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Home > Archive>Volume 22, Issue 3, 2021 >789-799. DOI:10.13430/j.cnki.jpgr.20210104003
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Creating Novel Glutinous Rice Germplasms by Editing Wx Gene via CRISPR/Cas9 Technology
Author:
Affiliation:

Key Laboratory of Plant Functional Genomics of the Ministry of Education/Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, College of Agriculture, Yangzhou University

Fund Project:

The National Natural Science Foundation of China（31825019），The National Special Project on Breeding New Varieties of Genetically Modified Organisms（2016ZX08001006-005），The Jiangsu Province Key Research and Development Program Modern Agriculture（BE2018357)

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    Abstract:

    The Wx gene，which controls the synthesis of amylose in rice endosperm，is the major gene that determines rice eating and cooking quality（ECQ）. It can significantly improve rice ECQ by moderately reducing amylose content（AC）of endosperm. Editing the C-terminal of Wx encoded GBSSI enzyme to fine-tune its enzyme activity and grain AC is expected to further improve rice ECQ. By analyzing the functional domain of GBSSI through bioinformatics websites，two target sites，T1 and T2（located in the 12th and 13th exons of the Wx gene，respectively），were subjected for CRISPR/Cas9-medicated editing. The homozygous mutants were validated by PCR and Sanger sequencing，followed by quantifying the apparent amylose content，gel-permation chromatography，western blot and qRT-PCR. A total of 8 homozygous lines，C1-C8，with retained major domains of GBSSI were obtained. Among which，the 518-550/551 codons were shifted in the predicted protein in C2 and C3 lines and the predicted protein in C1，C4-C8 were shifted after codon 551，517 or 518. The apparent amylose content of C1-C8 was significantly reduced from 16.79% to 4.44%-3.69%，but significantly higher than that of near-isogenic line（NIL）carrying the conventional wx allele（AAC=2.92%）. GPC results showed that there was no amylose synthesis in the selected mutants，but their chain length distribution of Ap2 was longer than that in NIL wx. The qRT-PCR and western blot suggested no significant difference on transcript of Wx gene but obvious reduction of GBSSI accumulation in the developing seeds in mutants. No GBSSI accumulation was detected in all the homozygous lines except for C2 and C3. As a result，this study generated glutinous rice lines with fine-tuned starch fine structure to conventional glutinous rice by CRISPR strategy，and provided evidence of the 518-551 amino acids on the formation of GBSSI enzyme activity. These results provide a reference for further analyzing the structure of GBSSI protein to achieve its precise editing.

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History
  • Received:January 04,2021
  • Revised:February 03,2021
  • Adopted:February 04,2021
  • Online: May 07,2021
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