【作者】 王月华；

【导师】 韩烈保；

【作者基本信息】 北京林业大学 ， 草业科学， 2007， 博士

【摘要】 赤霉素(Gibberellins，GAs)是一类重要的植物激素，参与调控多种植物发育和生理过程。GA20氧化酶是赤霉素生物合成途径中的关键调节酶，克隆和研究草坪草中的GA20氧化酶基因为草坪草矮化育种打下基础。本研究通过RT-PCR和RACE的方法从我国野生的结缕草中分离出GA20氧化酶基因，并分别构建了原核表达载体、适合于双子叶植物和单子叶植物遗传转化的正义和反义表达载体。探讨了GA20氧化酶基因在大肠杆菌、烟草、结缕草和高羊茅中表达，经过研究主要获得以下研究结果：1、根据已知几种禾本科植物的GA20氧化酶基因同源性设计简并引物，提取我国野生结缕草叶片总RNA反转录成cDNA为模板，利用RT-PCR和RACE法，从结缕草中克隆了GA20氧化酶基因的cDNA全序列。该基因全长1311bp，编码区1176bp，推测编码392个氨基酸。与小麦、大麦和水稻中的GA20氧化酶基因的核苷酸序列有55％-73％的同源性。结缕草GA20氧化酶基因在GenBank上核苷酸序列的登录号为：DQ645453。2、引入酶切位点，将克隆的结缕草GA20氧化酶基因构建到原核表达载体pET-30a中，经IPTG分别诱导1、2、3、4和5h，进行SDS-PAGE电泳，结果表明，在大约45kDa处可见一条外源蛋白表达带，并且诱导的蛋白量随着时间延长而增加，未经IPTG诱导的pET-30a(+)-GA20和pET-30a(+)在45kDa处未见条带。3、以pC1303为基础，引入BamH I和BisW I酶切位点，构建了适合于双子叶植物遗传转化的GA20氧化酶基因正义和反义表达载体，利用叶盘法将GA20氧化酶的正义基因和反义基因转化到烟草中。并对转基因的抗性植株进行了分子检测和生长观察。共获得抗潮霉素的再生植株28株，其中转正义基因的抗性植株共12株，转反义基因的抗性植株共16株，经PCR和PCR-Southern检测，转正义和反义植株均呈阳性的株数分别为8株和11株，转化频率为67.8％。经过对转基因植株生长速度的测定，总体上，转正义基因的植株生长速度和植株高度要高于转反义基因和未转化的，转反义基因的植株其生长速度和植株高度要低于未转化的植株。在试验过程中观察到，转正义基因植株其根生长量要少于转反义基因的，转正义基因植株呈GA过量表现型，生长瘦弱，细高，叶子变小，叶色变浅，长势较转反义基因烟草的弱；转反义基因的烟草长势较好，植株有明显的矮化效果，茎杆较对照和转正义基因的烟草粗，叶色也较大，叶色变深。4、以pC1303为基础，引入BamH I和BisW I酶切位点，构建了适合于单子叶植物遗传转化的GA20氧化酶基因正义和反义表达载体。根据本实验室已建立好的结缕草和高羊茅的再生体系获得了两个草种的胚性愈伤，利用基因枪的方法将GA20氧化酶基因分别转到两个草种中。并对转基因抗性植株进行了PCR和PCR—Southern检测，共获得了转GA20氧化酶正义基因结缕草植株2株，高羊茅39株，其中凌志为35株，千年盛世为4株；获得转反义基因高羊茅的植株数为30株，其中凌志为27株，千年盛世为3株。结缕草转正义基因的植株的表型和对照植株差别不大。高羊茅的两个转基因品种表现较明显，其转正义基因植株的表型呈GA过量表型，生长较快，叶色变浅，植株变细；转反义基因的高羊茅植株生长缓慢，叶子变粗，叶色变深。更多还原

