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標題: Confocal microscopic dissection and characterization of lignin-related gene expressions in a rootless rice mutant
作者: 曹莎
Sha Tsao
關鍵字: 水稻;根部發育;突變株;木質素;Rice;root development;mutant;lignin
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TNG67 與 SA1613.1 皆為外表型正常且產量豐富的水稻,但兩者雜交的 F 2 子代卻出現分蘗數及根部性狀分離現象。利用 F 2 子代進行初步基因座定位分析,得知有兩個主要的 QTL 候選基因座存在,分別於第 1 號與 11 號染色體上,故命名為q1PN 與q11PN。當 q1PN 與q11PN 基因型為[T,S] (逗號前後分別代表q1PN 與 q11PN;T : TNG67 同型接合,S : SA1613.1 同型接合,H : heterozygous 異型接合)組合時,植株外表形呈現弱小、單分蘗且幾乎無根,將此性狀稱為 rootless plant with tiny single tiller (rolts)。與[T,T]比較,[T,S]植株發芽、照光 5 天後即出現種子根長度縮短、不定根數下降的情況。前人以顯微鏡觀察[T,S]種子根表面,發現其延長區與表皮細胞長度皆縮短,為了進一步研究內部細胞型態,因此再以[T,S]種子根縱切面做觀察。[T,T]植株的細胞分裂與延長區約 1000 μm 左右,而[T,S]則縮短為 90 μm左右,代表[T,S]根部出現提早成熟的現象。兩者在分裂與延長區的細胞大小相似,但長度卻明顯縮短,表示[T,S]在這段區域的細胞數目大幅下降。於成熟區計算皮層薄壁組織細胞之長度,發現[T,S]的細胞平均長度僅[T,T]的 25%。以 Auramine O染色,[T,T]的木質素出現在木質部,而[T,S]於細胞分裂、延長區與側根根原皆出現木質素異位累積。前人研究有發現添加蔗糖可改善 rolts 性狀,進行根部長度統計與根部縱切面實驗也進一步證實蔗糖的效應。利用次世代定序(RNA Seq)與即時聚合?鏈鎖反應(Real-time PCR)分析[T,S]與[T,T] 5 天之根部基因表現量差異,發現兩者在木質素合成相關基因上有表現量差異,且澱粉與蔗糖代謝路徑中有許多基因表現量差異超過兩倍。根據以上結果推測 rolts 基因可能與細胞壁生合成或養分供給相關,進而導致根部細胞出現木質素異位累積。目前利用[q1PN,q11PN]基因型,將第 11 號染色體上的 rolts 基因座候選區間縮小至 1660 kb。選擇此區間表現量差異超過兩倍與有興趣之基因進行互補試驗(complementation test),將其於TNG67 中表現並觀察是否出現 rolts 性狀,希望藉此可找到 rolts 相關基因。

TNG67 and SA1613.1 both are normal rice lines with high yield. However, their F 2 progenies exhibited wide segregations in panicle number and root biomass. Primary mapping on a F 2 population revealed two major QTLs, q1PN and q11PN, explained ~89% phenotypic variation. When both q1PN alleles are from TNG67, allele of q11PN from SA1613.1 acts semidominantly and generates rootless plant with tiny single tiller, abbreviated as 'rolts'. Compared with [T,T]-type rice (indicate q1PN and q11PN in front and after comma, respectively), [T,S] seedlings exhibited a shorter seminal root and fewer adventitious root (crown root) early on 5 days after germination. Previous microscopic analyses on the seminal root suggest that the lengths of epidermis cell in elongation zone and were shorten for rolts. Their internal longitudinal sections of were further examined in this study. Sign of early maturation was observed for rolts as the lateral root primordium formed right above a small cell division/elongation zone, whitch is less than 90 μm in contrast to ~1000 μm in wild-type plant. As cell size is similar between two plants, it suggest a severe decrease of the cell number in rolts. In the maturation zone, cells in rolts were barely elongated, with average length for cortex cells about 25% to that of the wild type plant. Stained by Auramine O, ectopically accumulated lignin can be seen for various cells in the division/elongation zones as well as the protruding end of lateral root primordium. Moreover, a typical xylem, indicated by the surrounded lignin, formed just several cells above the quiescent center. Interestingly, the root elongation defect seem to be reversible by addition of sucrose in hydroponic culture. Longitudinal dissections further confirmed the effect of sucrose. Examined by both RNA-Seq and real-time PCR, we found various genes related to starch metabolism or lignin biosynthesis that putatively involved in cell wall formation and/or nutrition supply were differentially expressed between [T,S] and [T,T] plants. As rolts gene was mapped on a 1660 kb region of chromosome 11, several differentially expressed genes within this region and with interesting putative function will be chosen for a complementation test, theoretically turn TNG67 into the rolts phenotype.
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