【摘 要】
：
树莓(Rubus idaeus)作为第三代新兴水果,是美国最常食用的功能性果品,果实中富含大量有益健康的次生代谢产物.典型的覆盆子酮不仅具有优雅果香作为天然高档香料,其衍生物更具有多样的生物活性,对于减肥,糖尿病,流感等都有显著功效.植物类型III聚酮化合物合酶(PKS)基因超家族即查耳酮合酶(CHS)基因超家族在该类化合物生物合成起始反应中行使着关键作用.在漫长地进化过程中,CHS基因分化形成具
【机 构】
：
北京农学院植物科学技术学院 北京 102206;北京农学院农业部都市农业(北方)重点实验室 北京 102206
【出 处】
：
中国生物工程学会第六次全国会员代表大会暨第九届学术年会
论文部分内容阅读
树莓(Rubus idaeus)作为第三代新兴水果,是美国最常食用的功能性果品,果实中富含大量有益健康的次生代谢产物.典型的覆盆子酮不仅具有优雅果香作为天然高档香料,其衍生物更具有多样的生物活性,对于减肥,糖尿病,流感等都有显著功效.植物类型III聚酮化合物合酶(PKS)基因超家族即查耳酮合酶(CHS)基因超家族在该类化合物生物合成起始反应中行使着关键作用.在漫长地进化过程中,CHS基因分化形成具有新的底物选择性和产物特异性的“类CHS(CHS-Like)”基因.苯亚甲基丙酮合酶(BAS)是催化覆盆子酮生物合成的关键酶和限速酶.前期工作中,我们已经成功地从树莓中克隆到RinPKS1,系统发育分析可知仍属CHS超家族,酶功能测定结果表明RinPKS1具有CHS与BAS双重活性.为了从进一步从树莓中分离新的植物类型III PKS家族基因.本研究利用CODEHOP(Consensus-DegenerateHybrid Oligonucleotide Primers)block引物设计技术,以TAIL-PCR 和悬挂PCR(Overlap PCR)为技术手段,设计多对引物从树莓中克隆分离RinPKS2基因,GenBank登录号为KR697773.序列分析得知RinPKS2基因整个开放阅读框全长1 164bp,由两个外显子组成,其中外显子1长165 bp,外显子2长999 bp,共编码388个氨基酸,预测分子量(Mr)为42 680 Da.系统发育分析可知RinPKS2基因为CHS-Like基因,据我们所知,该基因是迄今为止树莓当中分离得到的第一个CHS-Like基因.以基因组DNA为模板,利用悬挂PCR技术成功将两个外显子连接起来,并分别在外显子1上游引物和外显子2的下游引物中分别引入EcoR I 和Xho I位点,利用酶切及连接反应将整个RinPKS2开放阅读框连接至原核表达载体pET30a质粒上,构建pET30a-RinPKS2原核表达重组质粒.将重组质粒转入大肠杆菌表达菌株Rosetta中,通过对蛋白诱导表达条件的筛选,得知OD600值为0.4时加入1.0 mmol·L-1 IPTG,在25℃下诱导表达4 h即可获得高表达量的可溶性蛋白,SDS-PAGE电泳分析得融合蛋白分子量约为43 kDa,与预期分子量相同.经Ni2+亲和柱纯化、PD-10柱脱盐后得到高纯度可溶性蛋白.为后期研究其酶学性质和动力学特性奠定了基础.
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