【作者】 汪得凯；

【导师】 孙宗修；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2005， 博士

【摘要】 随着水稻基因组测序计划的全面完成,阐明基因的生物学功能成为后基因组时代的重要研究内容,而水稻的株型是构成产量的主要因素,对水稻株型发育的分子机理的了解可以为育种提供理论基础。我们从T-DNA插入水稻突变体库中,筛选到三个与株型相关的突变体:短穗Bulrush-like panicle2（Bp2）、大叶角larger leaf angles（11a）和茎秆螺旋twisted-stem and tiller angle（ts-ta）,并对这3个突变体进行了形态学鉴定、遗传分析和初步的生理学研究,克隆了Bp2和11a基因并进行了部分功能研究。主要结果如下: 1.Bp2突变体表现为半矮化、穗型短而密集、叶片窄、抽穗延迟等特点,且花器官数目增加,结实率低,穗轴和枝梗伸长受到抑制,该突变体属于dn矮化类型,并发现突变体对温度敏感;遗传分析证实Bp2是稳定遗传的且受一对不完全显性核基因控制;Bp2和对照植株用GA3处理,野生型植株的株高GA₃处理比没有处理的增加17%,而Bp2处理与未处理植株的株高变化不显著,初步确定Bp2对外源GA₃不敏感; 2.对Bp2突变体自交和杂交后代植株进行Basta抗性分析,并用PCR和Southern杂交证明Bp2突变体的表型与T-DNA共分离;利用TAIL-PCR扩增Bp2突变体中T-DNA插入位置的旁侧序列,结果T-DNA插入到第5染色体的一个同源异型盒转录因子基因中;RT-PCR扩增Bp2基因的cDNA,并对Bp2编码产物进行了同源性分析;用RT-PCR分析了的器官表达,表明OsBp2在根、茎、叶、穗中呈组成型表达; 3.利用酵母自激活研究了转录因子OsBp2的激活功能,pGBKT-Bp2可以成功激活报告基因的表达;研究了其他禾谷类作物是否舆OsBp2的同源基因,结果野生稻中含有该同源基因,而小麦、玉米、高粱、甘蔗无明显的同源基因;构建了OsBp2基因的双链RNA干涉载体和超量表达载体,遗传转化正在进行; 4.对11a突变体的特征进行了鉴定,11a植株半矮化、叶角度大、叶片微卷和抽穗期延迟,突变体矮化类型也属于dn类型;GA₃处理的11a突变体株高可以部分恢复;11a可稳定遗传且受一对隐性核基因控制;Basta抗性分析和PCR分析表明11a的表型与T-DNA共分离;11a的旁侧序列分析发现T-DNA插入到第一染色体的一个WRKY转录因子基因中,根据已有报道,将该基因命名为OsWRKY11;用RT-PCR扩增11a的cDNA序列,并对11a编码产物进行了同源性分析;用RT-PCR分析了11a的器官表达特性,表明OsWRKY11在根、茎、叶、穗中呈组成型表达;用GFP进行亚细胞定位表明OsWRKY11在细胞核内发挥作用; 5.对ts-ta突变体进行了鉴定,突变体苗期叶鞘弯曲、螺旋生长,分蘖期表现为分蘖角度变大、半矮化、迟抽穗、育性较差等特点;苗期取弯曲生长的叶鞘进行石蜡切片,发现弯曲部位的细胞排列紧密;ts-ta是稳定遗传的且受一对隐性核基因控制;Basta抗性分析表明突变体与T-DNA不是共分离。更多还原

【Abstract】 With accomplish of the rice genome sequence project, elucidating the molecular basis of gene biological functions was the next challenge in post genomic era. Plant-type was the main factor in rice yield. Elucidated the molecular mechanisms of plant-type development will provide the theoretical fundament for rice breeding. Three mutants related to plant-type, i.e. Brush-like panicle2 （Bp2）, large leaf angles（lla） and twisted-stem and tiller angle（ts-ta） , were isolated from our T-DNA insertion mutant populations. The morphology , genetic and physiology of three mutants involved were analyzed. Mainly results were as follows:1. Bp2 showed semi-dwarf, short and dense panicle, narrow leaf, late heading and low fertility.Compared with the wild type, the floral organ number was increased in Bp2. Interestingly, Bp2 mutant showed thermo-sensitive. Genetic analysis of Bp2 mutant showed that Bp2 was stably inherited and the phenotype was regulated by a nuclear incomplete dominant gene. Bp2 and wild-type were treated with exogenous GA₃ at internodes elongation stages. The results demonstrated that Bp2 plants were insensitive to exogenous GA₃.2. The Basta reactions suggested that Bp2 mutant was co-segregated with the T-DNA insertion. PCR and Southern blotting confirmed the results. The sequence flanking T-DNA indicated that T-DNA inserted in a putative homebox transcription gene in rice chromosome 5. The cDNA of OsBp2 gene was amplified by RT-PCR. The OsBp2 transcriptions were detected in all organs which been tested.3. The OsBp2 ORF was fused in pGBKT7 DNA-BD sequence and transformed into yeast. The pGBKT7-Bp2 can successfully active the expression of report gene. The OsBp2 genes homologous were investigated in grass family. RNA interference vector and over-expressing vector were constructed.4. lla show semi-dwarf, large angles, rolled leaf and late heading. Bp2 and wild-type were treated by exogenous GA₃ at seedling and heading stage. Although the lla mutant sensitive to exogenous GA₃ at different concentrations, GA₃ tested plant height and leaf angles in lla couldn’t restore to the corresponding wild type. Genetic analysis of lla showed that lla can stable inherits and the lla mutant was regulated by a recessive nuclear gene. Basta reactions suggested that lla mutant was co-segregated with T-DNA insertion. PCR experiment confirmed the results. The sequence flanking T-DNA indicated that T-DNA inserted in a rice WRKY transcription gene in rice chromosome 1. The cDNA of LLA was amplified used by RT-PCR. The homologous proteins were also identified. The transcripts of OsWRKYll were detected in all organs which been tested. Subcellar location analysis showed that OsWRKY11 actives in nucleus.5. ts-ta showed twisted sheath at seedling stages and large tiller angles at adults, ts-ta also showed semi-dwarf, late heading and low fertility. The sections of sheath at seeding in ts-ta exhibits epidermis cells in twisted part was small, as a result the epidermis cells was arranged dense.更多还原