Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20938
標題: Isolation and characterization of rice embryo specific gene Ose731 and annalysis of Ose705, Ose712, Ose730 genes expression by in situ hybridization
水稻胚發育時期特有表現基因Ose731之選殖與分析及Ose705、Ose712、Ose730之原位雜交分析
作者: 羅慧如
Lo, Hui-Ru
關鍵字: coleoptile
芽鞘
shoot apex
胚芽
出版社: 分子生物學研究所
摘要: Ose731, a rice embryo specific and full-length cDNA clone, was isolated form a cDNA library made with poly(A) RNA from 10 DAP (days after pollination) embryo. The transcript of Ose731 was 1.2 kb in length. Tissue specific and temporal expression was analyzed by Northern blot hybridization. The mRNA expression of Ose731 was first detected at 7 DAP, then increased rapidly during 9 to 2.5 DAP and declined there after until barely detected in the mature embryo. The results also indicated that there was no mRNA deteced in roots, leaves, anthers and endosperm respectively. Western blot analysis revealed that the protein product was also detected in the rice embryo and the protein product reached the highest level at 30 DAP. The genomic Southern blot analysis revealed only one copy for Ose731 gene in the rice genome. A genomic clone osg731 has been isolated and partially sequenced. A fragment of 3085bp in length, consisting of a 5'' region of 1769bp, a 3'' flanking region of 496bp and a complete coding region of 820 bp (including a 103bp intron), has been sequenced. An unknown protein with 238 amino acid residues was deduced from this gene. In rice embryo sac, the zygote began cell division soon after fertilization. The zygote became a globular embryo containing five to eight cells within the first 48 hours. Organ development began with scutellum emergence in 3-day-old embryo. The shoot apex organized and the coleoptile developed from scutellum tissue at 4 DAP, the epiblast emerged at 5 DAP, and the vascular bundle and root apex differentiated at 6 DAP. In situ hybridization to detect the expression of mRNA was performed at various stages of developing embryos. The results revealed that the mRNA expression of Ose731 was first detected at globular stage. At 4 DAP, the hybridization signals were detected in a ventral region, where the shoot apical meristem and epiblast would later development, while less signal was detected in coleoptile. The first leaf primordium and the shoot apical meristem is flat at 5 DAP, at this stage, the signals were also detected. At 6 DAP, the second leaf primordium differentiated, and the Ose731 expression signal was observed both in the first and the second leaf primordia. A strong signal in shoot apical meristem was also detected at 6 DAP. At 10DAP, strong hybridization signals were observed in the radical, shoot and vascular bundles. However, the mRNA expression of three rice embryo-specific genes Ose712, Ose705 and Ose730 were also detected at various stages of developing embryos by in sity hybridization. The results revealed that the mRNA expression of these three genes was first detected at globular stage but less than Ose731. At 5 DAP, the hybridization signals of Ose712 were detected in shoot apex, root apex and first leaf primordium not in coleoptile and scutellum; wherease, at 10 DAP, the hybridization signals were only detected in basal part of coleotiple, shoot apex, first and second leaf primordia and less in root radicle not in other tissues. At 30 DAP, the hybridization signals of Ose712 were detected in coleoptile and root radicle. The hybridization signals in 5 DAP rice embryo of Ose705 were detected in coleoptile, shoot apex and root radicle but not in scutellum, at 10 DAP, the hybridization signals was detected in scutellum strongly and also in coleoptile and epiblast but not in shoot apex, first and second leaf pridomodia and root apex. At 5 DAP, the hybridizatin signals of Ose730 was also detected in coleoptile, shoot apex and root radicle;wherease, at 10 DAP, the mRNA expression of Ose730 were detected in shoot apex, first and second leaf primodria, radicle apex and coleorhiza of embryo organ and the signals are not strong.
以水稻授粉後10天之胚體建立一個cDNA基因庫,經由差異性篩選得到38個水稻胚特有表現選殖群組,Ose731是其中的一個選殖體,本論文針對此稻胚特有表現選殖體進行其基因組選殖系之篩選及分析。Ose731基因之cDNA全長為1023bp, 5''端非轉譯區含有26bp,轉譯區含有715bp以及3''端非轉譯區有262bp;該基因轉譯得到一個含有238個胺基酸之蛋白(分子量為26414.12)。經由篩選得到其基因體組選殖系,osg731,經過核酸定序分析共得到了3085bp,結果發現,5‘端啟動子區(promoter region)啟有1769bp,而且具有一個典型的G-box、C/A bybrid以及可能的“TATA“box、”CAAT”box; 3’下游區域有496bp及820bp的蛋白轉譯區(其中包括一個103bp之intron)。 根據北方墨點分析,Ose731基因之mRNA在水稻授粉後第7天就已經被偵測到,之後表現量持續增加,直到10DAP其表現量達到最高而且這種表現量一直持續到25DAP,之後則減少直到無法偵測到。Ose731基因所表現之蛋白也是水稻胚體特有,其表現量在11DAP即可被偵測到,直到25、30DAP此蛋白表現量最高,之後表現量又下降。 為了更清楚的知道Ose731基因及其所表現之蛋白在水稻胚體器官中的表現情形,故以原位雜交分析技術(RNA:RNA in situ hybridization)來偵測其mRNA在胚體中之表現,結果顯示,在球形胚體時期此基因之mRNA即有表現,隨著水稻胚體的發育在芽鞘(coleoptile)、胚芽(shoot apex)、胚根(root apex)、第一及第二葉原體(first and second leaf primodria)、外胚葉(epiblast)以及維管束(vascular bundle)中表現。再利用免疫組織化學染色法(immunohistochemistry staining)來偵測Ose731基因所產生之蛋白在胚體中之表現,結果此蛋白所表現之組織與mRNA的表現相同,但是在10DAP的胚體中此蛋白之表現組織卻與mRNA的表現不同,它在水稻胚體之芽鞘、子葉盤(scutellum)、外胚葉有表現,但是在胚芽、胚根及第一及第二葉原體卻沒有表現,根據上述結果,推測此蛋白會轉移至鄰近細胞。 另外,為了比較Ose731與其它水稻胚特有表現基因之不同,所以選擇了三個基因,分別為Ose712、Ose705以及Ose730進行原位雜交分析。結果顯示,這三個基因在水稻球形胚體皆有表現,但是表現量較Ose731少;在5DAP之胚體中,Ose712在胚芽及胚根有表現其它組織則無,而Ose705則表現在胚芽、芽鞘及胚根,Ose730也是表現在胚芽、胚根及芽鞘。在10DAP時期,Ose712只有表現在胚芽、芽鞘及第一及第二葉原體的基部,在胚根則有較少的表現;Ose705在此時期則很強的表現在子葉盤及外胚葉,在芽鞘也有較少的表現量,其它組織則無;Ose730表現在胚芽、芽鞘、第一及第二葉原體、胚根及胚根鞘,而且表現量較弱。Ose712在30DAP之胚體則表現在子葉鞘及胚根,其它組織則無。由以上分析結果可以知道,每一個基因在水稻胚體的表現都不同,可以用此來推測這些基因在水稻胚體所可能扮演的角色。
URI: http://hdl.handle.net/11455/20938
Appears in Collections:分子生物學研究所

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