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Molecular Cloning and Expression of ANS(anthocyanidin synthase) and GST(glutathione S-transferase) in Seed Coats of Soybean
本論文目的以選殖青仁烏豆種皮之ANS（anthocyanidin synthase）與GST（glutathione S-transferase）基因，以探討ANS基因與R基因座之關聯性，並希望能得到具有轉移花青素功能之GST。根據已發表的葡萄等物種之ANS核酸序列設計退化引子，以逆轉錄聚合酶鏈鎖反應策略，得到含預期片段之ANS4殖系，經篩選cDNA基因庫，共得到ANS23-1及ANS100二個完整之殖系。另將本實驗室先前所選殖的殖系ANS27-1合併探討，三殖系均可編碼由352個胺基酸所組成的蛋白質，其中ANS23-1與ANS27-1編碼相同之蛋白質。由南方雜交分析及基因組DNA聚合酶鏈鎖反應顯示，大豆基因組中有三套ANS基因，且無多型性。在Clark近同源品系所有的R與R* 或rm55基因型品系中，ANS基因之表現呈現兩個高峰期，而所有隱性r與r-m基因型則無第二高峰之表現。由逆轉錄聚合酶鏈鎖反應之結果，發現黑色種皮的UC9（iRT）和棕色種皮UC14（irT）品系於種子鮮重50-75毫克時期均有三種ANS基因之表現，而UC14大於400毫克之時期則缺少ANS23-1基因的表現，顯示ANS基因表現與色素產物及種皮顏色有關，但是，由本論文的試驗結果仍無法描述ANS基因與R基因座之關係。
以GST2 （Y10820）cDNA序列所設計的專一性引子，選殖到GST420殖系，並用以篩選基因庫，共得到二個全長之GST cDNA殖系GST11-1（926 bp）與GST70-1（930 bp），分別編碼具有219個及216個胺基酸的蛋白。由蛋白質結構分析顯示，二殖系均具有GST保留之 N端及C端domain及G-site。南方雜交分析結果顯示，於不同基因型之Clark近同源品系GST基因至多為二套，且無多型性之樣式。而不同組織表現分析中，GST在種皮的全生育時期及葉片等部位均有表現，而在花中並無偵測到基因的表現，而此二殖系是否扮演花青素轉移之”護衛”，仍需進一步試驗。
The purpose of this thesis is to clone the cDNA encoding anthocyanidin synthase (ANS) and glutathione-s-transferase (GST) from soybean seed coat to study their relationship between Clark near isogenic lines with various genes relation to pigment biosynthesis. Three complete cDNAs, ANS23-1 (1,252 bp), ANS27-1 (1,302 bp) and ANS100 (1,233 bp), coding for ANS were cloned from seed coats of the black soybean variety Chin-Ren-Woo-Dow (CRWD). Southern blot analysis indicated that ANS gene is presented as a small gene family in soybean genome and shows no RFLP difference. The deduced amino acid sequences of these three ANS cDNAs are with 80-74﹪identity to ten registered ANS proteins by BLAST analysis of NCBI. Two gene expression peaks, at seed fresh weight of 50-75 mg and >400 mg, of ANS gene are detected during seed development in the isogenic line containing a dominant R or rm55 genotype by northern hybridization. The ANS gene is not or only slightly expressed in the recessive r and r-m genotypes at late stage of seed development besides, no ANS23-1 signal is detected in the irT genotype by competitive RT-PCR. The result suggests that the gene encoded by the R locus is closed related with the ANS gene.
Two complete cDNA clones, GST11-1 (926 bp) and GST70-1 (930 bp), of type III GST were obtained by screening the cDNA library with a specific fragment amplified by primers designed from soybean GST2 (Y10820) sequences in Genebank. The deduced amino acid sequences show 100-41% identity to ten registered GST proteins by Blast analysis of NCBI. Southern blot analysis indicates that two copies of GST are presented in the genome of Clark isogenic lines and with no polymorphism. Northern hybridization shows that two GST genes are expressed in the seed coats and leaves, but not expressed in flowers. Further study will be conducted to realize the differential expression of the GSTs in these tissues.
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