利用CRISPR/Cas9技术编辑GmBADH1基因改变大豆耐盐性

Knockout of GmBADH1 gene using CRISPR/Cas9 technique to reduce salt tolerance in soybean

耐盐基因功能鉴定对于大豆品种改良以及盐碱地的开发利用至关重要。盐害胁迫下作物通过合成和积累甜菜碱作为渗透保护剂,减小盐害对作物产量的影响。BADH基因已在多种植物中被证实调节植物对逆境胁迫的响应,但在大豆中的调控机制尚不清晰。本研究克隆了GmBADH1基因,qRT-PCR显示该基因在根、茎、叶中均有表达,尤其在根中的表达丰度最高。通过农杆菌介导的大豆遗传转化技术将CRISPR/Cas9系统构建的表达载体转入到大豆品种JACK中,产生了3种可以调控大豆耐盐性状的靶向突变,均发生在编码区,分别为缺失19 bp、插入1 bp、插入9bp替换2 bp。前两者,过早出现的终止密码子,使其均产生截断的BADH1蛋白,后者发生移码突变。在出苗期和苗期分别对突变纯合植株进行盐处理,结果表明,出苗期耐盐性与野生型相比显著下降,苗期与野生型相比无明显差异。这说明GmBADH1基因可能主要调控出苗期的耐盐性状。本研究为深入挖掘大豆耐盐基因和培育大豆耐盐品种提供了依据。

Functional identification of salt-tolerant genes is crucial for the improvement of soybean varieties and the development and utilization of saline-alkali land.Under salt stress,crops reduce the effect of salt damage on crop yield by synthesizing and accumulating betaine as osmotic protective agent.BADH gene is been proved to regulate plant response to stress in many plants,but the regulation mechanism in soybean is not clear.In this study,GmBADH1 gene was cloned,and qRT-PCR showed that the gene was expressed stems and leaves,especially in roots.The expression vector constructed by CRISPR/Cas9 system was transferred into soybean variety JACK by Agrobacterium-mediated soybean genetic transformation technology,and three targeted mutations that could regulate the salt tolerance of soybean were produced.They all occurred in the coding region,which had three difference of deletion 19 bp,insertion 1 bp,and insertion 9 bp to replace 2 bp.The first two,premature termination codons,resulting in truncated BADH1 protein,the latter occurred frameshift mutation.The mutant homozygous plants were treated with salt at the emergence stage and seedling stage,respectively.The results showed that the salt tolerance of the mutant homozygous plants was significantly lower than that of the wild type at seedling stage,and there was no significant difference compared with the wild type at seeding stage,which indicating that GmBADH1 gene may mainly regulate the salt tolerance of seedling stage.This study provides a basis for further excavation of soybean salt-tolerant genes and cultivation of soybean salt-tolerant varieties.