Journal article
Efficient Inactivation of Symbiotic Nitrogen Fixation Related Genes in Lotus japonicus Using CRISPR-Cas9
Frontiers in plant science, Vol.7(2016), pp.1333-1333
08/31/2016
Handle:
https://hdl.handle.net/2376/100620
PMID: 27630657
Abstract
The targeted genome editing technique, CRISPR/Cas9 system, has been widely used to modify genes of interest in a predictable and precise manner. In this study, we describe the CRISPR/Cas9-mediated efficient editing of representative SNF (symbiotic nitrogen fixation) related genes in the model legume
Lotus japonicus
via
Agrobacterium
-mediated stable or hairy root transformation. We first predicted nine endogenous
U6
genes in Lotus and then demonstrated the efficacy of the
LjU6-1
gene promoter in driving expression of single guide RNAs (sgRNAs) by using a split yellow fluorescence protein (YFP) reporter system to restore the fluorescence in Arabidopsis protoplasts. Next, we chose a customized sgRNA targeting
SYMRK
(symbiosis receptor-like kinase) loci and achieved ~35% mutagenic efficiency in 20 T0 transgenic plants, two of them containing biallelic homozygous mutations with a 2-bp deletion near the PAM region. We further designed two sgRNAs targeting three homologous leghemoglobin loci (
LjLb1, LjLb2, LjLb3
) for testing the possibility of generating multi-gene knockouts. 20 out of 70 hairy root transgenic plants exhibited white nodules, with at least two
LjLbs
disrupted in each plant. Compared with the constitutively active CaMV 35S promoter, the nodule-specific
LjLb2
promoter was also effective in gene editing in nodules by hairy root transformation. Triple mutant knockout of
LjLbs
was also obtained by stable transformation using two sgRNAs. Collectively, these studies demonstrate that the CRISPR/Cas9 system should greatly facilitate functional analyses of SNF related genes in
Lotus japonicus
.
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Details
- Title
- Efficient Inactivation of Symbiotic Nitrogen Fixation Related Genes in Lotus japonicus Using CRISPR-Cas9
- Creators
- Longxiang Wang - State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural UniversityLonglong Wang - State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural UniversityQian Tan - State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural UniversityQiuling Fan - College of Life Science and Technology, Huazhong Agricultural UniversityHui Zhu - State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural UniversityZonglie Hong - Department of Plant, Soil, and Entomological Sciences and Program of Microbiology, Molecular Biology, and Biochemistry, University of IdahoZhongming Zhang - State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural UniversityDeqiang Duanmu - State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University
- Publication Details
- Frontiers in plant science, Vol.7(2016), pp.1333-1333
- Academic Unit
- Center for Reproductive Biology; Crop and Soil Sciences, Department of
- Publisher
- Frontiers Media S.A
- Grant note
- 31570233 / National Natural Science Foundation of China
- Identifiers
- 99900546527801842
- Language
- English
- Resource Type
- Journal article