【作者】 徐碧玉；

【导师】 金志强；

【作者基本信息】 华南热带农业大学 ， 作物遗传育种， 2005， 博士

【摘要】 香蕉和大蕉是热带、亚热带发展中国家的最重要作物之一。是全球约四亿人的日常食物,香蕉已成为世界第四大粮食作物。香蕉贸易是产蕉国家及地区换取外汇的主要方式。特别是发展中国家,从中获取85%,目前香蕉已是许多发展中国家农业的重要组成部分,正迅速发展。中国的香蕉产量居世界第四位,是热带农业的支柱产业。香蕉为典型的呼吸跃变型水果,果实采后不耐储藏。对其采后生理学及分子生物学研究已成为研究热点,目的在于深入了解该类果实后熟机理,为延长果实保鲜期提供理论依据及为创造保鲜新方法奠定基础。 微阵列是目前功能基因组学研究的重要手段之一,该方法可高通量、快速筛选目的基因,同时可研究基因在发育过程中的表达情况。我们首次利用此方法对通过抑制差减法获得的287个香蕉采后两天果实的cDNA片段进行了不同器官的表达研究,通过计算机自动扫描及软件分析,共获得32个果实特异的cDNA片段。进行Blastn和Blastx比较分析,结果表明获得32个果实特异表达cDNA片段,包括信号转导(4个)、成熟相关蛋白(4个)、蛋白激酶(3个)及物质代谢(9个)等相关基因。根据芯片扫描光吸收值得大小,选取大、中、小的三个克隆(编号BR24、BR47、BR63)进行Northern blot分析,结果证明三个克隆均为果实特异表达,表达量也与芯片一致。选取编号BR63的克隆利用RACE方法获得5′端500bp,3′端1800bp。测序结果表明该cDNA全长2443bp,含有一个完整的阅读框架,长1959bp,编码652个氨基酸。氨基酸同源性比较(BlastX)与小麦、水稻的淀粉磷酸化酶基因(α-1,4-葡聚糖磷酸化酶)同源72%。结合香蕉采后成熟过程中乙烯释放量及呼吸强度测定变化特点,选取采后成熟不同时间段的果实提取RNA反转录cDNA,利用RT-PCR方法对各时间段mRNA表达进行检测,结果表明,在采收前(0h)未有表达,采收后随着成熟度提高表达量逐渐下降。说明该基因的表达是在呼吸跃变之前,而在跃变开始时,已很少或不表达。 栽培香蕉基本是三倍体,常规育种较为困难。近年来随着生物技术,特别是转基因技术的不断成熟及香蕉再生体系的不断完善,利用生物技术培育香蕉新品种已成共识。同时由于香蕉自身的特点,将其作为生物反应器生产口服疫苗及有用的化合物也成研究的热点。外源基因特异高效表达,启动子的作用十分重要,香蕉为单子叶植物,更多还原

【Abstract】 Bananas and plantains （Musa spp） are globally important fruit crop, especially in developing tropic and sub-tropic countries, because they feed tens of millions in Central America and Asia. Besides that, banana trade is an essential source of income for many national economies. Therefore, the United Nations Food and Agriculture Organization ranks bananas as the world’s fourth most important food crop. Banana postharvest ripening plays an important role in influencing fruit quality and shelf life. As a result, many researches on molecular biological and physical mechanism of banana postharvest ripening have been carried out. Banana fruits are typical climacteric fruits, with many features in ripening like tomatoes, avocado, mango and others. Many experiments demonstrate that banana fruit ripening is regulated by ethylene, companied by aspiratory climacteric. In the view of molecular biology, protein de novo synthesis is necessary for many of physiological changes during postharvest. For the reason of that, by cloning the transcripts of the proteins related to fruit ripening, it would be possible to understand the genes related to fruit ripening and their expression patterns.Differential screening of cDNA libraries representing ripening banana pulp led to the isolation of groups of differential expressed mRNAs. Identification of these transcripts partial sequence analysis indicates that proteins encoded by these genes involved in ethylene biosynthesis, aspiration, starch mobilization, cell wall breakdown and other mobilizations. In this study, cDNA microarrays combined with suppression subtractive hybridizaiton （SSH） were carried out successfully for isolating fruit-specific expressed cDNA. 287 cDNA fragments from SSH were printed onto a glass plate so that a cDNA chip was made. By using labeled RNA from root, leaf and stem, and fruit as probes to screen the chip, as a form of reverse Northern blot, 32 cDNA clones were identified as fruit-specific expressed in banana. Sequencing of these 32 cDNAs showed that 4 cDNAswere homology to those genes related to signal transduction, 4 clones were homology with those genes encoding ripening associated protein, 3 clones were homology with those genes encoding protein kinase, 9 clones were homology with those genes encoding some enzymes which took part in some metabolisms in banana fruit ripening, 8 clones were homology with those genes encoding different proteins with different functions and also 4 clones had no homologues in NCBI. The fruit-specific expressed cDNA in the chip were confirmed to be fruit-specific by using Northern blot, in which 3 clones selected randomly were used as probes to hybridize to RNA from root, stem and leaf, and fruit. The results of Northern blot suggested that those cDNAs expressed not only specially in fruit, but also the quantity of expression were just similar to those in the chip. It is the first report that so many clones have been isolated from banana and so important cDNA have been obtained, because just total 36 clones had been obtained previously.The gene encoding a -1,4-glucan phosphorylase has been firstly cloned in this study. As for the importance in banana fruit ripening, the full length of the gene was obtained by using rapid amplification of cDNA 5’ and 3’ end and RT-PCR was used to examine the expression patterns in different organs and different stages of fruit ripening. In order to determine the ripening stages, ethylene biosynthesis and CO₂ production in postharvest banana were measured. Combining the result of RT-PCR with different ripening stages represented by ethylene biosynthesis and CO₂ production, it could be conclude that a -1,4-glucan phosphorylase gene expression happened much earlier than the bust of ethylene biosynthesis and CO₂ production. However, the function of this gene in fuit ripening was still needed to further reseach.A highly fruit-specific expressed promoter takes an important role in expressing foreign protein in fruit, which make banana be possible to be used as bioreactor to produce oral pharmaceutical proteins and chemicals in this way. For the reason of that a highly fruit-specific expressed gene promoter called BanLec gene promoter was cloned in this study. It was demonstrated tha BanLec gene expressed with a very high level in banana fruit in our previous reseach. BanLec gene promoter was isolated by using chromosome walking and a 670 nt DNA with basic elements of promoter, such as TATA-box, CAAT-box,更多还原