【作者】 张建业；

【导师】 陈力耕；

【作者基本信息】 浙江大学 ， 果树学， 2002， 博士

【摘要】 银杏(Ginkgo Biloba L．)，又名白果，著名的孑遗植物，被誉为活化石，是珍贵的食用、药用、材用、观赏用树种。银杏又是严格的雌雄异株，还没有发现雌雄同株现象，其雌雄株在染色体核型，过氧化物同工酶谱等都有较大差异。雌雄花形态也截然不同，有的专家还认为银杏有性染色体。银杏果实营养丰富，味道鲜美，遗憾的是银杏的童期很长，被称为公孙树，实生树的开花需要十几年，严重制约着银杏品种改良和经济利用。为缩短银杏童期，果树工作者进行了大量的研究，从栽培方式，传统育种方法进行了探索，但成效不大。 近年来模式植物拟南芥的开花机理研究取得了很大进展。至少有80个以上与拟南芥开花有关的基因和位点被识别出来。其中LEAFY(LFY)是一个花分生组织特征基因，控制着拟南芥营养分生组织向花分生组织的转变，也影响拟南芥的开花时间。LFY的过量表达可以促使拟南芥、水稻、欧洲山杨、柑桔提早开花。虽然银杏是较古老的裸子植物，但由于LEAFY基因的保守性，银杏LEAFY同源基因也可能调控着银杏开花的时间。同时，银杏的遗传转化研究极少，也需要加以研究。为此，本实验从银杏LEAFY同源基因的克隆入手，分析其雌雄株LFY基因结构差异，构建LFY基因的植物正义反义表达载体，建立矮牵牛遗传转化体系，以研究银杏LFY同源基因的功能，同时建立了银杏组织培养体系，为银杏的遗传转化和提早开花结果奠定基础。 1．常规的CTAB法提取的银杏基因组DNA呈黄色，不易被内切酶所消化，也不能作为PCR模板。我们将CTAB法稍做改良：研磨样品时加入适量的不溶性PVP，提高β-巯基乙醇在提取液中的比例，从而提取到了无色、可酶解、可作PCR模板的高质量的银杏基因组DNA。 2．首次用银杏栽培品种大佛手(Ginkgo biloba L．cv．Dafushou)雄株基因组DNA为模板，通过PCR扩增，获得两个银杏雄株LEAFY同源基因GinLFY和GinNdly基因。 结果分析表明，GinLFY DNA序列全长为3450个核苷酸。经同源性分析和DNA序列结构分析，推测该序列为银杏雄株LEAFY全长基因。该基因与文献报道的银杏雌株LEAFY基因相比，核苷酸序列同源性高达99％，蛋白质序列同源 浙江大学博士学位论文 摘要 性达99％，缺少了3个核昔酸，突变均在植物LEAF’Y基因的非保守区内。从内 含子边界特征和多种植物LEAFY基因同源性比较，确定了该基因的内含子位置。 该基因含 2个内含子，3个外显子。GinNap全长基因含 1493个核昔酸，也含有 2个内含子，3个外显子。与文献报道的银杏雌株 GinNap基因相比，碱基数少 了三个，对应地少了一个丙氨酸，核昔酸同源性为99．7％，氨基酸同源性为99．3％。 与拟南芥LEAF’Y基因相比较，银杏GinLFY和G毗基因没有做为转录激 活区域的位于氨基酸序列中间的酸性区和位于N端的富含脯氨酸区，但这两个 区域均位于LEAFY基因的非保守区。银杏雄株GinLFY和GinNdly之间氨基酸序 列同源性为 60．2％，这小于被子植物拟南芥 LFY与金鱼草 FLO之间的同源性（7 ％）。另外，与拟南芥L厂Y同源性分别为50刀％和扔＊％：与辐射松＊＊FL L同源 性分别为80．3％和58．二％；与辐射松NEEDLY同源性分别为58二％和75石％。 3．利用p8I121和pCAMBIA1301两个植物表达双元载体质粒构建了心”以沙 基因的反义与正义植物表达载体PZJI 17、PZJlls、PZJllg、PZJ120等四个质粒。 pCAMBIA1301所带的报告基因GUS含内含子，使得GUS只能在真核细胞中起显 色反应。但它的多克隆位点旁侧没有启动子与终止子。构建 Gll7Ndl基因的植 物表达载体前，先给PCAMBIA1301加上了启动子和终止子，形成PCAMBIA1301 一 355—P帖质粒。然后利用限制性内切酶双酶切，构建成 PZJI 19和 PZJ120 质粒。PBI 121表达载体构建方法是酶切去除GUS基因，利用T4连接酶代之以 GinNdl基因，构建成PZJll7和PZJI 18。通过PCR检测和酶切验证，证明质粒 构建正确，并将之转入根癌农杆菌EHA105儿BA4404、GV3101和发根农杆菌15834 中。 4．利用叶片建立了矮牵牛遗传转化体系。培养基MS＋BAZ．0＋NAAO．4适于矮 牵牛叶片诱导愈伤组织，再生不定芽，每块愈伤组织上的不定芽数可达12个以 上，并且源源不断地发出不定芽来。经叶盘的农杆菌敏感性、抗生素耐受性实验， 建立了以叶盘为受体的矮牵牛遗传转化体系。利用含GinNdP基因的正义植物 表达载体质粒PZJI 17的根癌农杆菌EHA105转化矮牵牛，获得了一些抗卡那霉 素的抗性芽，经PCR检测证明，CinNdy基因己经整合到矮牵牛的基因组中。 5．银杏的组织培养银杏的子叶，胚轴，胚根，胚乳，茎段，叶柄，叶片， 根都可以诱导出愈伤?更多还原

