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马铃薯块茎颗粒结合型淀粉合酶基因的克隆及其RNAi载体的构建
2016-10-19 11:22   审核人:

马铃薯块茎颗粒结合型淀粉合酶基因的克隆及其RNAi载体的构建

Cloning of Granule-Bound Starch Synthase Gene and Construction of Its RNAi Vector in Potato Tuber

 

刘玉汇1,2,王丽3,杨宏羽1,余斌1,2,李元铭4,张俊莲1,2,*,王蒂1,2,*

1 甘肃省作物遗传改良与种质创新重点实验室 / 甘肃农业大学农学院, 甘肃兰州7300702 甘肃省干旱生境作物学重点实验室, 甘肃兰州7300703 甘肃农业大学生命科学技术学院, 甘肃兰州7300704 甘肃农村发展研究院, 甘肃兰州730070

LIU Yu-Hui1,2,WANG Li3,YANG Hong-Yu1,YU Bin1.2,LI Yuan-Ming4,ZHANG Jun-Lian1,2,*,WANG Di1,2,*

1 Gansu Key Laboratory of Crop Genetic & Germplasm Enhancement /College of Agronomy, Gansu Agricultural University, Lanzhou 730070, China; 2 Gansu Key Laboratory of Aridland Crop Science, Lanzhou 730070, China; 3 College of Life Sciences and Technology, Gansu Agricultural University, Lanzhou 730070, China; 4 Gansu Rural Development Research Institute, Lanzhou 730070, China

 

 

 

摘要: GBSSI是马铃薯块茎中控制直链淀粉合成的关键酶, 为培育高支链淀粉含量或纯支链淀粉含量的转基因马铃薯材料, 根据GenBank登录号X58453设计特异引物, 采用RT-PCR技术获得马铃薯块茎GBSSI相似基因, 利用生物信息学相关软件分析, 预测GBSSI相似基因cDNA序列编码的蛋白质结构和功能。结果表明, 克隆的GBSSI相似基因与报道的GBSSI基因序列相似性达到99.78%, 其开放阅读框长1 824 bp, 编码607个氨基酸, 具有许多重要功能位点;三级结构预测结果表明该蛋白具有淀粉合成功能, 基因序列已注册到GenBank, 序列登录号为EU403426。以此基因CDS542 bp的靶标序列作为干扰区段, 扩增GBSSI的正反向基因片段, 并引入237 bp的内含子序列, 构建由Patatin启动子驱动的具有正义基因片段gbss A-内含子VP1-ABI3-like protein-反义基因片段gbss B的植物干扰表达载体pBI121g-PgABI, 将为淀粉合成的进一步研究和高支链淀粉含量或纯支链淀粉含量的马铃薯品种的培育奠定基础。

关键词马铃薯 颗粒结合型淀粉合酶 基因克隆 序列分析 RNAi载体

 

AbstractThere is about 17% starch in potato (Solanum tuberosum L.) tubers. Potato starch granules are composed of two polysaccharides, unbranched amylose (approximately 20% to 25%) and branched amylopectin (approximately 75% to 80%). To develop transgenic potato with high-amylopectin in tubers, we got a cDNA sequence of the potato GBSSI from the total RNA of potato tubers by RT-PCR using specific primers of conserved domain of GenBank Accession Number X58453 sequence.The GBSSI cDNA sequence shared 99.78% similarity with the GBSSI gene in GenBank (accession No. X58453). The full-length of cDNA was 1 824 bp, which contained 607 amino acids, three conserved domains and many important functional sites. The 3D structure of GBSSI was highly similar to that of the glycogen synthase, indicating that GBSSI has a function of starch synthesis. GBSSI similar gene obtained here was granule-bound starch synthase, and its sequence was submitted to GenBank, with the accession number of EU403426. On the basis of a 542 bp RNAi target region from the CDS of GBSSI, the sense and antisense fragments were amplified and separated by a 237 bp intron to construct the RNA interference expression vector pBI121g-PgABI containing “sense gbssA-VP1-ABI3-like protein intron-antisense gbss B” regulated by Patatin promoter, which will lay a solid foundation for the study on synthesis of starch and breeding of transgenic potato with high amylopectin content or pure amylopectin.

 

Key wordsPotato GBSSI gene Gene cloning Sequence analysis RNA interference vector

 

基金资助:本研究由国家科技支撑计划(2012BAD06B03), 国家现代农业产业技术体系建设项目(CARS-10-P18)和甘肃省重大专项(1102NKDM025)资助。

 

通讯作者: 张俊莲, E-mail: zhangjunlian99@yahoo.com.cn; 王蒂, E-mail: wangd@gsau.edu.cn   

 

 

引用本文: 刘玉汇,王丽,杨宏羽等. 马铃薯块茎颗粒结合型淀粉合酶基因的克隆及其RNAi载体的构建[J]. 作物学报, 2012, 38(07): 1187-1195.

LIU Yu-Hui,WANG Li,YANG Hong-Yu et al. Cloning of Granule-Bound Starch Synthase Gene and Construction of Its RNAi Vector in Potato Tuber[J]. Acta Agron Sin, 2012, 38(07): 1187-1195.

链接本文: http://211.155.251.148:8080/zwxb/CN/10.3724/SP.J.1006.2012.01187    http://211.155.251.148:8080/zwxb/CN/Y2012/V38/I07/1187
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