Bioinformatics Analysis of NbEHD1,Construction of CRISPR/Cas9 Gene Editing Vector and Genetic Transformation in Nicotiana benthamiana

doi:10.7668/hbnxb.20193839

Abstract

Abstract:

In order to study the biological function of NbEHD1 in Nicotiana benthamiana,its gene structure,conserved domains,phosphorylation sites,subcellular localization and evolutionary relationship were predicted by bioinformatics methods.The encoding sequence of NbEHD1 was 1 638 bp,and its genomic sequence contained 16 exons and 15 introns.NbEHD1 protein was predicted located in the cytoplasm,which had 42 phosphorylation sites and had no signal peptide and transmembrane region.NbEHD1 belonged to the P-loop_NTPase superfamily and had a conserved domain specific to the EHD family.Phylogenetic relationship showed that NbEHD1 was closely related to EHD sequences of Solanum tuberosum and Solanum lycopersicum.Blast analysis against N.benthamiana sub-data base in SNG was performed to obtain the predicted full length of NbEHD1,then its full nucleic acid sequence was amplified by gene-specific primers.After obtaining NbEHD1 CRISPR/Cas9 gene editing construct,the vector was successfully transformed into the leaves of N.benthamiana by Agrobacterium-mediated genetic transformation.Eventually 16 T₀-generation plantlets were successfully identified,which provided materials for further research to determine the biological function of NbEHD1.

Key words: Nicotiana benthamiana, NbEHD1, Bioinformatics analysis, CRISPR/Cas9

BAI Lu, XIN Cuihua, LIU Lele, WANG Junjie, JIAN Lei, SHAO Yutao, PEI Haixia, GUO Jiangbo. Bioinformatics Analysis of NbEHD1,Construction of CRISPR/Cas9 Gene Editing Vector and Genetic Transformation in Nicotiana benthamiana[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(5): 77-83. doi: 10.7668/hbnxb.20193839.

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Figures/Tables 12

Tab.1 Gene cloning primers

引物 Primer	序列(5'-3') Sequence(5'-3')
OXN578	ATGGAGTTTGATACTTGTCCCATTAATCGC
OXN579	GTCGTAAGGATTCCTGAAATTTTTGAGAAGTTC

Tab.2 Primers for CRISPR/Cas9-NbEHD1 vector construction

引物 Primer	序列(5'-3') Sequence(5'-3')
DT1-F0	GGGAAAAGCAGAGAACACAAGTTTTAGAGCTAGAAATAGC
DT2-R0	GAAGTTGCTCGCGTTTACATCAATCTCTTAGTCGACTCTAC
DT1-BsF-SL	CAGCTAGAGTCGAAGTAGTGATTGGGGAAAAGCAGAGAACACAA
DT2-BsR-SL	ACTTGCTATTTCTAGCTCTAAAACGAAGTTGCTCGCGTTTACAT
OXN974	TGTCCCAGGATTAGAATGATTAGGC
OXN1050	AGCCCTCTTCTTTCGATCCATCAAC

Tab.3 Primers for detection of positive plantlets

引物 Primer	序列(5'-3') Sequence(5'-3')
OXN1007	GAGAATCAGACCACGCAGAAGG
OXN1008	GCCTTGCCAATCTCCTGCTC
OXN1197	TCCTGTGAGTTATGCATCAATTTAC
OXN1364	GCAATTGTGTGGGTTGGTCAAC
OXN974	TGTCCCAGGATTAGAATGATTAGGC
OXN1050	AGCCCTCTTCTTTCGATCCATCAAC

Fig.1 Gene structure of NbEHD1

Fig.2 Conserved domain analysis of NbEHD1 protein

Fig.3 Predicted phosphorylation sites in NbEHD1 The abscissa is the sequence site and the ordinate is the phosphorylation potential.Red vertical line.Serine;Green vertical line.Threonine;Blue vertical line.Tyrosine;Purple horizontal line.Threshold.

Fig.4 The analysis of NbEHD1 protein interaction networks

Fig.5 Phylogenetic tree of NbEHD1 protein

Fig.6 Enzyme digestion of pKSE401-NbEHD1 recombinant plasmid M.250 bp DNA Ladder;1-5.Enzyme digestion of pKSE401-NbEHD1 recombinant plasmid;6.Enzyme digestion of pKSE401 empty vector.

Fig.7 pKSE401-NbEHD1 sequence alignment

Fig.8 Cultivation of CRISPR/Cas9 positive plantlets A.Callus induction;B.Rooting culture;C.Positive plantlets.

Fig.9 Detection plantlets containing the CRISPR/Cas9 vector A.Identification of Cas9 protein;B.Exon cloning.M.250 bp DNA Ladder;1-11.Partially transformed plantlets; 12.Positive control (recombinant vector);13.H₂O;14.Negative control (untransformed plantlet).

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