AcABI5a negatively regulates AsA synthesis in transgenic and gene editing kiwifruit lines. (IMAGE)

Nanjing Agricultural University The Academy of Science

AcABI5a negatively regulates AsA synthesis in transgenic and gene editing kiwifruit lines.

Caption

AcABI5a negatively regulates AsA synthesis in transgenic and gene editing kiwifruit lines. (A) Two AcABI5a-overexpression transgenic lines (OE-AcABI5a#8 and OE-AcABI5a#9) and two independent AcABI5a homozygous mutant lines (Abi5#2 and Abi5#9), WT: wild type. (B) The presence of the transgene in AcABI5a-overexpression lines was confirmed by PCR amplification and immunoblotting with FLAG antibody. The forward primer (F) for PCR amplification was derived from the 35S promoter sequence of the vector, and the reverse primer (R) was derived from the CDS sequence of AcABI5a. the length of the amplified fragment was 500 bp. The protein size of AcABI5a-3 × FLAG was 50.0 KDa. (C) RT-qPCR analysis gene expression level of AcABI5a of WT and wo AcABI5a-overexpression transgenic lines. (D) Leaf AsA content of OE-AcABI5a#8 and OE-AcABI5a#9 lines. (E)–(F) RT-qPCR analysis gene expression level of (E) AcMYBS1 and (F) AcGGP3 in (D). (G) A schematic map shown above represents the targeting sites in the exon regions (rectangle) of AcABI5a; the PAM motifs (NGG) are shown in the last three bases. Sequences and chromatograms of AcABI5a in ‘Donghong’ wild-type (wt) and two gene editing lines (Abi5#2 and Abi5#9), reflecting the specifics of the gene editing events. The target sequence is underlined, the PAM sequences (GGG and GGA) can be seen, and the dashes indicate deletions. (H) Transgenic positive lines of editing of ‘Donghong’ were detected by agarose gel electrophoresis. The amplified fragment was a Cas9-AcABI5a vector fragment with a length of 930 bp. (I) Gene expression (by RT-qPCR) of AcABI5a in two gene editing lines. (J) Leaf AsA content of AcABI5a gene edited of ‘Donghong’ (note that fruit was not available for these lines). (K)–(L) Gene expression (by RT-qPCR) of (K) AcMYBS1 and (L) AcGGP3 in (J). Values are means ±SD of at least three independent biological replicates. Significant differences were detected by t-test (*P < 0.05; **P < 0.01; ***P < 0.001).

Credit

Horticulture Research

Usage Restrictions

Credit must be given to the creator.

License

CC BY

Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.