【作者】 夏广清；

【导师】 何启伟； 赵双宜；

【作者基本信息】 山东农业大学 ， 园艺学， 2005， 博士

【摘要】 植物生长发育受遗传因素和内外环境的影响。两者共同调控植物的生长发育、形态建成及开花结实等发育过程。其发育过程中不同信号传导途径间及它们之间的Cross-talking已被人们认识。赤霉素在大多植物中参与成花转变的信号传导过程,但其具体作用因植物而异,多种情况下可加速成花转变。本研究以冬性不同的两个大白菜（Brassica Campestris L,ssp pekinesis）B-17和1039自交系为试材,分别研究了植物成花生理信号（赤霉素）以及成花关键基因（LFY）的表达调控及在植物成花转变过程中的作用机理;同时采用RT-PCR方法,从大白菜花蕾中克隆了开花关键基因LFY,并构建了其双链dsRNA表达抑制载体,通过农杆菌介导法将其转入冬性较弱的秋大白菜1039中,并对转基因植株进行了形态学、生理学和分子生物学的研究,主要试验结果如下: （1）GA处理后可以使不同生态型的大白菜开花明显提前,同时使植物体内物质代谢、酶活性、DNA甲基化水平和内源GA含量发生了改变,具体表现为:随GA处理浓度增加,两个大白菜试材中可溶性总糖、蔗糖、淀粉和可溶性蛋白及内源GA含量增加:POD、α-淀粉酶活性升高而EST活性变化不明显;DNA甲基化水平降低。然而大白菜的冬性不同,对GA处理浓度反应也不同,在相同的条件下,弱冬性1039大白菜试材开花明显先于冬性较强的B-17大白菜试材,因此对冬性较弱的大白菜,较低的GA处理浓度就可以促进其开花。 （2）利用Northern杂交和相对定量PCR（RQRT-PCR）技术分析了GA不同处理浓度对大白菜种子、幼苗、花蕾中LFY基因表达的影响及其与LFYmRNA表达之间的关系。结果表明,LFY基因的mRNA在GA不同处理浓度下均有表达,并且在一定范围内,其表达量和GA处理浓度正相关。表明GA处理后,由于促进了大白菜不同发育阶段LFY基因的表达活性和其表达量,进而促进植物开花。 （3）通过改变培养基pH值和琼脂糖浓度,获得了由大白菜子叶-子叶柄诱导胚状体和不定芽的培养基配方。其分化生长培养基的配方分别为:MS培养基+3%蔗糖+4.5mg. L^-1 6-BA+0.5 mg. L^-1 IAA, pH 6.0-6.2,琼脂糖浓度为1.0%-1.2%及MS培养基+3%蔗糖+4.5 mg. L^-1 6-BA+0.5 mg. L^-1 IAA, pH5.7-5.8,琼脂糖浓度为0.8%-0.9%。通过对本实验供试的三个不同大白菜试材的比较研究发现,当pH值在6.0-6.2,琼脂糖浓度在0.8%-0.9%时,1039大白菜试材中有胚状体的形成,其诱导频率为1%左右,而当pH值更多还原

【Abstract】 Plant growth and development can be affected by genetic factors, internal and external environments. Different signal transduction pathways and their Cross-talking have been discovered in plant development. In most cases, gibberellin is involved in floral transferring process, and performs diverse function. In this paper, two ecotypes of Chinese cabbage (Brassica Campestris L,ssp pekinesis) B-17 (late-bolting) and 1039 (early-bolting) imbred lines were selected to explore the mechanism of GA and LFY gene in flowering pathway. LFY gene fragment was isolated from the bud of Chinese cabbage by RT-PCR method, and double strand RNA interference expression vector was constructed. The transgenic plants of 1039 imbred line were obtained through Agrobacterium tumefacien transformation, and their morphology, physiology and molecular biology were analyzed. The main results were as follows:(1) Both ecotypes of Chinese cabbage showed earlier flowering with GA treatment, which changed in the substance of metabolism, enzyme activities, DNA methyl level and endogenous GA content. With GA concentration increased, the contents of total soluble sugar, sucrose, starch, soluble protein and endogenous GA increased as well as the activities of POD , a -amlyse activities, but the activities of esterase changed little, DNA methyl level is then reduced. Under the same GA treatment, 1039 line flower earlier than B-17.(2) The relationship between GA concentration and LFY gene expression were analyzed in seed, seedling and bud of Chinese cabbage by Northern blot and relatively quantitative PCR (RQRT-PCR). The result showed that the amount of LFY -mRNA expression was positively correlated with the concentration of GA treatment. GA treatment promoting plants to bloom was probably due to increase expression of LFY gene in different development stage.(3) Through changing pH value and agarose concentration of culture medium, we developed a method for embryo and adventitious bud induction. The MS medium supplemented with 6-BA 5mg.L-1, NAA 0.5mgL-1, pH 6.0-6.2, agrose 0.8-0.9% was found to be optimum for embryo inducing in 1039 imbred line, and the frequency of induction was about 1.0%. While pH 5.7-5.8, agrose 1.0-1.2% was found to be optimum for adventitious更多还原