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柑橘果糖激酶分子生理特性及功能研究

Studies on the Molecular and Physiological Characteristics and Function of Fructokinase in Citrus

【作者】 秦巧平

【导师】 张上隆;

【作者基本信息】 浙江大学 , 果树学, 2004, 博士

【摘要】 柑橘是世界性重要果树,我国柑橘栽培面积居世界首位,产量列第三,加入WTO后,我国柑橘业将面临与国际柑橘生产市场激烈竞争的局面,柑橘果实品质已成为竞争成败的关键。糖含量是柑橘果实品质的重要指标之一,且糖是酸、类胡萝卜素和其它营养成分及芳香物质合成的基础原料,糖含量直接影响果实食用价值及商品价值。因此,研究柑橘果实糖的运输、分配、积累的生理及分子生物学机制,可以为改良品种提高果实品质提供理论依据。 本研究以我国主栽品种温州蜜柑(Citrus unshiu Marc.)为试材,克隆到两个果糖激酶基因,对它们在不同器官中的差异表达进行了比较分析,在此基础上,通过RT—PCR及RACE技术,得到一个果糖激酶基因全长cDNA序列和全长基因组DNA序列;为了阐明果糖激酶在柑橘糖代谢中的作用机理,对果实发育进程中果糖激酶活性变化与糖积累的关系进行了研究,并根据分离的温州蜜柑果糖激酶基因,构建了反义植物表达载体和带内含子的反向重复果糖激酶基因植物表达载体,进行了遗传转化初步研究,主要研究结果如下: (1)、RNA提取是分子生物学的重要技术之一,果树组织成分复杂,提取困难,因此改进技术、提高RNA质量,成为果树分子生物学研究的关键,经反复比较测试,改进后的TRIzol法、CTAB法、SDS/酚法、异硫氰酸胍法所提取的柑橘不同组织RNA质量均明显提高,已能适应于后续研究的需要。 (2)、根据植物果糖激酶基因(FRK)保守区设计引物,以温州蜜柑基因组DNA为模板,采用PCR法扩增得到长度分别为1213 bp和793 bp的果糖激酶基因,分别命名为Cufrk1、Cufrk2。Cufrk1(GenBank:AY118083)编码205个氨基酸,含4个外显子和3个内含子;Cufrk2(GenBank:AF521003)编码204个氨基酸,含2外显子和1个内含子。序列分析表明,Cufrk1和Cufrk2与其他植物中已分离的果糖激酶基因氨基酸序列同源性为64~78%,Cufrk1与Cufrk2氨基酸序列同源性为68%。 (3)、Northern分析显示,在幼叶、发育初期果实(5月8日~6月2日)均检测到Cufrk1与Cufrk2较高的表达量;在花瓣中能检测到Cufrk1极小量的表达,而Cufrk2在花瓣中的表达量较高;在7月3日~9月12日的果实中这两个基因的mRNA均未检测到,在10月22日果实中仅检测到Cufrk1的表达;在茎及10月22日果皮中,均未检测到这两个基因的表达。 (4)、利用RT-PCR及RACE技术从温州蜜柑果实中分离到了编码果糖激酶基因的cDNA全长序列,命名为CuFRK1(GenBank:AY561840)。CuFRK1 cDNA全长为1459bP,含有一个完整的开放阅读框,编码350个氨基酸,含有2个果糖激酶糖特异结合域及3个ATP结合域。及护劫口与其他植物中己分离的果糖激酶基因氨基酸序列同源性为62~78%。 (5)、以叶片基因组DNA为模板,扩增得到伪月烈口基因的全长基因组DNA序列。全长序列为3169bP,由7个外显子和6个内含子组成,外显子部分(1161 bP)含有完整的开放阅读框,7个内含子大小为106bP~623bP,起始处序列均为GT,结束处序列为AG,这6个内含子的A十T含量都比较高(63.56%~73.59%),符合植物内含子的规律。 (6)、在测定时期内(5月18日一11月13日),随着温州蜜柑果实的发育,可食组织糖含量不断增加,果糖激酶活性逐渐降低,7月3日是酶活性由急剧下降到缓慢下降的转折点;在果实整个发育过程中可食组织果糖含量与果糖激酶活性以鲜重或蛋白质含量表示均成显著负相关:8月3日到10月22日,果皮组织中的3种糖含量不断增加,而在n月13日成熟果皮中蔗糖和葡萄糖含量略有下降;果皮果糖激酶活性(以鲜重表示)从8月3日到10月22日呈现快速下降,在n月13日略有升高。 (7)、果实膨大期后植株增施氮肥,在成熟期可食组织及果皮中蔗糖和果糖占总糖比例均有所下降,而葡萄糖所占比例升高:在测定时期内,可食组织果糖激酶活性以蛋白质含量为单位表示均明显高于对照果实,以鲜重表示则酶活性在8月3日低于对照,9月12日到成熟期酶活性高于对照;增施氮肥果皮组织果糖激酶活性以鲜重和蛋白质含量为单位表示与对照的差异不一致;Northern分析表明,增施氮肥能促进发育后期果实可食组织中cufl沂1基因的表达,但对cuf).kZ的表达无明显作用。 (8)、根据伪为咭召序列的外显子部分设计引物,通过RT一PCR从温州蜜柑幼叶中扩增到426 bp的cDNA片段(编码139个氨基酸,其中包括58个氨基酸的糖结合域)。将此片段反义插入到去掉乙配5,基因的双元表达载体pBI 121中,构建成反义植物表达载体浏万刁谓爪根据RNA干涉原理,经两次亚克隆得到1032 bp(含168、bp内含子)反向重复的FRK重组基因片段,插入到去掉‘召S基因的pBll21 355启动子下,构建成带内含子的反向重复FRK基因表达载体刀刀了一jhP丹?Ko将构建的表达载体通过农杆菌介导法,以草墓叶片圆盘为试材,进行了遗传转化初步研究。

