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|標題:||水稻 T-DNA 插入突變體 M38333 , M69202及M69217的特性分析|
Rice functional genomics study using T-DNA insertion mutants: Characterization of rice T-DNA mutants M38333, M69202 and M69217
|作者:||Joji Grace, C.Villamor|
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|摘要:||本研究透過順向遺傳學及逆向遺傳學研究策略，篩得一些T-DNA插入突變株。為了探討水稻轉錄因子APETALA2 (AP2) 基因的功能，利用逆向遺傳學的策略，篩得水稻T-DNA插入突變株M38333，其T-DNA插入水稻BAC OSJNBa0044E16第37,503 bp處，位於預測為AP2基因下游5,705 bp。M38333外表性狀與TNG67比較，呈現半矮性、開花延遲及在水稻劍葉lamina joint處傾斜下垂。分析T-DNA插入鄰近基因之表現，發現333-07 (unknown protein) 及333-09 (putative AP2 protein) 兩個基因有被活化大量表現，推測M38333的外表性狀特異可能藉由活化此兩基因所造成，目前正進一步構築大量表現此兩基因的水稻轉殖株，以證實M38333之外表性狀。另外，探討水稻Squamosa promoter binding like protein (SPL)基因功能，結合順向遺傳學及逆向遺傳學策略，篩得水稻T-DNA插入突變株M69202，其T-DNA插入水稻BAC OSJNBa0016N23第52,005 bp處，位於OsSPL14基因上游5,489 bp。M69202外表性狀呈現植株高度矮化、分蘗數較少、開花延遲及萌芽率較低現象。推測可能藉由活化202-06 (putative β-glucosidase isozyme 2 precursor) 和206-08(OsSPL14) 的大量表現所造成。另一方面，以順向遺傳學方式，篩得水稻T-DNA插入突變株M69217，其外表性狀為植株高度矮化、開花延遲及高分蘗數，其T-DNA插入水稻BAC OJ1297_C09第36,560 bp處，位於一個推測為hypothetical protein基因的第一個intron之間。經由基因表現分析得知，M69217突變株會造成217-07 (putative chorismate mutase )以及217-09 (putative cinnamoyl-CoA reductase) 兩基因的活化，然而其外表性狀與基因表現並非百分之一百的關連。本研究將藉由選殖突變株中被活化的基因，並建立能大量表現選殖基因的水稻轉殖株，以進一步探討基因功能。|
A number of rice T-DNA mutant lines were screened in this study using both reverse and forward genetics approaches. In attempt to study APETALA2 (AP2), a large super family of plant-specific transcription factors, the mutant line M38333 was acquired through reverse genetics. The T-DNA had inserted at 37,503 bp in BAC OSJNBa0044E16 5,705 bp downstream the TAA of a putative AP2 gene. M38333 shows distinct semi dwarf, delayed flowering and greater lamina joint inclination phenotype compared to TNG67 and RT-PCR analysis suggests that this phenotype may be due to the activation of flanking genes 333-07 (unknown protein) and 333-09 (putative AP2 protein), since it is highly co-segregated with the phenotype of the mutants. To further confirm these findings, genes 333-07 and 333-09 were cloned for over-expression in TNG67 however over-expression transgenic plants are yet to be characterized. Moreover, combined reverse and forward genetics was employed for the study of Squamosa promoter binding like protein (SPL) gene and the mutant line M69202 having a T-DNA inserted at 52,005 bp of BAC OSJNBa0016N23 5,489 bp upstream the OsSPL14 gene was acquired. The mutant exhibits short plant height, less tillers, delayed flowering and slow germination rate. The T-DNA insertion and the phenotype of M69202 is highly linked and suggests that the activation of 202-06 (putative β-glucosidase isozyme 2 precursor) and 202-08 (OsSPL14) may play a role on the observed phenotype of the mutants. On the other hand, M69217 which was acquired through forward genetics and characterized to be dwarf, delayed flowering and have high tiller number, has a T-DNA at 36,560 bp of BAC OJ1297_C09 located in the first intron of a hypothetical protein on chromosome 2 of rice genome. RT-PCR analysis revealed that 217-07 (putative chorismate mutase) and 217-09 (putative cinnamoyl-CoA reductase) are activated by the CaMV 35s enhancer of the T-DNA. The T-DNA insertion and the activation of these genes is highly linked to the phenotype observed in the mutants, although, not 100%. To further understand what roles these genes might have in the mutant, cloning of the said genes for over expression in transgenic rice is now on going. Preliminary data shown in this study suggests that these selected mutant lines are potential material that can be used for gene functional analysis in rice.
|Appears in Collections:||分子生物學研究所|
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