应用CRISPR-Cas9基因编辑技术改良传统优质糯稻品种

Improvement of a Traditional High-quality Glutinous Rice Variety by CRISPR-Cas9 Gene Editing System

靖西香糯是广西著名地方水稻品种,米饭水晶状、糯性强、香味浓、食味优,是优质糯稻的极品,主要缺点是高秆易倒伏,产量较低。为改良靖西香糯株高特性以及保持该品种的传统风味,本研究运用CRISPRCas9技术定向编辑调控水稻株高的SD1基因,在外显子区内设计了两个打靶效率高的靶序列,将这2个靶序列组装到PYLCRISPR/Cas9-MH上,通过农杆菌介导法转化靖西香糯,得到了株高显著变矮的半矮秆突变株系。结果表明,T0代共获得24株SD1突变株,转化植株突变率为85.7%,其中2个靶点同时突变频率占总突变的58.3%,双靶点纯合突变高达42.8%,其中3株(12.5%)是T-DNA-free纯合突变植株。利用T0代双靶点纯合突变T-DNA-free植株种子种植的T1代,检测到16个T-DNA-free植株,其中11个为半矮秆植株。结果还表明,在SD1基因第一外显子内单个碱基的替换或双等位的突变并没有对株高产生较大的影响,而第一外显子双位点缺失突变产生大片段缺失可导致内源GA水平显著降低,进而显著降低了株高。本研究为培育矮秆香糯水稻新品系提供了帮助,表明CRISPR-Cas9应用可高效地定向改良水稻品种,研究结果也为应用现代生物育种技术更好地利用传统资源提供了一个范例。

Jingxi is a famous local rice variety in Guangxi.The rice is crystal-like,strong-tasting,rich in aroma,and has excellent taste.It is the best quality japonica rice.The main drawback is that the tall culms are easy to fall down and the yield is low.In order to improve the plant height characteristics and maintain the traditional flavor of this species,we used CRISPR-Cas9 technology to edit and regulate the SD1 gene of rice plant height,and designed two target sequences with high target efficiency in the exome region.These two target sequences were assembled into PYL CRISPR/Cas9-MH,and Jingxi was transformed by Agrobacterium-mediated method to obtain a semi-dwarf mutant strain with a significantly shorter plant height.The results showed that 24 strains of SD1 mutants were obtained in the T0 generation,and the mutation rate of the transformed plants was 85.7%.The frequency of the two target mutations accounted for 58.3%of the total mutations,and the homozygous mutations of the two targets were as high as 42.8%,of which 3 strains(12.5%)is a T-DNA-free homozygous mutant plant.16 T-DNA-free plants were detected in the T1 generation planted with the T0 generation double-target homozygous mutant T-DNA-free seed plants,of which 11 were semi-dwarf mutants.The results also showed that single base substitutions or biallelic mutations in the first exon of the SD1 gene did not have a significant effect on plant height,whereas double-site deletion mutations in the first exon resulted in large fragment deletions.This can lead to a significant reduction in endogenous GA levels,which in turn significantly reduces plant height.This study has provided assistance for cultivating a new line of dwarf taro rice,indicating that CRISPR-Cas9 can be used to efficiently target and improve rice varieties.The research results also provide an example for the application of modern biological breeding technology to better utilize traditional resources.