【作者】 戚珠云；

【导师】 熊立仲；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 细胞分裂素对植物的生长、发育、细胞的分裂和分化、顶端优势、衰老、种子萌发、逆境胁迫的响应和氮代谢起着重要的作用。细胞分裂素主要在细胞分裂旺盛的组织产生,经过运输在植物的各个组织起作用。已经报道拟南芥存在AtENT和AtPUP这两个运输细胞分裂素的基因家族。对AtPUP的研究主要集中在其转运速率和底物选择性上,而对拟南芥生长发育方面的影响却未见报道。水稻是世界上重要的粮食作物之一。在水稻中只有OsENT基因家族被报道过可以转运细胞分裂素,PUP型基因家族还未见相关的研究报道。本文通过拟南芥中AtPUP基因家族氨基酸的序列比对,在水稻中筛选出一个具有12个成员的OsPUP基因家族。在对OsPUP家族12个成员进行生物信息学分析的同时对其中一个命名为OsPUP7的成员的功能进行了深入的研究。首先对OsPUP家族12个成员分析发现这些成员都有着较强的时空表达特异性。在水稻植株的不同组织和发育阶段,表达量变化较大。并且成员都具有较为保守的跨膜序列,多数成员具有10个跨膜结构。为了进一步研究该家族对植物生长发育的影响,从RMD获得了成员OsPUP7的T-DNA插入突变体(03Z11BF84),通过对突变体的共分离检测和突变体中的新转录本的序列分析发现新的转录本在突变体中起着显性负性作用。我们在检测OsPUP家族、ARR家族、CKX家族和IPT家族在突变体和对照植株中的表达差异的同时,完成的逆境胁迫和激素处理后OsPUP7表达量检测结果显示该基因表达会受到逆境和激素的诱导。根据上述表达量检测结果,对突变体进行了室内非生物逆境胁迫、激素处理和田间的干旱胁迫,结果显示OsPUP7基因在水稻的非生物逆境胁迫和激素响应中起作用。通过对突变体和对照植株未成熟种子和叶片中的细胞分裂素含量的测量,发现在突变体的未成熟种子中细胞分裂素含量较对照高,可能是OsPUP7突变体中新转录本转运功能丧失导致了突变体中细胞分裂素的积累。通过荧光定量PCR和GUS染色检测了OsPUP7成员的表达谱,显示其主要在维管束组织、幼穗和未成熟种子中表达量较高,与其转运的功能相符合。我们还通过OsPUP7转化酵母fcy2突变体的功能互补实验验证了OsPUP7转运咖啡因的功能。更多还原

【Abstract】 Cytokinins play important roles in plant growth, development and also in stress responses, such as cell division and differentiation, apical dominance, senescence, seed germination, nitrogen metabolism. Cytokinins are mainly synthesized in the tissues that have vigorous cell divisions and have effect on other tissue of plants by transportation. It was reported that there are two cytokinins transporting gene families (AtENT and AtPUP) in Arabidopsis. The study about AtPUP family mainly focuses on the transporting rate and substrate selectivity, while the effect on growth and development of Arabidopsis has not been reported. The rice (Oryza Sativa L.) is one of the important food crops in the worldwide. Only OsENT gene family has been reported that it has the ability to transporting cytokinins, however PUP-type gene family has not been reported before.In this study, by searching the rice genome using with BLASTP method using the amino acid sequences of Arabidopsis AtPUP gene family, we found there are12members of PUPs in rice. Besides the bioinformatics analysis of12members of the OsPUP family here, we mainly focus on OsPUP7for further functional analyses. At first, by analyses of12members of OsPUP family, they have specific temporal and spatial expression patterns. The expression levels have obvious variations at the different development stage and in different tissues of rice plant. All members have the consensus transmembrane sequence domain and most of them have10transmembrane helix regions.We want to find effect on growth and development of rice about this gene family, so obtained a T-DNA insertion mutant (03Z11BF84) from RMD. By analyses of co-segregation of mutant of OsPUP7and isolation and functional analyses of a new transcript of OsPUP7, We found the new transcript plays a dominant negative effect on mutant. We detection the expression level differences of OsPUP family, ARR family, CKX family and IPT family between the mutant and control. At the same time, the result of expression level examination about OsPUP7under the treatments of abiotic stress and hormone treatments show that the expression of this gene can be effected by abiotic stress and hormone treatments. From the results of expression examination, we finished the abiotic stress and hormone treatments in room and the drought stress test in the field. The results show that the OsPUP7have an effect on response to abiotic stress and hormone treatments. By the measurement of the cytokinins content in immature seed and leaf of mutant and control, we found that the cytokinins content in immature seed of mutant is higher than that in control. The accumulation of CTK in mutant may be caused by the lost transporting ability of the new transcript of OsPUP7mutant. The expression profiles about OsPUP7by Real-Time Quantitative PCR and GUS reporter analyses suggest that OsPUP7mainly express in vascular bundle, spikelet and immature seed, the expression model correspond with the transporting ability of OsPUP7. We also verified OsPUP7has the ability of transporting caffeine by the experiment of functional completementation through transforming the OsPUP7in the yeast fcy2mutant.更多还原