【作者】 刘永忠；

【导师】 邓秀新；

【作者基本信息】 华中农业大学 ， 果树学， 2006， 博士

【摘要】 奉节晚橙（Citrus sinensis Osbeck）是一个来源奉节72-1脐橙的晚熟芽变品种。这两个品种一起将构成一个很好的柑橘成熟机理研究体系。加强对奉节晚橙的研究，明确奉节晚橙晚熟芽变发生的机理，将为柑橘熟期育种奠定理论基础，最终有助于柑橘产业的持续健康发展。本文研究了奉节晚橙与奉节72-1脐橙在果实成熟过程中的糖酸、色素、以及转录水平的变化，探讨了晚熟芽变脐橙发生原因，取得如下主要结果：1．建立了一种适合于柑橘不同时期不同组织的RNA提取方法。利用该方法所提取的RNA的A₂₆₀／A₂₈₀在1.65-1.90之间，A₂₆₀／A₂₃₀大于2.0；其质量和浓度能够直接用于其它分子研究，如RT-PCR，cDNA-AFLP和Northern杂交（地高辛）实验。2．着色差异分析表明：与奉节72-1脐橙着色相比，奉节晚橙的转色期约延迟30天左右，造成这种着色延迟的原因主要是奉节晚橙果皮前期叶绿素含量减少进程和后期类胡萝卜素积累进程推迟所致。奉节晚橙果皮的叶绿素含量显著减少发生在11月上旬到11月下旬，迟于奉节72-1品种，后者叶绿素含量显著降低发生在10月中下旬到11月上旬；奉节晚橙果皮的类胡萝卜素含量显著积累始于12月中旬，而奉节72-1品种则始于11月初。另外奉节晚橙果皮中叶绿素含量在10至11月间显著高于原品种，类胡萝卜素含量在12至1月间显著低于原品种。基因表达分析发现，奉节晚橙果皮的类胡萝卜素合成酶的相关基因较强表达的时间也整体迟于奉节72-1脐橙；叶绿素水解酶基因表达在10至12月中旬其表达强度较奉节72-1脐橙弱，翌年1月份则有较强的表达。3．通过对果实成熟过程中的糖分积累分析表明，奉节晚橙和奉节72-1脐橙果实在成熟过程中两个品种的糖分变化差异主要发生在果肉组织，且奉节晚橙果肉中三种糖分含量在12月中旬之前是显著低于奉节72-1脐橙果肉中的糖分含量，而在元月份后则明显高于后者；果皮中奉节晚橙的蔗糖含量在果实成熟前期两个时期（10月11日和11月3日）显著低于奉节72-1脐橙。而蔗糖相关代谢酶基因表达分析表明，两个品种果肉中酸性转化酶和蔗糖合酶的基因表达表现明显差异，这种差异部分影响了奉节晚橙和奉节72-1果实间的糖分差异。4．奉节晚橙和奉节72-1脐橙果肉酸积累分析发现奉节晚橙在成熟前期柠檬酸含量显著高于奉节72-1脐橙，而在后期（11月底始）则低于后者；在果实成熟中期，奉节晚橙节果酸含量显著低于对照品种。柠檬酸代谢相关酶基因表达分析表明柠檬酸合成酶和顺乌头酸酶基因表达在两个品种间表现差异，而造成奉节晚橙柠檬酸含量与奉节72-1的差异可能与此时奉节晚橙果肉中柠檬酸合酶基因表达较强和顺乌头酸酶基因表达受抑制相关。5．利用分子标记技术，SSR和AFLP对两个品种的DNA差异进行分析，未发现两个品种间在DNA水平上可重复的差异。6．采用cDNA-AFLP技术分析了两个品种的果实在成熟过程中转录水平的表达，并对cDNA-AFLP的结果进行了进一步验证。实验筛选了120对选择性引物，总共获得近20000多条清晰带，结果发现其中有34对引物出现差异带，检测到144条清晰的转录差异带（TDFs）。在成功回收的129个TDFs中，通过测序和序列比对（Blastx）分析表明表达差异片段主要与信号转导、转录因子、成熟衰老、抗逆性、核糖体蛋白和DNA复制等有关。7．利用RACE技术，以cDNA-AFLP技术获得的差异片段为基础克隆了6个与成熟相关的基因，分别命名为CitPG（EF185420）、CitCAD（EF185415）、CitMads（EF185417）、CitMAP（EF185418）、CitNAC（EF185419）、CitEREBP（EF185416），除了MADs-box基因外，其它5个目前在柑橘中还未见报道，且CitMads与目前报道的柑橘Mads-box基因序列不同。表达分析表明在果实成熟过程中，他们在两个品种的果皮或果肉组织中有表达差异，可能与晚熟性状的表现有关。以上研究结果表明，相对奉节72-1脐橙，奉节晚橙在许多方面发生了改变，这种性状的改变很显然不是因为某一相关指标因子发生突变而引起局部性状发生改变，而确实是果实成熟过程或发育过程整体延迟所致。而导致这种整体改变可能是在表达水平上而非在DNA水平发生改变。由于果实成熟是一个综合而又复杂的过程，而非跃变型柑橘果实是一个渐进式的程序性过程，因此要使奉节晚橙整体协调发生改变，那么这种改变的发生很有可能在果实发育过程或成熟过程发生的上游某个阶段，即可能是启动果实成熟开始的一个‘开关’发生变化，从而导致奉节晚橙成熟过程整体表现延迟。更多还原

