Cloning and Characterization of Pigment Transportation Gene －Glutathione S-transferase (GST) from Soybean Seed Coat

Abstract

利用大豆EST基因庫中穀胱苷肽轉移酶(glutathione-S-transferase)(GST2，Y10820)基因序列設計特異核酸引子組，進行逆轉錄酶鏈合反應(RT-PCR)，獲得GST2之基因片段GST420 殖系。經序列分析及基因庫比對確認後，用以篩選黑色(iRT 基因型)大豆青仁烏豆(CRWD)種皮之cDNA 基因庫，共得到二個完整的 GST cDNA 殖系 GST11-1(AY382831, 926 bp)與 GST70-1(AY382832,930 bp)，分別編碼具有 219 個及 216 個胺基酸的蛋白質。由蛋白質結構分析顯示，二殖系均具有 GST 共同之 N 端與 C 端domain 及與 glutathione 結合之 G-site 等三個保留區，依特性分類應屬於第三群之GST基因。以七個不同I, R, T 基因型組合Clark近同源品系進行南方雜交(Southern hybridization)結果顯示，在大豆基因組中GST基因至多為二套，且未發現多型性。由北方分析(Northern hybrid-dization)結果發現，此二個GST基因在種皮的全生育時期及葉部均有表現，但在花及豆莢組織中並未偵測到基因的表現或是表現量極低。由於在大豆EST基因庫中仍有其他GST基因相關殖系存在，因此在花瓣組織中是否另有專一性表現之GST基因，扮演花青素轉移之角色仍有待研究。 Primers specific for GST gene (GST2, Y10820) were synthesized according to the results of soybean EST database search. Expected cDNA fragment GST420 was amplified from total RNA extracted from soybean seed coats of CRWD variety with iRT genotype for experiment. Sequence analysis of GST cDNA fragment confirmed that a partial GST gene fragment was cloned. Two complete cDNA clones, GST11-1 (926 bp, AY382831) and GST70-1 (930 bp, AY382832) of type III GST were obtained by screening the cDNA library with GST420 fragment. The deduced amino acid sequences showed 41-100% identity to ten registered GST proteins by Blast analysis of NCBI. Southern blot analysis indicated that two copies of GST were presented in the genome of Clark isogenic lines with no polymorphism. Northern hybridization showed that these two GST genes expressed in the seed coats and leaves, but not expressed in flowers. Further experiment will be conducted to study the function of GST genes in these tissues