两种拟南芥CBF2基因突变体的鉴定与分析

Analysis and Identification of Two CBF2 Gene Mutants in Arabidopsis

低温是主要的逆境胁迫之一,限制了作物的地理分布和产量。筛选理想的抗冻靶基因用于分子育种对于稳定农业生产具有重要意义。CBF调节子在植物抗冻反应中扮演主要的角色,CBF2是CBF调节子的组成成分之一,在抗冻反应中扮演负调控的作用。采用qRT-PCR和常规农艺性状测定法对2种CBF2基因突变体cbf2-1和cbf2-2进行了多项指标的鉴定,结果发现在这2种突变体中CBF2基因的表达存在不同程度的缺陷,cbf2-1中CBF2基因受低温诱导表达,但表达量与对照相比,明显下降;而cbf2-2中CBF2基因诱导表达的效应完全丧失。抗冻性检测结果显示CBF2基因的表达量越低,其突变体植株的抗冻性越强,同时冷胁迫后冷响应的下游基因表达量也越高,更重要的是,虽然这2种CBF2基因突变体抗冻性明显增强,但与野生型植株相比,其开花时间和单株种子产量并没有明显变化。以上结果表明CBF2是一种可以利用基因组编辑技术进行作物抗冻育种的理想候选靶基因,可以利用最新的基因组编辑技术进行作物抗冻育种研究。

Cold stress is one of the major abiotic stress,which limits the geographical distribution and yield of crops. CBF regulon plays a major role in freezing tolerance. CBF2,one of the components of the CBF regulon,has a negative role in freezing tolerance. In this study,two SALK mutants whose T-DNA insertion are both within the promoter of CBF2 gene,cbf2-1 and cbf2-2 were identified by qRT-PCR method. Results showed that the level of CBF2 gene expression in the two mutants reduced. CBF2 gene expression was induced by low temperature in cbf2-1 mutnat. However,the degree of induction was lower in cbf2-1compared with the control. The effect of induction by low temperature was lost in cbf2-2. Analysis of freezing tolerance showed that the less degree of induction,the stronger freezing tolerance of plants and the higher the expression of downstream cold-responsive genes. More important thing was that although freezing resistance of the two CBF2 gene mutants were improved,their flowering time and seed yield per plant did not change significantly compared with the wild type plants. All the results indicate that CBF2 may be an ideal candidate target gene for crop breeding of freezing tolerance by genome editing technology.