Regulation of Tubulin Gene Expression During Soybean Internode Development

Fosket博士分离并测序了两个大豆β-微管蛋白基因(SB1和SB2)，这两个基因表现出不同的发育和器官特异性表达模式。然而，这两个基因都在伸长的节间表达，当黄化幼苗暴露在光下时，它们在转录水平上强烈而迅速地下调。他将确定控制SB1和SB2节间表达的顺式作用元件的性质。构建了由SB1和SB2启动子驱动的无启动子大肠杆菌β-葡萄糖苷酶(GUS)报告基因的载体，并通过农杆菌介导法转化烟草，并在转基因烟草中表达了嵌合基因。SB1和SB2可能的启动子区域的缺失和突变对转基因烟草中GUS报告基因表达的影响将被确定，以确定它们在伸长节间表达和光诱导下调的调控元件。GUS酶活性测定和S1核酸酶保护实验将用于验证GUS酶活性是嵌合基因转录活性的反映。GUS活性在转基因烟草植株中的精确细胞定位将通过组织化学方法确定，而SB1和SB2基因在大豆节间的细胞特异性表达将通过使用标记的基因特异性探针进行原位杂交来确定。此外，还将产生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。在大豆节间组织中，调控SB1和SB2微管蛋白基因转录的反式作用因子将通过足迹和凝胶滞留分析来确定。这些DNA结合调节蛋白将通过亲和层析分离，并将研究它们与SB1和SB2启动子元件相互作用的性质。针对这些DNA结合蛋白产生的抗体将用于从lambda gt11表达文库中分离编码它们的cDNA。这些DNA将被用作探针，以检测编码这些DNA结合蛋白的基因的表达调控。在植物中，微管是一个重要的亚细胞成分，参与决定细胞如何膨胀，从而决定组织如何生长。这项研究将阐明微管蛋白在植物生长和对光的反应中是如何表达的。

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.