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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (2): 354-364.doi: 10.3724/SP.J.1006.2023.22002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Function analysis of OsPIN5c gene by CRISPR/Cas9

YANG Xiao-Yi1(), WANG Hui-Hui1, ZHANG Yan-Wen1, HOU Dian-Yun1, ZHANG Hong-Xiao1, KANG Guo-Zhang2, XU Hua-Wei1,*()   

  1. 1College of Agriculture, Henan University of Science and Technology, Luoyang 471000, Henan, China
    2National Key Laboratory of Wheat and Maize Crop Science, Henan Agricultural University, Zhengzhou 450046, Henan, China
  • Received:2022-01-13 Accepted:2022-06-07 Online:2022-07-08 Published:2022-07-08
  • Contact: XU Hua-Wei E-mail:1961827259@qq.com;xhwcyn@163.com
  • Supported by:
    Natural Science Foundation of Henan Province(182300410012);Natural Science Foundation of Henan Province(202300410151);Natural Science Foundation of Henan Province(202300410340);Open Research Fund of National Key Laboratory of Wheat and Maize Crop Science(SKL2021KF03)

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Abstract:

Polar auxin transport (PAT) plays a key role in plant growth and development, and auxin efflux carriers PIN-FORMED (PIN) are the crucial proteins controlling PAT. Although the functions of some OsPIN genes have been reported, the function of OsPIN5c gene is still unclear. In this study, the target site was designed at the first exon of OsPIN5c and the recombinant CRISPR/Cas9 vector of OsPIN5c was constructed. Twenty-four independent transgenic rice lines were obtained by transformation using Nipponbare (Oryza sativa L. ssp. japonica) as the materials. PCR product sequencing indicated that 15 lines were identified as mutants and the corresponding mutation rate was 62.5%, the mutation type were biallelic heterozygous mutations. Three independent ospin5c homozygous mutants were further obtained in T1 generation lines and named as ospin5c-1, ospin5c-2, and ospin5c-3, respectively. Sequence alignment analysis showed that the three types of mutations resulted in frame-shift mutation and premature translation termination, which were shortened from 398 amino acid (aa) in wild-type (WT) plants to 109 aa, 106 aa and 250 aa, respectively. Transmembrane helices (TMH) indicated that the TMH of these three mutation proteins were disappeared totally. Protein structure demonstrated that the helix of three mutation proteins were obviously reduced than the native OsPIN5c protein. Phenotype structure indicated that, compared to the WT, the shoot height, root length and adventitious root number were significantly decreased in ospin5c mutants at seedling stage. Tissue-specific analysis showed that OsPIN5c was highly expressed in roots, and OsPIN genes (OsPIN1a and OsPIN5b), OsYUC genes (OsYUC1, OsYUC4, OsYUC6, and OsYUC7) were up-regulated significantly in ospin5c mutants. The gravitropism response of ospin5c mutants was partially inhibited. In conclusion, three ospin5c homozygous mutants were obtained via CRISPR/Cas9 technology and the function of OsPIN5c was investigated in this study, providing the potential gene resources for crop genetic improvement by using OsPIN5c gene.

Key words: OsPIN5c, CRISPR/cas9, gene function, rice

Table 1

Primers and sequences in this study"

引物名称Primer name 正向引物Forward sequence (5'-3') 反向引物Reverse sequence (5'-3')
OsPIN5c-CRISPR TGTGTGGGTTCTCGTGGTGCATCAC AAACGTGATGCACCACGAGAACCCA
HPT CTGAACTCACCGCGACGTCTGTC TAGCGCGTCTGCTGCTCCATACA
OsPIN5c-Assay CCTCGTCGCCTGCTTCGCCG CCACACCGCTCTCACCAGCGG
OsPIN1a-qPCR CCTGAAATCCATCTCCATCCTC AACGTCGCCACCTTGTT
OsPIN5b-qPCR GCAAAGGAGTATGGGCTTCA GCAATCAGAATCGGCAGAGA
OsYUC1-qPCR AGGTGTTGGTCGTGGGATGCG GCGATGCCGAACGTGGATAGA
OsYUC4-qPCR CCTCGACCTCTGCAACCACAATG CGACAACAGGAGTACCAGCCAATC
OsYUC6-qPCR GGATACCAAAGCAACGTCCCC TGAAGCCAACAGAGTAGAGCCCTG
OsYUC7-qPCR ACCGGCTACCGCAGCAATGTG CGTACAGCCCCGACTCACCCT
OsACTIN1-qPCR CTTCATAGGAATGGAAGCTGCG CACCTTGATCTTCATGCTGCTA

Fig. 1

Schematic diagram of OsPIN5c target site and screening for positive clones A: OsPIN5c target site in schematic diagram; PAM: protospacer adjacent motif; black line indicates PAM sequences. B: positive clones by PCR. M: DL2000 DNA marker; 1-12: PCR products."

Fig. 2

Screening and analysis of mutation lines in T0 generation A: PCR amplification of OsPIN5c genomic fragments; M: DL2000 DNA marker; 1-7: PCR products. B: mutation type analysis. PAM: protospacer adjacent motif; black line indicates PAM sequence."

Fig. 3

Screening for ospin5c homozygous mutants in T1 generation PAM: protospacer adjacent motif; black line indicates PAM sequence; red arrows show the mutation sites."

Fig. 4

Screening for transgene-free lines A: PCR screening results; M: DL2000 DNA marker; 1-25: HPT PCR amplification results; P: positive control; N: negative control. B: hygromycin sresistance assay of detached leaves. 1-25: hygromycin sresistance assay of ospin5c detached leaves."

Fig. 5

Schematic representations of amino acid sequence change of OsPIN5c mutant proteins A: amino acid change of the target sites; PAM: protospacer adjacent motif; underlined amino acids indicate mutant amino acids; asterisk indicates the end of translation. B: blast analysis of the mutant proteins."

Fig. 6

Transmembrane helices (TMH) and tertiary structure analysis of OsPIN5c mutant proteins A: transmembrane helices (TMH) analysis; B: tertiary structure analysis."

Fig. 7

Phenotype of ospin5c mutants at seedling stage A: phenotypic analysis of 7-day-old ospin5c mutants. B: phenotypic analysis of 14-day-old ospin5c mutants (n ≥ 15). **: P < 0.01."

Fig. 8

Relative expression patterns of genes A: tissue-specific analysis of OsPIN5c. B: the relative expression levels of OsPIN and OsYUC genes. *: P < 0.05; **: P < 0.01; ***: P < 0.001."

Fig. 9

Root gravitropism analysis of WT and ospin5c mutants A: the phenotypes of WT and ospin5c mutants after gravity stimulus; 0-24 h indicate time after gravity stimulus. B: statistical analysis of root reorientation angle in WT and ospin5c mutants after gravity stimulus (n ≥ 12). *: P < 0.05; **: P < 0.01."

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