【Abstract】 Ginkgo Biloba L, with a common name of white fruit, is considered as a living fossil. It is used as food, medicine, wood and ornamental tree. It is a dioecious plant. The female tree is different from the male in many aspects , such as karyotype analysis, pattern of peroxidase isozymes and shape of flower. Some experts think Ginkgo has a sexchromosome. Seeds of Ginkgo is used as a dryfood and is full of nutrition. Its juvenile period is too long and seedling will flower after more than ten years. Long juvenile period severely restricts the progress of its breeding and reduces its economic value. To shorten Ginkgo juvenile period , numerous studies on its culture and conventional breeding technology has been done, but success is very little. Ginkgo will come to bearing slightly early by drawfing the tree and its intensive culture. The best way is to breed earlier flowering varieties through biotechnology.Recently, studies on the floral mechanism in Arabidopsis have made great progress. About 80 genes that control or related to plant flowering were identified. LEAFY(LFY), one of floral meristem identity genes, which confers transition from vegetative to floral meristem identity in Arabidopsis, and affects its flowering time. Overexpression LFY gene promotes plant earlier flowering. Successful experiments have been demonstrated in Arobidopsis , aspen, rice and citrus.Although Ginkgo is an old gymnosperm, its LEAFY ortholog will perhaps controls flowering time. Many studies show that LEAFY is high homolog even among distantly related plant species. Exception of these, little studies on tissue culture and transformation of Ginkgo have been done.This paper emphasizes on the isolation , cloning and analysing two Ginkgo orthologs of LEAFY from the male tree. The results obtained were summarized as bellow:1. The genomic DNA of Ginkgo extracted by CTAB method is light brown in color. It is neither digested with restriction endonucleases nor acted as template for polymerase chain reaction (PCR). The method was modified by adding suitable insoluble PVP and higher concentration of p-mercapto ethanol in the extract solution. Using the modified method, high quality genomic DNA prepared from ginkgo, grape and myrica are colorless and are susceptible to digestion with the restrictionendonucleases and can be used as template for PCR.2. GinLFYand GinNdly gene were obtained from the leaf genomic DNA of the male tree of Ginkgo cultivar Dafushou in the mid-April through PCR.The total length of 3450bp of five segments DNA were amplified by PCR. The phylogenic relation and similarity of the gene is full length of LFY-like gene, which is 99% similar both in nucleic acid sequence and amino acid sequences with that of GinLFY f rom the female. Compared with the GinLFY of female, the gene from the male tree is deficient in 3 bp, and mutantion sites exists on the varied region of LFY-like. The intron of the gene have similar characteristics with the LFY like of many plant species. The gene contain two introns and three exons. The full length of GinNdly from the male tree is 1493bp, which is 3 bp lower than the gene from the female tree. Here too the gene has two introns and three exons.LFY and LFY-like proteins from angiosperm contain a proline-rich region near the amino terminal and an acidic central region which is typical for transcriptional activators and may be important for the function of LFYlike protein. The two domain exists in the variable regions of all LFY-like proteins. Unlike angiosperm LFY-like proteins, GinLFY and GinNdly of male tree does not contain the two domain. The similarity of amino sequences between the GinLFYand GinNdly is 60. 2%, which is 9. 8% lower than LFY and FLO (70%). GinLFY(male) amino sequence similarity with LFY, PRFLL (pinus), NEEDLY (pinus) is 50.0%, 80.3% and 58.2%, respectively. GinNdly (male) amino sequence similarity with LFY, PRFLL (pinus), NEEDLY (pinus) is 45. 3%, 58. 2% and 75. 6% respectively.3. Two sense plant expression vectors and two antisense vectors were cons更多还原