【Abstract】 Gibberellins(GAs) are important plant hormones, involved in various developmental and physiological processes of plants.GA20 oxidase is the key regulatory enzyme in GA biosynthesis and metabolism pathway. Cloning and studying the function of GA20 oxidase gene from turfgrass was a basic work for turfgrass dwarf breeding.The GA20 oxidase was obtained from wild Zoysiagrass(Zoysia japonica steud.) by the means of RT-PCR and RACE, the pET-30a-GA20 vector, pC35S1303GA20+and pC35S1303GA20- vectors, pC1303GA20+ and pC1303GA20- vectors were constructed respectively. The expression of GA20 in E.Coli, tobacco, tall fescue (Festuca arundinacea L.) and zoysiagrass were discussed and the conclusions showed as followings:1、 A pair of degenerate primers were designed according to the conserved sequence of GA20 oxidase of several gramineous plants the GA20 oxidase was isolated from wild Zoysiagrass by the method of RT-PCR and RACE. The gene cDNA full-length was 1311bp, containing a 1176 bp open reading frame, encoding a putative polypeptide of 392 amino acids. The GA20 oxidase in Zoysiagrass shared 55%-73% homology with wheat, barley, Oryza sativa and Lolium perenne. The GenBank accession number of GA20 oxidase gene isolated from Zoysiagrass is DQ645453.2、 The GA20 oxidase gene was cloned into the prokaryotic expression vector pET-30a and expressed in E. Coli BL21(DE3) strain under induced 1h, 2h, 3h, 4h and 5h by IPTG The product was identified by SDS-PAGE and the results showed that the specific expression of 45kDa protein was achieved, and the protein weight was increasing with the induce time increasing. 3、 The restriction eddonuclease BamH I and BisW I were used to construct sense expression vector pC35S1303GA20+ and antisense expression vector pC35S1303GA20- adapted to dicotyledonous plants genetic transformation. Ttansgenic tobacco plants obtained from leaf discs applying Agrobacteriummediated gene transfer carrying pC35S1303GA20+ and pC35S1303GA20-. 28 individuals hygromycin-resistant plants were recovered involving in 12 individuals transformed pC35S1303GA20+ and 16 individual transformed pC35S1303GA20- hygromycin-resistant plants. Hygromycin-resistant plants were identified by PCR and PCR-Southern blotting respectively, 8 individuals with pC35S1303GA20+ were obtained and 11 individuals with pC35S1303GA20-were obtained. The putative transgenic plants frequency was 67.8%. Transgenic tobacco growthrate was detected. As a whole, the growth rate faster degree as follows: pC35S1303GA20+ transgenic plants >control > pC35S1303GA20- transgenic plants. The root quantity of pC35S1303GA20+ transgenic plants was less than that of pC35S1303GA20- transgenic plants. These pC35S1303GA20+ transgenic plants displayed a phenotype that may be attributed to the overproduction of GA. The phenotype included a longer hypocotyl, less and lighter-green leaves, increased stem elongation and slimmer, and pC35S1303GA20- transgenic plants displayed a phenotype that may be attributed to the reducing production of GA, the phenotype included stem turned shorter and thicker, larger and darker-green leaves.4、 The restriction eddonuclease BamH I and BisW I were used to construct sense expression vector pC1303GA20+ and antisense expression vector pC31303GA20- adapted to monocotyledon plants genetic transformation. Plant regeneration system and Biolistic gene deliver system constructed by our laboratory was used in the experiments. Embryonic callus of tall fescue and zoysiagrass were used as material for genetic transformation with the sense and antisense GA20 oxidase gene by particle bombardment. The transgenic plants were obtained. Through PCR and PCR-Southern blot analysis, 2 zoysiagrass individual and 39 individuals tall fescue transformed pC1303GA20+ were obtained, including in Barlexus 35 individuals and Millennium 4 individuals; 30 individuals tall fescue transformed pC1303GA20- were obtained, including in Barlexus 27 individuals and Millennium3 individuals. There was not remarkbale dissimilarity in the phenotype between pC1303GA20- transgenic of zoysiagrass and control. Tall fescue pC35S1303GA20+ transgenic plants displayed a phenotype that may be attributed to the overproduction of GA. The phenotype included a longer hypocotyl, less and lighter-green leaves, increased stem elongation and slimmer. And tall fescue pC35S1303GA20- transgenic plants displayed a phenotype that may be attributed to the reducing production of GA, the phenotype included stem turned shorter and thicker, larger and darker-green leaves.更多还原