白菜型冬油菜RuBisCo蛋白亚基基因rbcL和rbcS的克隆及其在干旱胁迫下的表达  

Cloning of RuBisCo Subunits Genes rbcL and rbcS from Winter Rapeseed(Brassica rapa) and Their Expression under Drought Stress

作　　者：米超 赵艳宁 刘自刚[2,1] 陈其鲜 孙万仓[2,1] 方彦[2] 李学才[2,1] 武军艳[2,1] MI Chao;ZHAO Yan-Ning;LIU Zi-Gang;CHEN Qi-Xian;SUN Wan-Cang;FANG Yan;LI Xue-Cai;WU Jun-Yan(Gansu Research Center of Rapeseed Engineering and Technology/Improvement and Key Laboratory of Crop Genetics and Germplasm Enhancement of Gansu Province/Gansu Provincial Key Laboratory of Arid Land Crop Sciences,Lanzhou 730070,Gansu,China;College of Agronomy,Gansu Agricultural University,Lanzhou 730070,Gansu,China;Gansu General Station of Agro-technology Extension,Lanzhou 730070,Gansu,China)

机构地区：[1]甘肃农业大学农学院,甘肃兰州730070 [2]甘肃省油菜工程技术研究中心/甘肃省干旱生境作物学重点实验室/甘肃省作物遗传改良与种质创新重点实验室,甘肃兰州730070 [3]甘肃省农业技术推广总站,甘肃兰州730040

出　　处：《作物学报》2018年第12期1882-1890,共9页Acta Agronomica Sinica

基　　金：国家自然科学基金项目(31660404);国家重点基础研究发展计划项目(2018YFD0100502);甘肃省高校科研成果转化培育项目(2018D-13);甘肃省现代农业产业技术体系建设专项资金;国家现代农业产业技术体系建设专项(CARS-13);甘肃省科技重大专项项目(17ZD2NA016-4)资助~~

摘　　要：应用双向电泳结合液相色谱-质谱联用(LC-MS)分析,筛选得到干旱胁迫下白菜型冬油菜与光合作用相关差异蛋白质RuBisCo小亚基。根据已发表白菜型油菜(Brassicarapa)RuBisCo亚基rbcL和rbcS保守序列设计引物,采用RT-PCR扩增陇油7号的cDNA,获得rbcL和rbcS基因开放阅读框,长度分别为1095 bp和549 bp,编码364和181个氨基酸的蛋白质。生物信息学分析结果显示, rbcL与大白菜(Brassica rapa subsp. chinensis)的蛋白质同源性达到99%以上,其保守序列属于RuBisCoLarge超家族,该蛋白质理论相对分子量及等电点分别为40.29kDa和6.70,不稳定指数(instabilityindex,II>40为不稳定蛋白质)为41.67,属于不稳定蛋白;脂融指数(aliphaticindex)为83.63,平均亲水性值(grand average of hydropathicity)为-0.232,属于亲水性蛋白质;二级结构包括38.74%α-螺旋(alpha helix)、10.99%延伸链(extendedstrand)及50.27%自由卷曲(randomcoil);三级结构具有5个不同的活性口袋。rbcS与甘蓝(Brassicaoleracea)蛋白质同源性达到99%,其保守序列具有rbcS超家族和RuBisCo Small Like超家族,理论相对分子量为20.32 kDa,理论等电点为8.23,不稳定指数为33.66,属于稳定蛋白质;脂融指数为74.86,平均亲水性值为-0.142,属于亲水性蛋白质;二级结构包括16.02%α-螺旋、28.73%的延伸链及55.25%自由卷曲;三级结构具有4个不同的活性口袋。实时荧光定量和半定量分析显示,干旱胁迫下,白菜型冬油菜叶片rbcL和rbcS基因表达量下调是光合作用下降的原因。并且,冬油菜叶片Pn下降与RuBPCase表达抑制和活性降低有关,非气孔限制是Pn下降的主要因素。In this study,we used the two-dimensional gel electrophoresis(2D-DIGE)technology combined with the liquid chromatography-mass spectrometry(LC-MS)technology to filtrate the differential protein small subunit of RuBisCo which was related to photosynthesis in winter rapeseed(Brassica rapa)under drought stress.According to the published Brassica rapa RuBisCo subunit conserved sequences of rbcL and rbcS,we designed the primers and used the reverse transcription-polymerase chain reaction(RT-PCR)technology to amplify the cDNA sequence in Longyou 7.We obtained the open reading frame(ORF)of the RuBisCo subunit rbcL and rbcS,which had the length 1095 bp and 549 bp,and encoded the proteins contains 364 and 181 amino acids,respectively.The results of bioinformatics analysis showed that compare with Brassica rapa subsp.chinensis and Brassica rapa,the protein homology of rbcL and in Longyou 7 was above 99%,with the conserved domain sequence belonging to RuBisCo large superfamily.The theoretical relative molecular mass and isoelectric point of rbcL were 40.29 kDa and 6.70,respectively.And the instability index was-41.67(II>40 was considered as unstable protein),showing that it was an unstable protein.The aliphatic index was 83.63 and the grand average of hydropathicity was 0.232,which indicated that it was a hydrophilic protein.The secondary structure included 38.74%alpha helix,10.99%extended strand and 50.27%random coil.And the tertiary structure contained five differential activity pockets.The protein homology of rbcS and Brassica oleracea in Longyou 7 was 99%,that conserved domain sequence was contained the rbcS superfamily and the RuBisCo Small like superfamily,which theoretical relative molecular mass and isoelectric point were 20.32 kDa and 8.23,respectively.The instability index was 33.66,showing that was a stable protein,and the aliphatic index was 74.86,the grand average of hydropathicity was-0.142,showing a hydrophilic protein.The secondary structure included 16.02%alpha helix,28.37%extended strand and 55.25%random co

关 键 词：白菜型冬油菜 RBCL RBCS 干旱胁迫 

分 类 号：S565.4[农业科学—作物学]