Generation of MpROP Loss-of-Function Mutants

Loss-of-function mutants of MpROP were generated with the CRISPR/Cas9 system as described previously (62 (link)). CRISPR target sites were predicted on the website (http://crispr.hzau.edu.cn/CRISPR2/), and two target sites on the first (Mprop-2) and third exon (Mprop-1) were selected. Synthetic oligos DNA for Mprop-1 named MpROPcas9.1-F(En_01)/MpROPcas9.1-R(En_01) and for Mprop-2 named Mpropcrispr.6-En01-F/Mpropcrispr.6-En01-R were annealed, inserted in the entry vector pMpGE_En01 (62 (link)) with the in-fusion HD cloning kit (Clontech), and then introduced into the destination vector pMpGE010 (62 (link)) by using LR clonase II Enzyme mix (Thermo Fisher Scientific). The reconstructed vectors were introduced into regeneration thalli of WT by Agrobacterium GV3101, and transformants were selected with 10 mg/L hygromycin (Roche). The transgene was confirmed by sequence analysis using genomic DNA. Then the target region was amplified by PCR (Biosune, Fuzhou) to sequence for the respective target sites.

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Rong D., Zhao S., Tang W., Luo N., He H., Wang Z., Ma H., Huang Y., Yao X., Pan X., Lv L., Xiao J., Liu R., Nagawa S, & Yamamuro C. (2022). ROP signaling regulates spatial pattern of cell division and specification of meristem notch. Proceedings of the National Academy of Sciences of the United States of America, 119(47), e2117803119.

Publication 2022

in-fusion HD cloning kit LR clonase II Enzyme mix hygromycin

Agrobacterium Crispr Enzyme Exon Genomic Hygromycin Oligos Regeneration Sequence analysis Transgene Vectors

Corresponding Organization :

Other organizations : Fujian Agriculture and Forestry University, Hunan University of Technology, Chinese Academy of Sciences, Shenzhen Institutes of Advanced Technology, University of California, Riverside

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Variable analysis

independent variables

Loss-of-function mutants of MpROP generated with the CRISPR/Cas9 system

dependent variables

Phenotypic outcomes of the loss-of-function mutants

control variables

Wild-type (WT) plants

controls

Positive control: Wild-type (WT) plants
Negative control: Not explicitly mentioned

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