脱落酸受体及其基因的分子免疫学研究(英文)

An Immunological Approach to ABA Receptor and Its Gene

发展了研究脱落酸（ＡＢＡ）结合蛋白的两类免疫探针。其一，用ＡＢＡ－Ｃ１－ＢＳＡ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析柱纯化出ＡＢＡ结合蛋白，聚丙烯酰胺凝胶电泳显示该蛋白分子量为５６ＫＤ的一条带，它具有特异结合ＡＢＡ的能力（Ｋｄ＝２．０×１０－９ｍｏｌ／Ｌ）。当用蛋白水解酶Ｋ水解该蛋白，并用１％琼脂糖凝胶电泳时，发现其中存在约３００个核苷酸的ｒＲＮＡ分子。用识别ＡＢＡ结合蛋白的抗体筛选ｃＤＮＡ表达文库，从２００，０００个独立噬斑中获得１２０个编码玉米１７ｓＲＮＡ的ｃＤＮＡ克隆和１个ｃＤＮＡ编码结合蛋白的ｃＤＮＡ克隆（２４ｃＤＮＡ）。２４ｃＤＮＡ有１０７５个碱基对，含编码２５４个氨基酸的开放阅读框架。其二，制备了识别抗ＡＢＡ单克隆抗体的独特型抗体（ａｎｔｉ－Ｉｄ），它具有模拟并竞争ＡＢＡ的能力。用上述两类免疫探针定位了植物细胞中的ＡＢＡ结合蛋白。发展了研究脱落酸（ＡＢＡ）结合蛋白的两类免疫探针。其一，用ＡＢＡ－Ｃ１－ＢＳＡ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析柱纯化出ＡＢＡ结合蛋白，聚丙烯酰胺凝胶电泳显示该蛋白分子量为５６ＫＤ的一条带，它具有特异结合ＡＢＡ的能力（Ｋｄ＝２．０×１０－９ｍｏｌ／Ｌ）。当用蛋白水解酶Ｋ水解该蛋?

Abstract Two types of immunological probes, anti-ABBP Abs, have been developed. The purified ABBP from ABA-C 1-BSA-sepharose 4B column was identified by PAGE and appeared in one band of about 56KD, as well as showed a specific binding ability and a high affinity for ABA (Kd2.0×10 -9 mol/L). Une〖HJ*3/7]xpectedly, the existence of rRNA with a length of around 300 nucleotides could be found, when the ABBP was digested with proteinase K and identified by electrophoresis on an agarose gel(1%). As a result, about 120 cDNA clones coding maize 17s RNA and only one cDNA clone coding ABBP (24cDNA) were obtained from 200,000 separated phage plaques by the anti-ABBP pAbs. 24cDNA had 1075bp and contained an open reading frame coding 254 amino acids. The anti-idiotypic Ab raised against an ABA MAb showed the ability of either mimicking ABA or competing with ABA. The localization of ABBPs in plant cell was investigated.