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SCIENCE CHINA Life Sciences, Volume 60, Issue 5: 524-527(2017) https://doi.org/10.1007/s11427-017-9031-y

Increased lateral root formation by CRISPR/Cas9-mediated editing of arginase genes in cotton

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  • AcceptedMar 9, 2017
  • PublishedMay 1, 2017

Abstract

There is no abstract available for this article.


Funded by

National Natural Science Foundation of China(31301373,31601349)

Ministry of Agriculture of China(2016ZX08009003-003-004)


Acknowledgment

We are grateful to Tao Zhou, Wenzhong Deng and Congwei Wang all from Biotechnology Research Institute, Chinese Academy of Agricultural Sciences for technical assistance. This work was supported by the National Natural Science Foundation of China (31301373, 31601349), and the Ministry of Agriculture of China (2016ZX08009003-003-004).


Interest statement

The author(s) declare that they have no conflict of interest.


Supplement

SUPPORTING INFORMATION

Figure S1 Sequencing results of 15 cotton callus lines created using sgRNA1.

Figure S2 Sequencing results of 15 cotton callus lines created using sgRNA2.

Figure S3 Sequencing results of 15 T0 mutant cotton plants induced by sgRNA1.

Figure S4 Sequencing results of 15 T1 mutant cotton plants induced by sgRNA1.

Figure S5 Arginase activity of T1 transgenic cotton seedling roots.

Figure S6 NOS activity of T1 transgenic cotton seedlings.

Figure S7 NOx content in the roots of T1 transgenic cotton seedlings.

Table S1 PCR primers

The supporting information is available online at life.scichina.com and www.springerlink.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


References

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  • Figure 1

    CRISPR/Cas9 editing of GhARG homologs increased lateral root development in cotton seedlings. A, Target sites of sgRNA (blue letters) in GhARG’s first exon. Red letters indicate PAM sequence. Highlighted letters in purple and yellow are two copies GhARG in A- and D-genomes, respectively. B, Genetic map of CRISPR/Cas9 constructs. PNtU6, promoter comes from Nicotiana tabacum L. C, Gel analysis of cotton callus edits efficiency. Lanes 1 to 15 are Bsl I-digested PCR products of transgenic callus, lanes 16 and 17 are digested and undigested PCR products of wild-type callus. Numbers under image indicate target site edit efficiency for sgRNA1 and sgRNA2, calculated by the band intensities. GhARG gene has been successfully edited in the randomly selected 15 calluses of sgRNA1 and sgRNA2, both with efficiency higher than 10%. D, Sequence alignment to identify edit types of sgRNA1 target sites in T0 seedlings. The top 2 are unedited sequences from the A- and D-chromosomes. Deletions and insertions are shown by dashes and purple letters, respectively. Alignments indicated that sgRNA1-6 cotton lines have 7 edit types, while sgRNA1-9 cotton lines have three edit types. E and F, Images of T1 seedling root systems in high and low level nitrogen medium. WT, wild-type R18 cotton; L24 and L28, T1 transgenic cotton. G and H, Statistical analysis of lateral root development including number of roots and total root surface area. GhARG knock outs have significantly increased numbers of lateral roots and total root surface area. WT, wild-type R18 cotton; L24 and L28, T1 sgRNA1 transgenic cotton plants; HN, high nitrogen level medium; LN, low nitrogen level medium. Error bars indicate the standard deviation of three independent experiments. (*, P<0.1; **, P<0.05).

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