2004-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/704917摘要：pPZP202:BAR:SK是一個全新的、具有便利性及高效率的T-DNA Tagging載體。這個載體具有抗“Basta”殺草劑之基因-BAR，是目前最經濟、方便及有效率的轉殖植物篩選基因。因此，針對一個具有特殊變異表現型之T-DNA tagged knockout植株，利用plasmid rescue的方法，可得知是那一個基因遭受破壞所致。這種reverse genetics的手段，是目前研究基因與其功能，最直接而且有效的一個策略。在我們的先前實驗中，己經順利地產生五仟多個T-DNA Tagging植株，其中篩選到了至少23個具有葉部形態產生變異之變異株。在TP15及TP17這二個變異株中，以Southern blotting分析，確定各只含有一個T-DNA插入，利用plasmid rescue的方法己找到其插入點的基因。經分析、比對的結果TP15變異株，植株具有葉片向下捲曲，花穗增長，種子數目減少的特性，其T-DNA是插入在clathrin heavy chain基因的coding region之中，這個基因在動物系統中是扮演蛋白質sorting及endocytosis的功能；至於<br> Abstract: A convenient and economic gene knockout method using a de novo T-DNA tagging vector containing herbicide Basta resistant gene has been used to generate transformed Arabidopsis plants. Null mutations are useful because they provide direct evidences for making strong arguments that the phenotype is indeed caused by the mutation. The T-DNA tagged gene can be recovered using plasmid rescue. During the passed year, we generated around 5,000 T-DNA tagged lines. At least 23 mutants with various leaf form mutation have been isolated. One copy of T-DNA insertion was shown in TP15 and TP17 by Southern blot analyses. Mutant TP15, with a knocked out clathrin heavy chain gene and which is functional with protein trafficking and endocytosis in animal system. TP15 demonstrate phenotypes of epinasty, long inflorescent stalk, and reduce seed set. In TP17, a gene with homology as mouse mSin3A gene was knockouted, which functional as a regulator in histone deactylase complex. TP17 with serrated life margin and reduced seed. We are also proceeding to identify and characterize genes for the rest of interesting mutants. We also propose to generate a T-DNA insertion pool containing 100,000 to 150,000 independent lines to cover the whole Arabidopsis genome. The pool will be open to the academics and research institutes for screening mutants of their interest.T-DNAKnockoutLeaf MorphogeneisReverse Genetics