Regulation of Tubulin Gene Expression During Soybean Internode Development

Fosket博士已经分离出两种大豆β微管蛋白 基因（SB1和SB2）表现出不同的模式， 发育和器官特异性表达。然而，这两种基因 在伸长的节间中表达，它们强烈地， 在转录水平上迅速下调， 将黄化的幼苗暴露于光。他将决定 控制节间顺式作用元件的性质 SB1和SB2的表达。构建了无启动子的E. 大肠杆菌β-葡萄糖苷酶（GUS）报告基因是由SB1 和SB2启动子已经被制备并转移到烟草中， 农杆菌介导的转化，并且嵌合基因是 在转基因烟草植物中表达。的影响 SB1推定启动子区的缺失和突变 和SB 2对GUS报告基因在大肠杆菌中表达的影响 转基因烟草将被确定，以确定监管 负责它们在伸长过程中表达的元件 节间和它们的光诱导下调。Gus 酶活性测定和S1核酸酶保护实验 将被用来验证GUS酶活性是一种 这反映了嵌合基因的转录活性。 GUS活性在细胞内的精确定位 转基因烟草植物将用组织化学方法测定， 而SB1和SB2基因在细胞中的特异性表达， 大豆节间将通过在 使用标记的基因特异性探针进行原位杂交。 此外，将生成SB1和SB2特异性抗体 并用于确定哪些微管阵列包含这些 微管蛋白转录调控因子 SB1和SB2微管蛋白基因在大豆节间组织中的表达 将通过足迹法和凝胶阻滞法进行识别 测定。这些DNA结合调节蛋白将通过以下方法分离： 亲和色谱法及其与 将研究SB1和SB2启动子元件。产生的抗体 这些DNA结合蛋白将用于分离cDNA 从Lambda GT11表达式库编码它们。的cdna 将被用作探针来检查表达的调节， 编码这些DNA结合蛋白的基因。 %%% 在植物中微管是一种重要的亚细胞成分 参与决定细胞如何扩张， 组织如何生长这项研究将阐明如何表达 微管蛋白在植物生长过程中表达， 对光的反应。

Dr. Fosket has isolated and sequenced two soybean beta-tubulin genes (SB1 and SB2) which exhibit different patterns of developmental and organ specific expression. However, both genes are expressed in elongating internodes and they are strongly and rapidly down regulated at the transcriptional level when etiolated seedlings are exposed to light. He will determine the nature of the cis-acting elements controlling the internodal expression of SB1 and SB2. Constructs in which a promoterless E. coli beta-glucosidase (GUS) reporter gene is driven by the SB1 and SB2 promoters have been made and transferred to tobacco by Agrobacterium mediated transformation, and the chimeric genes are expressed in the transgenic tobacco plants. The effect of deletions and mutations of the putative promoter regions of SB1 and SB2 on the expression of the GUS reporter gene in transgenic tobacco will be determined to identify the regulatory elements responsible both for their expression in elongating internodes and for their light induced down regulation. GUS enzymatic activity assays and S1 nuclease protection experiments will be used to verify that GUS enzymatic activity is a reflection of the transcriptional activity of the chimeric genes. The precise cellular localization of GUS activity in the transgenic tobacco plants will be determined histochemically, while the cell specific expression of the SB1 and SB2 genes in soybean internodes will be will be determined by means of in situ hybridizations, using labeled, gene specific probes. Additionally, SB1 and SB2 specific antibodies will be generated and used to ascertain which microtubule arrays contain these tubulins. The trans-acting factors regulating the transcription of the SB1 and SB2 tubulin genes in soybean internodal tissues will be identified by means of footprinting and gel retardation assays. These DNA binding regulatory proteins will be isolated by affinity chromatography and the nature of their interaction with SB1 and SB2 promoter elements will be studied. Antibodies raised to these DNA binding proteins will be used to isolate cDNAs encoding them from a lambda gt11 expression library. The cDNAs will be used as probes to examine the regulation of expression of the genes encoding these DNA binding proteins. %%% In plants microtubules are an important subcellular component involved in determining how cells expand and as a consequence, how tissues grow. This study will elucidate how the expression of a microtubule protein is expressed during plant growth and in response to light.