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葉綠體psaA、psaB和rps14基因共同組成一個操縱子；psaA和psaB基因分別為第一光合系統P700反應中心之apoprotein A1和A2的基因，rps14則為核糖體蛋白S14 的基因。前人的實驗已在此操縱子上游段動子區域發現了site A及site B兩個蛋白質結合位。分析菠菜psaA-psaB-rps14操縱子上游啟動子區域核酸序列，可見其基本架構非常類似於其他高等植物相對應的區域。比較水稻和菠菜siteA蛋白質結合位，可發現有75%的相似性。Primerextension實驗亦指出該操縱子的體內轉錄起始點，在菠菜和水稻中位於相同的位置。本論文依據菠菜相對應水稻siteA位置的核酸序列合成一段35mer的探針，與菠菜葉綠體蛋白質萃取液進行gel retardation assay，發現了耐熱性siteA-specific DNA結合蛋白；且該結合蛋白和site B結合蛋白並不是同一種蛋白質。熱穩定實驗顯示，耐熱性site A結合蛋白在經過70℃熱處理15分鐘後依然能夠與探針結合。利用Superose 12膠體過濾層析法，site A結合蛋白的分子量估計為64kDa；SDS-PAGE和Soluthwestern blot的結果則顯示site A結合蛋白次單元體為34kDa，故推測site A-specific DNA結合蛋白乃是一種由兩個34kDa次單元體所組成分子量64kDa的DNA結合蛋白。本研究以硫酸銨分劃(25-65%飽合度)處理葉綠體 蛋白質高鹽萃取液，利用heparin-agarose親和性管柱層析法，以及熱處理(65℃，15分鐘)做蛋白質的部份純化；再進一步使用site A-specific DNA親和性層析法，可大幅提高茨菜葉綠體site A-specificDNA結合蛋白的純度。
The spinach plastid genes psaA, psaB and rps14 encoding the P700 chlorophyll a apoprotein of the photosystem I reaction center and the ribosomal protein S14 are organized into a transcription unit. Previous studies indicated that there are two protein-binding sites, site A and site B, in the promoter region. Nucleotide sequence analysis revealed that the arrangement of the operon upstream promoter region is conserved among spinach and other higher plants. There is 75% homology in the protein-binding site A sequences between rice and spinach. Primer extension analysis indicated that the in vivo transcription start site is located at the same position in spinach and rice. Competitive gel retardation assay of spinach chloroplast proteins using spinach site A sequence (35mer) as probe indicated that the existence of a heat-stable site A-specific DNA-binding protein which is different from site B-binding protein. Thermostability analysis indicated that the site A binding protein was still active to bind the probe after treatment at 70℃ for 15 minutes. Gel-filtration chromatography with Superose 12 column estimated the molecular weight of the site A-specific binding protein to be approximately 64kDa. Combined data from SDS-PAGE and Southwestern blot analysis suggested that the site A binding protein is composed of two 34 kDa-subunits. After ammonium sulfate fractionation(25-65%) of the spinach chloroplast high salt extracts, followed by heparin-agarose column chromatography and heat-treatment at 65℃ for 15minutes, the partially purified site A binding protein was further subjected to site A-specific DNA affinity column chromatography. The spinach chloroplast site A-specific binding protein has been purified to pretty high homogeneity.
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