关键词: 大豆, WUS基因, CRISPR/Cas9, 根瘤发育

Abstract:

The nodulation and nitrogen fixation of legumes have a great significance in agriculture, such as production, nutrient efficiency, and soil improvement. WUS gene plays an important role in meristem function in plants. The bioinformatics analysis shows that the protein encoded by the soybean WUS gene （GmWUS） and the ArabidopsisWUS gene （AtWUS） share a high conserve amino acid sequence, even though there is a big difference at C-terminal. The promoter sequence GmWUS2pro, 3000 bp upstream of the transcription start site, was amplified from the genome of soybean Tianlong 1, and fused with the reporter gene GUS to obtain the GmWUS2pro:GUS expression vector. qRT-PCR analysis revealed that GmWUS2 gene was highly expressed in soybean flowers and nodules. Through hairy root transformation mediated by Agrobacterium rhizogenes and GUS staining, it was found that the GmWUS2 promoter was active in both the roots and the nodules in soybean. The knockout vector of GmWUS2 was constructed using CRISPR/Cas9 technology, while overexpressing vector was constructed at the same time, and both vectors were transformed into soybean hairy roots, which was further inoculated with rhizobia HH103. The phenotypic differences between Gmwus2 mutant hairy roots and GmWUS2 overexpression hairy roots were statistically analyzed compared to an empty vector. It was found that the knockout of GmWUS2 significantly promoted the nodule and leaf development, suggesting the GmWUS2 gene affected growth of both the above-ground and underground parts of plants. Our results indicate that the WUS gene plays an important role in soybean nodule development, providing useful clues for both studying soybean nodulation and improving biological nitrogen fixation in agricultural production.

Key words: soybean, WUS gene, CRISPR/Cas9, nodule development