大量表現NTPHAN、SCR及WOX3基因於菸草對葉片形態之影響

Abstract

植物葉片為進行光合作用及氣體交換的主要器官，是由莖頂分生組織（shoot apical meristem，SAM）周圍區域（peripheral zone）的幹細胞改變細胞分裂的速度及方向，並轉變為具有葉片特性的細胞，而在葉片發育形成過程有許多轉錄因子參與調控，使葉片得以順利發生並延展。本論文主要探討三個與發育相關基因— NTPHAN、SCARECROW（SCR）及WOX3，對葉片形態的影響。其中，金魚草的PHANTASTICA（PHAN）基因和野生菸草的同源基因NSPHAN皆參與葉片向軸面發育的調控。SCR基因參與調控阿拉伯芥根及莖部皮層細胞的平周分裂，形成內皮和皮層。阿拉伯芥的PRS/WOX3基因參與子葉原基邊緣細胞專一性的決定，及調控分生組織邊緣細胞分裂的速度。本試驗藉由基因轉殖技術，增加上述三個基因的表現後，觀察其對菸草葉片形態的影響。 以雙偶載體pCAMBIA1302為骨架，構築以CaMV 35S啟動子驅動基因持續性表現的載體，利用農桿菌轉殖法將NTPHAN、WOX3及SCR基因分別或共同轉殖到菸草（Nicotiana tabacum, Wisconsin38），試驗結果得到表現SCR基因的轉殖株1株，表現WOX3基因的轉殖株1株，以及同時表現NTPHAN和SCR的轉殖株4株。觀察轉殖株外表形態及葉片的組織切片，持續表現WOX3基因的植株具有明顯且穩定的形態變異，轉殖株的葉片由於上表皮細胞無法延展，造成向上捲曲的情形，且葉肉細胞排列緊密，形態也異常，而葉片主脈有扭曲的現象，組織切片結果顯示，維管束的分布及排列發生異常；表現SCR基因的轉殖株在組織培養階段有異位葉片發生，部分葉肉細胞有增加平周分裂的情形，移植到土盆後，葉片較非轉殖株狹長，但解剖構造上沒有明顯變異；同時表現NTPHAN和SCR基因的轉殖株在發育初期會有較狹長葉片發生，而後會葉片會延展，邊緣呈波浪狀，外表形態與非轉殖株沒有明顯差異，由葉片的組織切片結果顯示，葉肉細胞排列相當緊密，且葉片組織細胞沒有明顯的向軸－背軸極性。檢測轉殖株葉片向軸－背軸極性，結果顯示轉殖菸草葉片中，調控向軸極性建立之基因－NtPHAV的表現增加。 綜合以上論述，NTPHAN基因的表現影響葉片極性的建立，SCR基因的表現會影響葉肉細胞的分裂，而WOX3基因的表現則會造成菸草葉片的形態及解剖構造上顯著的變異，顯示這些基因雖然參與調控不同階段或器官的發育，但可能都會影響菸草葉片形態發育和極性建立之相關基因的表現，進而改變葉片的外表形態及構造。

Leaves are the major photosynthesis and gas exchange organ of plants. They originate from the peripheral zone of the shoot apical meristem (SAM), stem cells change the rates and the planes of cell division, and are recruited into the leaf primordia. A number of transcription factors are involved in the regulation of leaf development and extension. In this thesis, three genes related to the development of plant organs were studied, which are NTPHAN, SCARECROW (SCR) and WOX3. PHANTASTICA (PHAN) from Antirrunhum and NSPHAN, the Nicotiana ortholog of PHAN, regulates the development of adaxial part of leaves. SCR regulates the periclinal cell division in the cortex of roots and stems to form cortex and endodermis. WOX3 specifies the cell fate at the margins of cotyledonary primordia and regulates the rate of cell division in the margins of meristem in Arabidopsis. The effects on the leaf morphology of these three genes were studied by transformation into tobacco. These genes, driven by CaMV 35S promoter, were constructed on the binary vector, pCAMBIA1302. The construct was individually or co-transformed to tobacco (Nicotiana tabacum) by Agro-transformation method. Three kinds of transgenic lines were obtained that overexpressed SCR, or WOX3, or NTPHAN and SCR. The transgenic plant of over-expressed WOX3 had upwardly curled leaves caused by disrupting the extension of upper epidermis. Also it had distorted veins and abnormal mesophyll. The transgenic plant of over-expressed SCR had an ectopic leaf from the vein at the abaxial side. Abnormal cell division can be found at some regions of mesophyll. Its leaves had larger ratio of length and width than non-transgenic plants’. The transgenic lines that over-expressed NTPHAN and SCR developed narrower leaves at first and then the leaves expanded but with wave-like edge. Cells in the mesophyll were arranged compactly without adaxial-abaxial polarity. It was also found that in most transgenic plant leaves, the expression of NtPHAV, which promotes the adaxial identity in the development leaf, was enhanced. This result suggests that these three genes involved in development at the different growth stages or in different organs possibly by affecting the expression of leaf developmental genes.