Fig. 4: Influence evaluation of PsCPT, PsSRPP and PsREF on rubber biosynthesis in P. sepium via CRISPR-Cas9 genome editing.

a Schematic structure of the T-DNA region in the binary vector used for CRISPR-Cas9 genome editing of PsCPT, PsSRPP and PsREF; RB, right border; AtU6-P, Arabidopsis thaliana U6 promoter; 3×tRNA-sgRNA-scaffold, a tandemly arrayed three tRNA-sgRNA architecture containing tRNA-sgRNA1-scaffold-tRNA-sgRNA2-scaffold-tRNA-sgRNA3-scaffold of each PsCPT, PsSRPP or PsREF gene, respectively; Pol III-T, RNA polymerase III poly (T); AtUBQ-P, A.thaliana ubiquitin 10 promoter; F, 3×Flag-tag peptide; SV40 NLS, nuclear localization signal of simian virus 40 large T antigen; Cas9, endonuclease from the Streptococcus pyogenes type II CRISPR-Cas system; nucl NLS, bipartite nuclear localization signal from nucleoplasmin; AtHSP-T, A.thaliana heat shock protein 18.2 terminator; 35S-P, cauliflower mosaic virus (CaMV) 35S promoter; sGFP, synthetic green-fluorescent protein with S65T mutation; NOS-T, nopaline synthase terminator; 35S-E-P, CaMV 35S promoter with enhancer; NPT II, neomycin phosphotransferase; 35S poly A, CaMV 35S polyA terminator; LB, left border. b Three representative transgenic plants with mutations via CRISPR-Cas9 genome editing of each PsCPT, PsSRPP and PsREF gene, namely, transgenic PsCPT-06, PsCPT-41, and PsCPT-63; PsSRPP-31, PsSRPP-34, and PsSRPP-36; PsREF-17, PsREF-18, and PsREF-59, showed reduced total cis-polyisoprene contents to different degrees compared with that of the wild-type (nontransgenic negative, WT) control; Data represent mean ± standard deviation, n = 3; Different letters indicate significant differences at p < 0.01. c, d The transgenic plants with mutations exhibited the high-molecular-weight at approximately 10⁴–10⁶ (Peak3) shifted forwards and smaller or completely absent; These results indicated that mutagenesis of these three genes, PsCPT, PsSRPP and PsREF, can down-regulate the biosynthesis of cis-polyisoprenes in P. sepium.