ZHANG Ting,MA Li-xin,LIU Jun,et al.Soybean phyA Downstream Gene Prediction and Expression Analysis and Its Knockout Vector Construction[J].Soybean Science,2021,40(03):309-318.[doi:10.11861/j.issn.1000-9841.2021.03.0309]
大豆phyA下游基因预测和表达分析及敲除载体构建
- Title:
- Soybean phyA Downstream Gene Prediction and Expression Analysis and Its Knockout Vector Construction
- Keywords:
- Soybean; PIF3; LAF1; FHY1; FHL; Tissue expression analysis; CRISPR/Cas 9; Gene knockout vector
- 文献标志码:
- A
- 摘要:
- 为了进一步研究大豆光周期调控通路中E3和E4的下游基因，为获得其突变体植株奠定基础，以拟南芥远红光响应受体phyA下游的重要信号传递因子PIF3、LAF1、FHY1和FHL的序列作为参考序列，在Phytozome 12数据库中查找其相似序列，进行序列比对和进化树分析，确定它们在大豆中的同源基因并采用qRT-PCR方法进行组织表达分析，使用CRISPR/Cas 9系统设计同源基因的敲除靶点并通过发根系统验证靶点的有效性。结果显示：AtPIF3在大豆中有6个同源基因，AtLAF1在大豆中有4个同源基因，AtFHL在大豆中有2个同源基因，而AtFHY1在大豆中没有同源基因。4个LAF1基因主要在顶端生长点表达，6个PIF3和2个FHL基因主要在荚中表达。共鉴定出12个有效靶点，能够分别将大豆中6个PIF3、4个LAF1和2个FHL基因成功敲除。研究结果为进一步获得稳定的大豆突变体材料和大豆phyA下游基因的功能研究提供了理论基础。
- Abstract:
- To further study the downstream genes of E3 and E4 in the photoperiod regulatory pathway of soybean, and lay a foundation for obtaining its mutant soybean，we focusd on PIF3, LAF1, and FHY1/FHL, which are important signaling factors downstream of Arabidopsis far-red light receptor phyA. We used genome sequences of Arabidopsis PIF3, LAF1 and FHY1/FHL as references for searching soybean homologs in Phytozome 12 database, made sequence alignment and phylogenetic tree to analyze the homologs in soybean, and performed real-time quantitative PCR to detect the expression patterns of these soybean homologs in various tissues. We applied CRISPR/Cas 9 system to knockout these homologous genes, and verified the effectiveness of each target through the soybean hair root system. The results showed that: AtPIF3, AtLAF1, and AtFHL has six, four, and two homologs in soybean, respectively. And AtFHY1 doesn′t have homolog in soybean. Four LAF1 homologs were mainly expressed in shoot apical meristem, six PIF3 homologs and two FHL homologs were mainly expressed in pods. According to the CAISPR/Cas9 knockout results, a total of twelve effective targets were identified, which can successfully knock out six PIF3, four LAF1, and two FHL homologs in soybean. This research provides a theoretical basis for obtaining stable soybean knockout mutant, which pave a way for studying the functions of soybean phyA downstream genes.Keywords:
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备注/Memo
收稿日期：2020-12-22