摘要
CRISPR/Cas9基因编辑系统已成为许多作物基因功能研究和品种改良的重要工具。脯氨酸(Pro)在植物响应干旱胁迫方面起着重要作用,脯氨酸脱氢酶ProDH是脯氨酸分解途径中的限速酶。为了研究StProDH1在马铃薯脯氨酸代谢中的作用,本研究以StProDH1为靶基因,构建了表达马铃薯脯氨酸脱氢酶g RNA的CRISPR/Cas9基因敲除系统,在StProDH1基因5'端第一个外显子上为靶标区域选择设计sgRNA,以p P1C.4为模板,克隆得到sgRNA克隆框,将得到的sgRNA克隆框通过同源重组技术连接到pP1C.4载体中,获得pP1C.4-Cas9-ProDH1-g RNA载体。通过设计引物特异性扩增以及测序进一步确定sgRNA准确地连入载体,这对马铃薯中脯氨酸累积及其培育抗旱马铃薯新品种具有重要意义。
CRISPR/Cas9 gene editing system has become an important tool for gene function study ahd varieties improvement in many crops.Proline plays an important role in plant response to drought stress,While Proline de- hydrogenase (ProDH)is a rate-limit enzyme in proline decomposition pathway.In order to study the effects of StProDH1 on proline metabolism in potato,StProDH1 as a target genewas used to generate a CRISPR/Cas9 knockout system expressing the ProDHI-gRNA of potato.On the first exon of 5'end of the StProDHI gene,the sgRNA was designed for the target region and pPIC.4 as the template was used to clone sgRNA cloning box;the obtained sgRNA cloning box was connected to pPIC.4 by homologous recombination technology,and the pP1C. 4-Cas9-ProDHI-gRNA vector was obtained.To further validated the sgRNA whether accurately connected to the vector by designing specific primers amplification and sequencing.It would of great significance for proline accumulation and cultivation of new drought-resistant varieties in potato.
作者
李世贵
杨江伟
朱熙
王树林
张宁
司怀军
Li Shigui;Yang Jiangwei;Zhu Xi;Wang Shuling;Zhang Ning;Si Huaijun(College of Life Science and Technology,Gansu Agricultural University,Lanzhou,730070;Gansu Provincial Key Laboratory of Aridland Crop Sci- ence,Gansu Key Laboratory of Crop Improvement &Germplasm Enhancement,Gansu Agricultural University,Lanzhou,730070)
出处
《分子植物育种》
CAS
CSCD
北大核心
2019年第3期841-845,共5页
Molecular Plant Breeding
基金
国家自然科学基金(31660416
31460370)
甘肃农业大学"伏羲人才"计划项目(FXRC20130102)共同资助