【Abstract】 ’Fengjiewancheng’（FJWC, Citrus sinensis Osbeck） is a novel late-ripening navel orange mutant of’Fengjie72-1’navel orang（FJ72-1）. Academically, FJWC might constitute a useful experiment system with its parental line for the study of citrus ripening mechanism. To get the knowledge or information as much as possible about the occurrence mechanism of this mutant will play a great role in citrus fruit-mature-time breeding via gene engineering, and at last in maintaining consecutive and healthy development of citrus industry. In order to give clues as to what mechanisms are monitored in the mutant, difference of FJWC and FJ72-1 in coloration or pigmentation, sugar, acid and mRNA transcripts during fruit ripening were compared, and the occurrence mechanism of FJWC was discussed in the end. The main results were as follows:1. An efficient RNA isolation method was established, which was proved to be widely applicable in different citrus tissues at different developmental stages. The A₂₆₀/A₂₃₀ of RNA was over 2.0, and the A₂₆₀/A₂₈₀ ranged from 1.65-1.90, indicating low levels of polysaccharide and good purity of the RNA. The quality of the RNA was further proved by means of RT-PCR, DIG Northern blot analysis and cDNA-AFLP.2. Coloration analysis during fruit ripening indicating that FJWC’s coloration was delayed about 30 days or so, which was because that the distinctive decrease of chlorophyll in the peel of FJWC occurred from the early of November to the late of November, later than that of its original cultivar which occurred from the middle of October to the early of November, and the obvious accumulation of carotenoid in the peel of the mutant began on Dec. 12th, while its original cultivar on Nov. 3rd. What’s more, analysis of independent-samples T test showed that the chlorophyll content in the peel of FJWC from October to November was significantly higher, and the carotenoid content from December to January was significantly lower than that of the parental line, FJ72-1 respectively. Gene expression of some carotenoid biosynthetic genes and cholorophyllase gene using RT-PCR technique were showed that the time of some carotenoid biosynthetic gene expression reaching a maximum in the peel of the mutant were postponed for one month comparing to those of its original cultivar, while the gene expression of cholorophyllase in the peel of FJWC reached a maximum in January, and kept slightly lower level from October to December than that of FJ72-1.3. Analysis of sugar seasonal changes in the pulp and peel of both FJWC and FJ72-1 during fruit ripening discovered a major difference of sugar contents in the pulp between the two cultivars. Sugar contents of sucrose, glucose and fructose in FJWC pulp were significantly lower than those in FJ72-1 before the middle December, and became higher after then. In fruit peel, only sucrose contents in FJWC were significantly lower than those in FJ72-1 at the two early sampling dates during fruit ripening. Transcript analysis of sucrose metabolic gene showed that CitAI（citrus acid invertase gene） and CitSS1（citrus sucrose synthase genel） had different expression patterns in the pulp between the two cultivars during fruit ripening which were related with the sugar content difference of them.4. Acid contents of FJWC during fruit ripening were also changed comparing with FJ72-1. The citric acid contents in the pulp of FJWC were obviously higher than those in FJ72-1 pulp during the early ripening stages, while lower than the latter after the late November. In the middle of fruit ripening, malic acid contents in FJWC were significantly lower than those of FJ72-1. Transcript comparison of citric metabolic gene showed that the difference of citric acid between the two cultivars might be related with the high transcript level of citrate synthase gene and with the block of cytosolic aconitase gene in the pulp of FJWC during the early ripening stages.5. No reliable differences on DNA level between the two cultivars were discovered by SSR and AFLP analysis.6. Expression profiles of the two cultivars’ fruit during fruit ripening were analyzed using cDNA-AFLP. After screening 120 pairs of selective primers which produced about 20000 clear transcript bands, only 144 clear transcript different fragments（TDFs） were found in 34 pairs of primers. However, only 129 TDFs were successfully recovered, sequenced and then blasted by blastx tool. The blastx results showed that most of the TDFs were related with signal transduction and transcription factors, senescence or maturation, stress reaction, ribosome protein and DNA replication.7. Using the sequence of TDFs, six genes related with fruit maturation were successfully cloned using RACE technique. There were named as CitPG（EF185420）, CitCAD（EF185415）, CitMads（EF185417）, CitMAP（EF185418）, CitNAC（EF185419） and CitEREBP（EF185416）. RT-PCR indicated that they expressed differently in fruit peel or pulp between the two cultivars, which implicated that these gene might be related with the occurrence of late-ripening phenotypes.Taken together, many aspects of FJWC were changed compared with FJ72-1, the late-ripening phenotypes of FJWC is obviously not the results of some factor mutation or alteration, but is involved in delay of ripening progress. Furthermore, the alteration of transcript level might be one of the reasons of the late-ripening phenotypes.Fruit ripening is a comprehensive and complicated progress. Unlike climacteric fruit, non-climacteric fruit ripening, such as citrus, experienced a gradual and programmed progress. Hence, what made FJWC coordinate changes must located in the upstream stages of fruit ripening or development, that is, a factor having the function to trigger the onset of fruit ripening might be altered, which lead to the coordinate delay of FJWC fruit ripening progress.更多还原