【Abstract】 Citrus is one of the most important fruits in the world. The cultivated area of citrus in China is maximum in the world. After entry of China into WTO, our domestic market faces the competition from abroad. Therefore, the breeding and production of high quality citrus fruit is urgent. Sugars play an essential role in flavor characteristics of the citrus fruit and are also a commercial measure of fresh fruit quality. Sugars are not only the substance that affect sweetness of fruit, but also from the material basis for synthesis of organic acid, carotenoids, and other nutrient and aromatic ingredients of citrus fruit. Studying the physiological and molecular mechanism of sugar transporting and accumulating process in tissues of developing citrus fruit will supply an important basis for scientific regulating citrus quality by genetic tools.In order to clarify the mechanism of sugar accumulation in citrus fruit, we cloned two fructokinase genes from satsuma mandarin (Citrus unshiu Marc.) and analyzed the different expression in various organs. Based on these results, we isolated the full-length cDNA and genomic DNA of one fructokinase gene. To elucidate the function of fructokinase on sugar accumulation in citrus fruit, the relationship of fructokinase with development of satsuma mandarin fruit and sugar accumulation was studied. On the side, an antisense and an intron-spliced RNAi expression vectors were constructed and preliminary study on transformation was carried out. The results were summarized below.(1). RNA isolation is one of the most important techniques in the studies of molecular biology. The cell composition in fruit trees is more complex so it’s hard to isolate high quality RNA from those tissues. Therefore, technique improving and isolating high quality RNA become the key points in the studies of molecular biology in fruit trees. Four total RNA extraction methods including modified TRIzol, CTAB, SDS/phenol, and single-step method were applied to isolate total RNA from various citrus tissues. The quality of RNA isolated by the four modified methods was improved markedly after our efforts and the RNA was fit for the latter studies.(2). Two Citrus unshiu Marc, fructokinase genes were isolated from genomic DNA using degenerate primers: Cufrkl (GenBank: AY 118083) 1213 bp in length, encoded a deduced protein of 205 amino acids, including 4 exons and 3 introns; Cufrk2 (GenBank: AF521003) 793 bp in length, encoded 204 amino acids, including 2 exons and 1 intron. Both amino acid sequences possessed sugar-binding domains and were 64~78% identical with previously characterized fructokinase genes of other plants. The amino acid sequences encoded by Cufrkl and Cufrk2 were 68% identical.(3). Northern blot showed that the transcripts of Cufrkl and Cufrk2 were distinct to a certain extent. Both transcripts could be detected in young leaves and in fruit at early developmental stages (from 8th May to 3rd Jun). Transcripts of Cufrkl could be detected in petals at a very low level but those of Cufrk2 were at a high level. Only transcripts of Cufrkl were detected in fruit on 22nd Oct. Transcripts of both genes were undetectable in stems and in the peel from ripe fruit.(4). One complete cDNA of fructokinase gene from citrus (Citrus unshiu Marc.) were isolated byusing of PCR, RT-PCR and rapid amplification of cDNA ends (RACE), named CuFRKl (GenBank: AY561840). The full-length cDNA of CuFRKl was 1459 bp in length and contained a complete ORF from 168 to 1220 bp, encoding 350 amino acids with two sugar-binding domains and three ATP-binding domains. The deduced amino acids of CuFRKl were about 62% to 78% identical to the previously characterized fructokinase genes from other plants.(5). The full-length genomic DNA of CuFRKl was isolated from satsuma mandarin leaves genomic DNA. The sequences were 3169 bp in length containing 7 exons and 6 introns. The 7 exons 1161 bp in length included a complete ORF. The size of introns ranged from 106 to 623 nucleotides. As in most plant introns, the content of A+T w

  • 【网络出版投稿人】 浙江大学
  • 【网络出版年期】2004年 03期
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