Figure 2.
Molecular Cloning and Expression Analysis of HTB
(A) Cloning of the htb-1 allele identified a G-to-A mutation in the acceptor site of the first intron of Carubv10008238, which disrupts the splicing of the first intron and results in a 7-bp deletion in the second exon, generating a premature stop codon in exon 2. The htb-2ge allele was generated by CRISPR with a single-base-pair deletion in the exon 2, resulting in a frameshift giving rise to a 77-amino-acid (aa) protein. The guide RNAs and PAM sequences were indicated by red and blue characters, respectively.
(B–G) GUS staining of pHTB:GUS line showing the dynamic expression of HTB during fruit development. Uniform expression of HTB is detected in inflorescence tissue (B) and in the gynoecium at stage 11 (C) and 12 (D). A stronger HTB expression is detected in the developing fruit shoulders in stages 13 (E) and 14 (F). At stage 15, only residual HTB expression is observed in the fruit (G).
(H and I) Subcellular localization of HTB:GFP protein in the roots of pHTB:HTB:GFP line.
Scale bars in (B)–(I) represent 100 μm.
(J) Comparative analysis of SUMO conjugates in total protein extracts from leaf, inflorescence (inflo.), and stage-13 (S13) and stage-15 (S15) fruits between WT and htb-1. The α-tubulin was immunoblotted as a loading control.
See also Figure S2.