为了研究中间锦鸡儿Caragana intermedia纤维素合酶基因(cellulose synthase,CesA)的结构及表达特性,通过同源克隆及RACE(rapid amplification of cDNA end)技术,获得1条中间锦鸡儿CesA基因的全长cNDA序列.NCBI Blast结果显示该基因与野生大豆Glycine soja基因GsCesA3相似度最高(94%),因此命名为CiCesA3.CiCesA3基因cDNA全长为3 475 bp,其中5'UTR长为175 bp,3'UTR长为76 bp,开放阅读框长度为3 225 bp,编码1 075个氨基酸.CiCesA3蛋白预测分子质量为119.89 ku,第16位到第79位氨基酸为CesA蛋白保守的锌指结构(Cx2Cx12FxACx2CxEx5Gx3Cx2C).CiCesA3蛋白C端有6个跨膜结构域,为亲水性蛋白.qRT-PCR检测不同组织中基因表达量的结果表明CiCesA3在叶中表达量最高.以上结果证明中间锦鸡儿CiCesA3基因与其他植物CesA基因相似,并无变异,为阐明中间锦鸡儿纤维素的合成提供了依据.
This research aims to figure out the structure and expression pattern of cellulose synthase gene(CesA)in Caragana intermedia. The full-length cDNA of CesA was obtained by homologous cloning and RACE from C. intermedia. NCBI Blast indicated the CesA shared the highest identity with GsCesA3 from Glycine soja(94%), and was named as CiCesA3. The length of CiCesA3 cDNA was 3 475 bp, the open reading frame was 3 225 bp, encoded a polypeptide of 1 075 amino acids. The length of 5 UTR and 3 UTR was 175 and 76 bp, respectively. The predicated molecular weight of CiCesA3 was 119.89 ku,and 16—79 amino acids contained a zinc-finger motif(Cx2Cx12FxACx2CxEx5Gx3Cx2C)which is a conserved- DOI:10.3969/j.issn.1000-1565.2020.04.010中间锦鸡儿CiCesA3的克隆及表达分析朱宏1,柴文娟2,杨飞芸2, 3,王瑞刚 2, 3(1. 哈尔滨师范大学 生命科学与技术学院,黑龙江 哈尔滨 150080;2. 内蒙古农业大学 植物逆境生理与分子生物自治区重点实验室,内蒙古 呼和浩特 010018;3. 内蒙古自治区植物基因资源挖掘与分子育种工程技术研究中心,内蒙古 呼和浩特 010018)摘 要:为了研究中间锦鸡儿Caragana intermedia纤维素合酶基因(cellulose synthase,CesA)的结构及表达特性,通过同源克隆及RACE(rapid amplification of cDNA end)技术,获得1条中间锦鸡儿CesA基因的全长cNDA序列.NCBI Blast结果显示该基因与野生大豆Glycine soja基因GsCesA3相似度最高(94%),因此命名为CiCesA3.CiCesA3基因cDNA全长为3 475 bp,其中5'UTR长为175 bp,3'UTR长为76 bp,开放阅读框长度为3 225 bp,编码1 075个氨基酸.CiCesA3蛋白预测分子质量为119.89 ku,第16位到第79位氨基酸为CesA蛋白保守的锌指结构(Cx2Cx12FxACx2CxEx5Gx3Cx2C).CiCesA3蛋白C端有6个跨膜结构域,为亲水性蛋白.qRT-PCR检测不同组织中基因表达量的结果表明CiCesA3在叶中表达量最高.以上结果证明中间锦鸡儿CiCesA3基因与其他植物CesA基因相似,并无变异,为阐明中间锦鸡儿纤维素的合成提供了依据.关键词:中间锦鸡儿;纤维素合酶;CiCesA3;基因克隆中图分类号:Q785 文献标志码:A 文章编号:1000-1565(2020)04-0399-10Cloning and expression analysis of CiCesA3 from Caragana intermediaZHU Hong1, CHAI Wenjuan2, YANG Feiyun2, 3, WANG Ruigang2, 3(1. College of Life Sciences and Technology, Harbin Normal University, Harbin 150080,China; 2. Key Laboratory of Plant Stress Physiology and Molecular Biology, Inner Mongolia Agricultural University, Hohhot 010018, China; 3. Inner Mongolia Engineering Research Center for Plant Gene Resources Mining and Molecular Breeding, Hohhot 010018, China)Abstract: This research aims to figure out the structure and expression pattern of cellulose synthase gene(CesA)in Caragana intermedia. The full-length cDNA of CesA was obtained by homologous cloning and RACE from C. intermedia. NCBI Blast indicated the CesA shared the highest identity with GsCesA3 from Glycine soja(94%), and was named as CiCesA3. The length of CiCesA3 cDNA was 3 475 bp, the open reading frame was 3 225 bp, encoded a polypeptide of 1 075 amino acids. The length of 5 UTR and 3 UTR was 175 and 76 bp, respectively. The predicated molecular weight of CiCesA3 was 119.89 ku,and 16—79 amino acids contained a zinc-finger motif(Cx2Cx12FxACx2CxEx5Gx3Cx2C)which is a conserved- 收稿日期:2019-11-16 基金项目:内蒙古自治区科技计划项目(2019GG007);内蒙古自然科学基金重大项目(2019ZD05);内蒙古自治区科技创新引导项目(KCBJ2018012) 第一作者:朱宏(1968—),女,黑龙江齐齐哈尔人,哈尔滨师范大学教授,博士,主要从事植物细胞生物学研究.E-mail: zhuhong10000@163.com 通信作者:王瑞刚(1972—),男,内蒙古赤峰人,内蒙古农业大学教授,博士生导师,主要从事植物生物化学与分子生物学研究.E-mail: ruigangwang@126.com第4期朱宏等:中间锦鸡儿CiCesA3的克隆及表达分析motif of CesA. CiCesA3 was a hydrophilic protein, and six transmembrane domains existed in the C-terminal. Real-time quantitative RT-PCR of CiCesA3 in C. intermedia different tissues showed the highest expression level of CiCesA3 was from leaf. The results demonstrated that CiCesA3 was similar to CesA of other plants, and provide some foundation to clarify the cellulose synthesis of C. intermedia.
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