Molecular Genetics of Arabidopsis Titan Mutants

[91754]拟南芥泰坦（ttn）突变体在种子发育过程中表现出显著的有丝分裂和细胞周期控制。这些突变体的共同特征是胚乳核变大和高度多倍体。突变胚胎在细胞大小、形态和活力上不同，取决于所涉及的位点。四年前，Meinke实验室首次发现了三种具有不同表型的泰坦突变体。在之前的资助期间，与诺华公司（先正达）合作，又发现了10个泰坦突变体，并克隆了5个泰坦基因。其中一些基因编码染色体凝聚和剂量补偿所需的凝聚蛋白（SMC）。另一个基因产物与与囊泡运输相关的ARF类GTP结合蛋白有关。其余的基因与其他细胞过程有关。在种子发育过程中，这些不同的蛋白质如何相互作用影响核分裂和细胞活力仍有待确定。该项目将使继续分析突变体成为可能，并确定种子发育过程中基因功能、核动力学和细胞生长之间的关系。重点将放在SMC类的泰坦上，因为这些蛋白质似乎与突变表型最直接相关。项目目标是通过反向遗传学鉴定剩余的SMC基因敲除，通过荧光和电子显微镜更详细地表征核和细胞骨架缺陷，分析多个SMC敲除的表型，并确定SMC在植物中的功能如何与其他生物中的功能进行比较。将首先通过尝试用映射到ttn1区域的ARF- gap来补充ttn1来检查ARF类泰坦。这将完成在上一个拨款期间启动的基于地图的克隆工作。另一类泰坦将通过分析novartis收集的更多标记的胚乳突变体来部分检查，包括那些具有中间泰坦表型的胚乳突变体。这些额外的突变体将与现有的巨体进行比较，以建立影响胚乳发育的复杂基因网络。泰坦表型表明拟南芥胚乳组织在有丝分裂和细胞周期控制中为异常核功能的增殖提供了理想的环境。对这些突变体的详细分析将为胚乳发育和进化过程中核动力学的新特征提供有价值的见解。长期目标是了解被子植物胚乳形成的复杂机制。

0091754MeinkeThe titan (ttn) mutants of Arabidopsis exhibit dramatic alterations inmitosis and cell cycle control during seed development. The commonfeature among these mutants is that endosperm nuclei become enlarged andhighly polyploid. Mutant embryos differ in cell size, morphology, andviability, depending on the locus involved. Three titan mutants withdistinct phenotypes were first identified in the Meinke laboratory 4 yearsago. During the previous grant period, 10 additional titan mutants wererecovered and 5 TITAN genes cloned in collaboration with Novartis(Syngenta). Several of these genes encode condensin (SMC) proteinsrequired for chromosome condensation and dosage compensation. Anothergene product is related to the ARF class of GTP binding proteinsassociated with vesicle trafficking. The remaining genes are involvedwith other cellular processes. What remains to be determined is how thesediverse proteins interact to influence nuclear division and cell viabilityduring seed development. This project will make it possible to continue the analysis of titanmutants and determine the relationship between gene function, nucleardynamics, and cell growth during seed development. Emphasis will beplaced on the SMC class of titans because these proteins appear to be mostdirectly linked to the mutant phenotype. Project objectives are toidentify the remaining SMC gene knockouts through reverse genetics,characterize nuclear and cytoskeletal defects in more detail throughfluorescence and electron microscopy, analyze phenotypes of multiple SMCknockouts, and determine how SMC functions in plants compare with thoseestablished in other organisms. The ARF class of titans will be examinedfirst by attempting to complement ttn1 with an ARF-GAP that maps to theTTN1 region. This should complete a map-based cloning effort initiatedduring the last grant period. The other class of titans will be examinedin part by analyzing more tagged endosperm mutants from the Novartiscollection, including those that have intermediate titan phenotypes.These additional mutants will be compared with existing titans toestablish the complex network of genes that influence endospermdevelopment. The titan phenotype demonstrates that Arabidopsis endosperm tissueprovides an ideal environment for proliferation of aberrant nucleidefective in mitosis and cell cycle control. Detailed analysis of thesemutants should provide valuable insights into novel features of nucleardynamics during endosperm development and evolution. The long-term goalis to understand the complex mechanisms that regulate endosperm formationin angiosperms.