explain the large difference in carotenoid content between the two species.(4) Two CpPsyl sequences were isolated from grapefruit cv. Marsh, and named as CpPsy1a and CpPsy1b. The peptide sequence of CpPsyla was identical to that of CsPsy1a, and CpPsy1b was99%identical to CsPsy1b. The number of residues encoded by these three genes (CsPsy1a, CsPsy1b and CpPsy1b) varies slightly (respectively,437,438and439, due to polymorphism in the number of AAT triplet present. Ten predicted peptide differences were found among the three genes, eight of them were generated by SNP and the other two by different number of AAT repeat units within a microsatellite.(5) The full-length coding region of three genes (CsPsy1a, CsPsy1b and CpPsy1b) were amplified using PCR and further inserted into pET-28a (+), resulting in the prokaryotic expression vector pET-CsPsy1a, pET-CsPsy1b and pET-CpPsy1b, respectively. After transformation of E. coli strain BL21which produces the PSY substrate GGPP, the HPLC profile of both types of transgenic cell included a peak with a retention time of19.0-21.5min which was not detectable in the empty vector control. Interesting, there was a demonstrable negative correlation between the number of microsatellite repeat units present and PSY activity.(6) An E. coli expression platform was used to test the effect of site-directed mutagenesis in CsPSYla on enzymatic activity, particularly that of the number of microsatellite repeat units presented. Four different variants were generated (pET-CsPSY1a-1, pET-CsPSY1a-2, pET-CsPSYla-3and pET-CsPSYla-4). After transformation of E. coli strain BL21which produces the PSY substrate GGPP, functional analysis suggested that there was a clear correlation between the number of microsatellite repeat units present and PSY activity.2Identification and functional characterization of the CsPsyla and CsPsylb promoters from sweet orange (1) The promoter of the sweet orange gene CsPsy1a of sweet orange was isolated using chromosome walking. Its sequence included a number of regulatory elements predicted to be responsive to light, hormone and other stress cues. The promoter’s transcription start site was determined using5’RACE based on RNA extracted from the pulp of sweet oranges cv. Anliu. Two sites were identified, the first a cytidine (C)186bp upstream of the translation initiation codon and an adenine (A)518bp upstream of translation initiation codon. PLACE and PlantCARE analyses suggested a potential TATA box551bp from the5’end of the ATG and33bp upstream of the potential TSS (’+1’) that was further from the translation initiation codon. In addition, a possible CAAT box was located at-83bp.(2) The926-bp region upstream of CsPsyla was fused to the β-glucuronidase (GUS) reporter gene, and introduced as a transgene into Arabidopsis thaliana. The pattern of GUS expression in the transgenic plants showed that the CsPsy1a promoter drove expression in the young seedlings, the leaves, the roots and in parts of the flower, in a developmentally regulated fashion. These results included that the CsPsy1a promoter drove expression in photosynthetic and non-photosynthetic tissue, and was induced by developmental cues.(3) Five5’-deletions expression vectors named V1～V5were constructed, and transformed into Arabidopsis. A promoter deletion analysis revealed that the region-479nt to-306nt positively regulated expression, the region-306nt to-93nt was associated with negative regulation of expression and the region-46nt to+21nt maintained basal promoter activity. The transgenic Arabidopsis seedlings were treated with different light quality during de-etiolation, A promoter deletion analysis revealed that Two highly repetitive sequences (Spl and A-box), located in the region from-479nt to-306nt, may act as light-responsive elements regulating CsPsy1a transcription during photomorphogenesis. The transgenic plants carrying V5::GUS were treated with various stresses and hormones treatment, of these, sucrose treatment showed a significantly different value from the control by one-sided t tests. The promoter deletion constructs were used to explore the sucrose induction of the CsPsy1a promoter, the results showed that the cis-acing element(s) responsive to sucrose must be located in the region between-479nt to-306nt.(4) The promoter of the sweet orange gene CsPsy1b of sweet orange was isolated using chromosome walking. Its sequence included a number of regulatory elements predicted to be responsive to light, hormone and other stress cues, as well as the CsPsy1a promoter sequence. There were many different cis-elements in the two sequences, for example, many light responsive element (G-box) were present in the CsPsy1b promoter sequence, but none was discovered in the CsPsyla promoter sequence. In a word, the bioinformatics analysis indicated that the two promoters was mainly regulated by light, and induced by various stresses.(5) The CsPsy1b promoter was fused to the P-glucuronidase (GUS) reporter gene, and introduced as a transgene into Arabidopsis thaliana. The pattern of GUS expression in the transgenic plants included that the CsPsy1b promoter drove expression in photosynthetic and non-photosynthetic tissue, and was induced by developmental cues, as well as the CsPsy1a promoter.(6) The transgenic Arabidopsis seedlings carrying CsPsy1ap::GUS and CsPsy1bp::GUS were treated with different light quality treatment (red, far red, blue and white), the results showed that two promoters were induced by different light quality treatment, however, there were some difference maybe due to the different light responsive elements in their promoters; The transgenic plants carrying CsPsy1ap::GUS and CsPsy1bp::GUS were treated with different light intensity treatment, the results showed that CsPsy1a promoter activity was enhanced by dim light, and the CsPsy1b promoter had no response to different light intensity; The transgenic plants carrying CsPsy1ap::GUS and CsPsy1bp::GUS were treated with different photoperiod treatment, CsPsy1a promoter activity was enhanced by long-day, and the CsPsy1b promoter had no response to different photoperiod.更多还原