摘要
为建立糯稻品种培育新体系,本试验研究了利用CRISPR/Cas9基因编辑技术对Waxy(Wx)基因定向突变进而培育糯稻的效果。在Wx基因的第2、第7、第8、第10、第11和12外显子区域设计6个靶位点,构建CRISPR/Cas9基因编辑表达载体,采用农杆菌介导法将基因编辑载体转入粳稻品种哈勃601,获得11~88株转基因再生植株。突变分析表明,转基因株突变率总体偏低(0%~30.77%),仅在第2、第7和第11外显子上发生突变,以1~2 bp的插入缺失为主,都为纯合或双等位突变。5份纯合T2突变体的稻米呈蜡质状,直链淀粉含量为由亲本的15.5%降至2.0%~2.6%,接近或达到糯稻水平,其RVA谱与糯稻对照相仿,淀粉粘滞性较亲本哈勃601显著下降。上述结果表明,通过利用CRISPR/Cas9可对Wx基因进行定向突变从而获得糯性材料,但在利用该技术培育新品种时,需要选择适当的靶位点并培育较多的突变体,才能从中选到符合国家标准的糯稻品种。本研究对利用基因编辑技术培育水稻新品种提供了科学依据。
To establish a new system for the breeding of waxy rice,this research studied the effect of directional mutation of Waxy（Wx） gene by using CRISPR/Cas9 gene editing technology to cultivate waxy rice.In total,six target sites were designed at the 2nd,7th,8th,10th,11th and 12th exon of the Wx gene,and the gene editing expression vector of CRISPR/Cas9 was constructed.Then,it was transformed into rice cultivar Hupper 601 via Agrobacterium-mediated transformation,which produced 11 to 88 transgenic plants.Mutations analysis indicated that the mutation rate of transgenic plants was generally low（0%～30.77%）,and the mutations occurred only on the 2nd,7th and 11th exon.The insertion deletion of 1～2 bp was the dominant,which were mainly homozygous or biallelic mutation.The milled rice of 5 homozygous mutant lines were opaque and the amylose contents accounted for 2.0%～2.6%（decreased from 15.5% of Hupper 601）,which was similar to the level of typical waxy rice.The RVA profiles of the mutant lines were similar to waxy rice,and the viscosity of starch was significantly lower than that of Hupper 601.Put together,our present study demonstrated that waxy rice could be generated through CRISPR/Cas9-based targeted mutagenesis of the Wx gene.However,when it was used for breeding waxy rice,appropriate targets should be selected and sufficient mutant plants should be produced,only then could we select the varieties of glutinous rice that meet the national standards.This study provided scientific evidence for using genome editing technology to cultivate new rice varieties.
作者
周鑫
邓丽
汪庆
刘洋洋
张渭章
舒庆尧
谭瑗瑗
Zhou Xin;Deng Li;Wang Qing;Liu Yangyang;Zhang Weizhang;Shu Qingyao;Tan Yuanyuan(State Key Laboratory of Rice Biology and Zhejiang Provincial Key Laboratory of Crop Germplasm,Institute of Crop Science,Zhejiang University,Hangzhou,310058;Wuxi Hupper BioSeed Industry Technology Institute,Wuxi,214100;Zhejiang Zhijiang Seed Company,Hangzhou,311107;The New Countryside Development Institute at Zhejiang University,Hangzhou,310058)
出处
《分子植物育种》
CAS
CSCD
北大核心
2018年第17期5608-5615,共8页
Molecular Plant Breeding
基金
杭州市重大科技创新项目(2015012A09)
浙江省农业(粮食)新品种选育重大科技专项(2016C02050-2)共同资助
