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標題: 利用五個玉米自交系以cDNA-AFLP技術分離玉米B染色體轉錄子
Isolation of B chromosome transcripts by using cDNA-AFLP analysis in five maize inbred backgrounds
作者: 林煥智
Lin, Huan-Zhi
關鍵字: 玉米;Zea mays.;B染色體;cDNA-AFLP;轉錄子;B chromosome;cDNA-AFLP;transcripts
出版社: 農藝學系所
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為了瞭解玉米B染色體的轉錄能力,本研究以cDNA-AFLP(complementary DNA amplified fragment length polymorphism)技術進行B染色體轉錄體的研究。利用帶與不帶B染色體的5個不同自交系,進行cDNA-AFLP分析,總共在28組引子組合中擴增到1,230個明顯的條帶,其中15個條帶(1.22%)至少在2個自交系中具有B染色體專一性,顯示B染色體具有微弱的轉錄能力。接著,選殖6個至少在4個自交系中具有B染色體專一性的cDNA-AFLP標誌,並進行南方雜合(Southern hybridization)與螢光原位雜合(fluorescence in situ hybridization)分析,發現與A染色體上的序列具有高相似度,但在B染色體上具有額外的拷貝數。進一步以RT-PCR(reverse transcription-polymerase chain reaction)分析證明4個標誌具有轉錄活性,且具多腺苷酸的尾端(polyadenylated tail),其中2個B染色體專一性cDNA-AFLP標誌B3547-179與B3849-212是B染色體專一性轉錄子且其表現量具有B染色體劑量補償現象(B-dosage compensation)。最後,將此6個標誌轉換成SCAR(sequence characterized amplified region)標誌,並利用4個B-10L易位染色體進行實體定位,將具有B染色體專一性的B4049-180gs SCAR標誌定位在DH2與DH3區域,而具B染色體劑量效應的B3849-212gs SCAR標誌定位在短臂至近端常染色質區間。另外,分別選殖DH2與DH3區域的B4049-180gs序列進行分析,顯示DH2區域具有較大的序列變異,支持B染色體由DH2區域演化出DH3區域的演化模式。

In this research, the cDNA-AFLP (complementary DNA amplified fragment length polymorphism) protocol was applied to the transcriptome of maize B chromosome for understanding the transcriptional activity of the B chromosome. A total of 1,230 clearly visible bands were amplified from 28 cDNA-AFLP primer sets by using five maize inbred lines with and without the B chromosome. Fifteen bands (1.22%) of which were shown the B-specificity in at least two inbred lines, suggesting the presence of weakly transcriptional activity of the B chromosome. Subsequently, six B-specific cDNA-AFLP markers with the B-specificity in at least four inbred lines were cloned, and analyzed by Southern hybridization and fluorescence in situ hybridization. These markers showed highly homology with sequences of A chromosomes, but had additional copies on the B chromosome. Only four markers were proven to have the transcriptional activity by RT-PCR (reverse transcription-polymerase chain reaction), and two of which were B-specific transcripts and reeked of the B-dosage compensation. Finally, the six B-specific cDNA-AFLP markers were transferred to SCAR (sequence characterized amplified region) markers and used to physically map their positions by four B-10L translocations. The B-specific SCAR marker, B4049-180gs was mapped on DH2 and DH3 regions, and the B-dosage SCAR marker, B3849-212gs was mapped between the short arm and the PE regions of the B chromosome. Furthermore, analysis of B4049-180gs sequences, cloned from DH2 and DH3 separately, exhibited that sequences from the DH2 had more variations, supporting the B evolution model in which the DH2 gave rise to the DH3.
其他識別: U0005-1908201317554000
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