Functional Analysis of the BAS1 Gene and Its Product: CYP 72B1

油菜素内酯是一种促进生长的激素，参与调节植物的可塑性以应对环境的变化。以前对油菜素内酯的研究主要集中在这些激素的代谢或接受途径上。最近发现了一个影响植物油菜素内酯反应的新基因CYP72B1。该基因似乎参与了油菜素内酯的失活，而不是生物合成或感知，作为油菜素内酯羟化酶，将最活跃的激素——油菜素内酯分解为不活跃的形式——26-羟基油菜素内酯。CYP72B1通过调节特定组织或特定时间的油菜素内酯活性水平，可能在植物发育过程中作为油菜素内酯反应的调节剂。一种假设是CYP72B1使用油菜素内酯作为酶底物产生活性较低的油菜素内酯26-羟基油菜素内酯。CYP72B1在酵母中的异源表达结合放射性标记油菜素内酯的饲养实验，将检测CYP72B1是否以油菜素内酯为酶底物。生理分析也将用于进一步表征26-羟基油菜素内酯活性。第二个假设是CYP72B1表达通过调节油菜素内酯水平影响拟南芥的生长。CYP72B1基因的T-DNA敲除突变将允许详细分析该基因在发育中所起的作用。第三种假设是CYP72B1活性在组织特异性水平上受转录和翻译后调节。RT-PCR实验表明，CYP72B1转录本在莲座中的积累量大于发育中的下胚轴。CYP72B1转录物积累的测量将扩展到许多不同的幼体和成年植物的组织。CYP72B1与报告基因的翻译融合也将用于检测植物发育过程中不同组织中CYP72B1蛋白的水平。总之，这项研究将增加对CYP72B1在植物发育过程中调节油菜素内酯水平和感知中的作用的理解。除了培养一名研究生和一名博士后外，还有几名本科生可能参与该项目。在适当的时候，这项研究的结果将被纳入由首席研究员Michael Neff教授教授的DNA操作本科实验课程。

Brassinosteroids are growth-promoting hormones involved in modulating plant plasticity in response to changes in the environment. Previous studies of brassinosteroids have concentrated on either the metabolic or reception pathways for these hormones. A novel gene affecting brassinosteroid responses in plants, CYP72B1, has recently been identified. This gene appears to be involved in brassinosteroid inactivation rather than biosynthesis or perception acting as a brassinosteroid hydroxylase that catabolizes the most active form of the hormone, brassinolide, into an inactive form, 26-hydroxybrassinolide. By regulating the active levels of brassinolide in certain tissues or at certain times, CYP72B1 may be acting as a modulator of brassinosteroid responses during plant development. One hypothesis is that CYP72B1 uses brassinolide as an enzyme substrate generating the less-active brassinosteroid, 26-hydroxybrassinolide. Heterologous expression of CYP72B1 in yeast coupled with feeding experiments using radiolabeled brassinolide will test whether CYP72B1 uses brassinolide as an enzyme substrate. Physiological analysis will also be used to further characterize 26-hydroxybrassinolide activity. A second hypothesis is that CYP72B1 expression affects growth in Arabidopsis by regulating brassinolide levels. A T-DNA knockout mutation in the CYP72B1 gene will allow a detailed analysis of the role this gene plays in development. A third hypothesis is that CYP72B1 activity is regulated transcriptionally and post translationally at the tissue specific level. RT-PCR experiments show that CYP72B1 transcripts accumulate greater in the rosettes vsersus the hypocotyls of developing seedlings. CYP72B1 transcript accumulation measurements will be expanded to many different tissues of both juvenile and adult plants. CYP72B1 translational fusions with reporter genes will also be used to examine CYP72B1 protein levels in different tissues during plant development. Together, this research will increase the understanding of CYP72B1's role in modulating brassinosteroid levels and perception during plant development. In addition to the training of one graduate student and one postdoctoral fellow, several undergraduates are likely to participate in this project. Findings from this research will be incorporated, when appropriate, into an undergraduate laboratory course on DNA manipulation taught by the Principal Investigator, Professor Michael Neff.