MOLECULAR ANALYSIS OF FLOWERING GENES OF PHARBITIS NIL

本研究的目的是为了了解其发病的分子机制。 植物成花诱导。光周期，一个关键的控制因素 调节短日照植物Pharbitis nil is的开花诱导 调查的对象。几个cdna文库将 构建和筛选代表以下各项的克隆 子叶和顶端分生组织mRNAs的丰度受 感光周期。MRNAs的时空表达 所代表的cDNA克隆将通过斑点杂交、Northern 杂交和原位杂交程序。的5‘和3’端 MRNAs将由引物延伸和S1核酸酶决定。 保护性化验。这些cdna克隆的核苷酸序列为 被确定用于分析编码的多肽。 与这些cdna克隆相对应的基因组克隆将通过 以cDNA克隆为探针筛选基因组文库。实物地图 的基因组克隆将通过限制消化来构建 核酸内切酶。Southern印迹杂交分析将用于 识别基因的转录和非转录区域。DNA 5‘非转录区和其他感兴趣区域的序列(S) 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。 基因转移程序将被用来研究其功能和 目的基因(S)的表达调控。对于功能型 研究将确定异常表达的影响。编码 序列将在任何一种意义上被克隆(以过度生产蛋白质 产物)或反义(以产生互补RNA)定向 在农杆菌Ti质粒中的强植物启动子下游 派生的二进制向量。嵌合基因将被重新引入到 通过农杆菌介导法进行基因转移的植物。表型 由构成成分引起的转化植株中发生的变化(完) 编码基因产物或反义RNA的生产将 要下定决心。用于监管研究的基因融合包含 目的基因的启动子-调节区(S)和一个报告基因 (例如，大肠杆菌β-葡萄糖醛酸酶，GUS)将被构建。跟随 转移到植物细胞的GUS活性将在转化子中进行测量 植物来描述调控序列。顺序删除将 被构建和分析，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因子 由既定程序决定的因素。 将使用Southern杂交来确定序列是否 在长日照植物中存在与近疫霉cdna克隆同源的克隆。 拟南芥等植物。如果是，我们将隔离 并对拟南芥基因的表达和功能进行了分析。 类似的程序。 开花诱导是被子植物生活史中的一个关键过程。 了解光周期是如何控制这一过程的将大大拓宽 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.