甘藷中SPORAMIN及SP-B相似基因的選殖與序列分析

Abstract

SP-B cDNA是以澱粉磷解?（starch phosphorylase; SP）抗體做?探針（probe），對甘藷（Ipomoea batatas cv. Tainong 57）lambda-gt 11 expression library進行澱粉磷解?基因篩選時，一同被篩選出的。SP-B基因在甘藷的表現具有組織專一性，其在塊根（tuberous root）中表現最強。 將SP-B cDNA序列與軟體DNA bank及protein bank內的資料比對，並沒有發現與其具有高同源性的DNA序列或蛋白質。故推測其可能是一種尚未被研究的基因．但另一方面卻發現SP-B cDNA序列與甘藷中水溶性蛋白sporamin基因的A，B亞群（subfamily）的反義股（antisense strand）分別具有91％及80％的同源性。根據cDNA序列的比對，SP-B可能扮演sporamin基因的反義基因（antisense gene）的角色，此二基因可能位於DNA上同一區域，但轉錄的方向相反；此外SP-B基因亦可能?一獨立表現的基因。?對此做進一步的探討，吾人以SP-B cDNA做?探針（probe），對甘藷基因庫進行篩選，並對篩選出的選殖株做核?酸序列分析，論文中首先分析的二個選殖株分別稱?gSPOR5-31及gSPB1-1。gSPOR5-31選殖株具有2248個鹼基對，此基因經由核?酸序列比對，發現其與日本Nakamura等人所發表的gSPO-A1基因轉譯區（coding region）具有93.5％的同源性，但5'及3'端的非轉譯區則只有80％與70％同源性，因此gSPOR5-31應?sporamin A次基因群中一個新的基因，分析其5'及3'端，發現皆具有起動子（promoter）相似序列，經由primer extension的方法得知其轉錄起始點位於起始密碼上游33個鹼基處。而gSPB1-1具有2017個鹼基對，且其與SP-B cDNA具有97.3％的同源性，其5'端具有TATA及CAAT等類似起動子序列，而其轉錄起始點位於起始密碼上游56個鹼基處。 經由DNA序列的比對發現sporamin及SP-B基因的反義股同源性極高，且經由核?酸序列分析發現gSPOR5-31基因不但5'端具有起動子序列，其3'端亦具有一反向的起動子相似序列，因此更提高了sporamin基因與SP-B基因是位於同一基因座的可能性。未來可對目前所篩選到的基因進行起動子功能測試以更進一步確定是否sporamin（或SP-B）基因的兩端皆具有起動子功能，而使得此基因座的兩股皆可進行轉錄。

SP-B cDNA was co-isolated with a starch phosphorylase (SP) cDNA from sweet potatoes (Ipomoea batatas cv. Tainong 57) expression library by an anti-SP antibody. The size of the SP-B cDNA is 827 bp and the nucleic acid sequence has 91% and 80% homology to the antisense strands of sporamin A and B subfamily genes. Basing on the preliminary investigation by Yeh, et al., SP-B could work as an antisense gene against sporamin genes. In order to understand whether the sporamin gene and SP-B gene were transcribed from opposite strands of the same DNA locus or not, we analyzed the sporamin and SP-B gene. We reported two genomic clones, designated as gSPOR5-31 and gSPB1-1, respectively isolated from sweet potato genomic libraries using SP-B cDNA as a probe. The nucleotide sequences were determined and analyzed. The gSPOR5-31, contains 2248bp. The transcriptional start site of the gSPOR 5-31 was located 33 bases upstream from the coding region. It has 93.5% homology with gSPO-A1(17) in coding region, 81.7% homology with gSPO-B1(17) in coding region Therefore, the gSPOR5-31 should be a novel gene family member of sporamin A. The 5' flanking and 3' flanking of the gSPOR 5-31 had putative promoters. The orientation of the two putative promoters were opposite. On the other hand, the gSPB1-1 containing 2017bp in size, has 97.3% similarity to the SP-B cDNA in coding region and there is a putative TATA box and a putative CAAT box in the 5' flanking region. The transcriptional start site of the gSPB1-1 was located 56 bases upstream from the coding region. The 5' flanking and 3' flanking sequences of the two genes will be constructed in pBlue-CAT respectively, in the future, transferred the chimeric plasmid to sweet potato or tobacco protoplasts, and assay for their promoter activity, if both of the 5'and 3' flanking of the genes have promoter function, it will demonstrate that both strands of the gene are able to be transcribed, possibly under some condition.