草坪遗传改良基因资源的发掘及草坪型高羊茅抗逆基因工程的初步研究

【作者】 曹雅君；

【导师】 蒯本科；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 叶片衰老是一个高度有序的调控过程,涉及到复杂的基因调控和多种生理生化特征的变化。我们以黑暗为选择因子,从经快中子诱变的拟南芥Columbia型M₂代种子中,筛选获得了耐受黑暗胁迫的突变体,将其中一株生长发育延迟而莲座叶早衰的突变体,命名为fnb45（fast neutron b45）。fnb45的突变造成了植株在形态、生长、发育等方面的变异,具有一因多效性。表型上,fnb45的子叶小而薄;莲座叶边缘为锯齿型,早期叶色灰绿,伴随着生长发育,叶片逐渐变为紫色,衰老死亡;植株顶端优势明显,结实率低,种子不饱满。另外,fnb45的发芽率较低（42.7%）,萌发速率慢（8天左右）;幼苗成活率仅30.1%;幼苗生长缓慢,20天左右才长出第一片真叶,营养生长期长,开花延迟（77天左右）,植株寿限延长（4个月）。遗传分析表明,fnb45是核遗传、单基因隐性突变位点。利用图位克隆的方法,fnb45被定位于第3条染色体长臂的两个分子标记T16K5和F3A4-1之间约50Kb的区间内,是一个新的调控拟南芥叶片衰老的遗传位点。我们研究了fnb45对外源激素、糖源和活性氧的响应,发现其对外源IAA、ABA、高浓度糖（蔗糖、葡萄糖）和PQ较敏感。同时,fnb45的突变还造成了营养生长阶段,植株体内的游离IAA、ABA、可溶性糖和花青素含量的急剧增加,破坏了细胞内的氧化还原平衡,导致活性氧清除系统中的SOD和CAT的活性增强,诱导细胞进入凋亡。高温（29℃）可诱导fnb45下胚轴伸长,但与Col野生型之间无明显差异,表明fnb45对IAA的敏感性是自身基因突变诱导的间接反应。我们检测了衰老和氧化胁迫相关基因的表达,发现突变体中ACS4,GD的表达水平上调,GS基因的表达下降。我们推测,fnb45位于叶片衰老调控途径的上游,该突变导致下游众多信号转导途径被改变,造成植株发育延迟、叶片早衰。更直接的证据还有待于基因的克隆和进一步的分析。更多还原

【Abstract】 Leaf senescence is a highly regulated progress that involves orderly,sequential changes in cellular physiology,biochemistry and gene expression.To screen stay-green mutants from mutagenized Arabidopsis seedlings(Col),mutants showing higher survivability under darkness stress were obtained.One of the mutants,which showed slow growth and premature rosette leaves,were named fnb45(fast neutron b 45).The mutant exhibited a pleiotropic phenotype,including small and thin cotyledons,slight opened hooks in etiolated seedlings,premature rosette leaves, increased apical dominance,decreased seeds.In addition,fnb45 showed delayed seed germination process(8 days) and reduced seed germination rate(42.7%),reduced plant stature,delayed flowering time(77 days) and prolonged lifespan of inflorescences(4 months).Genetic analysis showed the mutation was single recessive and mapped to the middle of chromosomeⅢ.Fine mapping results showed the mutation was located in a region of 50kb between T16K5 and F3A4-1,a novel locus involved in the regulation of leaf senescence in Arabidopsis.Compared with the wild type,fnb45 was hypersensitive to the exogenous IAA, ABA,high concentration of sugars and PQ.Also there were a 8-fold increase in free IAA level and an 4-fold increase in ABA level in the mutant.The increased endogenous level of IAA in fnb45 plants is probably indirectly affected by the FNB45 mutation.Sucrose metabolism and/or signal transduction were also found being altered,inducing increased levels of anthocyanin.And the activities of SOD and CAT in seedlings were higher than wild type.When checking the expression pattern of several senescence and oxidative stress related genes,we found that ACS4,GD,GS have apparent changes in expression.更多还原