应用CRISPR/Cas9系统编辑水稻SBE3基因获得高抗性淀粉水稻新品系

Obtain of New Rice Variety with High Resistant Starch Based on the Edition of Rice SBE3 Gene by CRISPR/Cas9 System

SBE3是位于水稻第2号染色体上的水稻淀粉分支酶基因,该基因表达量的降低引起水稻抗性淀粉(resistant starch,RS)含量的提高。本研究应用CRISPR/Cas9系统对SBE3基因进行编辑,根据基因编码区序列(CDS)在第8外显子区域设计长度为20 bp的引导序列,化学合成引导序列并构建成sgRNA,将其插入到Cas9质粒骨架载体中,转化农杆菌。通过农杆菌介导的遗传转化方法将构建好的p CAMBIA1300:SBE3sgRNA:Cas9表达载体转入水稻受体材料沪LPR18中。使用PCR扩增转基因阳性植株中含有CRISPR/Cas9靶点的片段并测序验证,确定12个突变单株,其中2个是纯合型,通过后代分离,得到没有潮霉素基因筛选标记的SBE3纯合突变单株,测定其抗性淀粉含量高达10%以上。以上结果表明,本研究已经通过CRIPR/Cas9技术获得稳定的高抗性淀粉水稻纯合突变体材料,为高抗性淀粉育种提供了新的种质资源和创建方法。

SBE3 is one of starch branching enzymes in rice located on rice chromosome 2. Reduced expression of SBE3 could increase the resistant starch （resistant starch, RS） content of rice. In this study, SBE3 gene was edited by CRISPR/Cas9 technology. 20 nt guide RNA （gRNA） target on the eighth exon of SBE3 were designed and inserted into the CRISPR/Cas9 plasmid backbone vector. The vector was transferred to Agrobacterium tumefaciens, and positive clones were verified by sequencing. The constructed pCAMBIA1300：SBE3gRNA：Cas9 expression vector was transferred into Hu LPR18 which was registered by Agrobacterium mediated transformation method. We amplified fragments containing CRISPR/Cas9 target of positive transformants using the PCR method and sequenced the PCR frangments. At last, we identified 12 To mutant plants, of which 2 were homozygous mutants. Homozygous mutant plants without hygromycin （HPT） gene were identified by segregation analysis. The RS content of the homozygous mutant plants was as high as 10%. The above results showed that we had obtained homozygous mutant materials of high resistant starch rice using CRIPR/Cas9 technique, which might provide new germplasm resources and method for the breeding of high resistant starch flee.