MOLECULAR ANALYSIS OF FLOWERING GENES OF PHARBITIS NIL

本研究的目标是了解 植物的成花诱导 光周期如何，一个关键的控制 因子，调节短日照植物牵牛花的诱导， 调查的对象。 几个cDNA文库将被 构建并筛选用于分离代表 子叶和顶端分生组织的mRNA，其丰度受一种 诱导光周期 mRNA的时空表达 将通过斑点印迹、北方印迹和DNA印迹来确定由cDNA克隆代表的DNA序列。 印迹和原位杂交程序。 的5'和3'端， 通过引物延伸和S1核酸酶测定mRNA 保护测定。 cDNA克隆的核苷酸序列将是 确定用于分析所编码的多肽。 将通过以下方法分离对应于cDNA克隆的基因组克隆： 以cDNA克隆为探针筛选基因组文库。 物理图 将通过限制性内切酶消化构建基因组克隆的 核酸内切酶。 Southern印迹杂交分析将用于 识别基因的转录区和非转录区。 的DNA 5 ′非转录区和其它感兴趣区的序列 也将被确定。 这些研究将确定启动子- 调节区序列，并定义内含子-外显子边界，如果 任何. 基因转移程序将用于研究功能和 目的基因表达的调控。 用于功能 研究将确定异常表达的影响。 编码 序列将被克隆在任何一个意义上（过量生产蛋白质 产物）或反义（产生互补RNA）方向 在农杆菌Ti质粒中的强植物启动子下游 派生的二元向量。 嵌合基因将被重新引入 通过农杆菌介导的基因转移程序将植物转化为植物。 表型 植物中发生的组成性（过度）变化 编码基因产物或反义RNA的产生将 被确定。 对于调控研究，含有 目的基因的启动子调控区和报告基因 (e.g. E.大肠杆菌β-葡萄糖醛酸酶，GUS）。 以下 转移到植物细胞的GUS活性将在100 ℃下测量。 植物来描绘调控序列。连续删除将 构建和分析用于调控序列的精细定位。 我们还将鉴定、分离和纯化反式作用调节蛋白， 按既定程序考虑。 Southern印迹杂交将用于确定序列是否 长日照植物中存在与P. nil同源的cDNA克隆 拟南芥和其他植物物种。 如果是，我们将隔离 分析了A. thaliana genes by 类似的程序。 开花的诱导是被子植物生命周期中的一个关键过程。 了解光周期如何控制这一过程将大大拓宽 我们对植物发育过程的了解。

The goal of this research is to understand the molecular mechanism of floral induction in plants. How photoperiod, a crucial controlling factor, regulates floral induction in a short-day plant Pharbitis nil is the subject of the investigation. Several cDNA libraries will be constructed and screened for the isolation of clones that represent cotyledon and apical meristem mRNAs whose abundance is affected by an inductive photoperiod. The temporal and spacial expression of the mRNAs represented by the cDNA clones will be determined by dot blot, northern blot, and in situ hybridization procedures. The 5' and the 3' ends of the mRNAs will be determined by primer extension and S1 nuclease protection assays. The nucleotide sequence of the cDNA clones will be determined for the analysis of the encoded polypeptides. The genomic clones corresponding to the cDNA clones will be isolated by screening a genomic library using cDNA clones as probe. A physical map of the genomic clone will be constructed by digestion with restriction endonucleases. Southern blot hybridization analysis will be used to identify the transcribed and nontranscribed regions of a gene. The DNA sequences of the 5' nontranscribed region and other region(s) of interest will also be determined. These studies will identify the promoter- regulatory region sequences, and define the intron-exon boundaries, if any. Gene transfer procedures will be used to study the function and regulation of expression of the gene(s) of interest. For functional studies the effect of aberrant expression will be determined. The coding sequences will be cloned in either sense (to overproduce the protein product) or antisense (to produce complementary RNA) orientation downstream from a strong plant promoter in a Agrobacterium Ti plasmid derived binary vector. The chimeric gene will be reintroduced into plants by Agrobacterium-mediated gene transfer procedures. Phenotypic changes occurring in transformant plants resultant of constitutive (over) production of either the encoded gene product or the antisense RNA will be determined. For regulatory studies gene fusions containing the promoter-regulatory region of he gene(s) of interest and a reporter gene (e.g. E. Coli beta-glucuronidase, GUS) will be constructed. Following transfer to plant cell GUS activity will be measured in transformant plants to delineate the regulatory sequences. Sequential deletions will be constructed and analyzed for fine mapping of regulatory sequences. We will also identify, isolate and purify the trans-acting regulatory factors by established procedures. Southern blot hybridization will be used to determine if sequences homologous to the P. nil cDNA clones are present in the long day plant Arabidopsis thaliana and other plant species. If yes, we will isolate and analyze the expression and function of the A. thaliana genes by similar procedures. Induction of flowering is a critical process in angiosperm lifecycle. Understanding how photoperiod control this process will greatly broaden our knowledge on developmental processes in plants.