Predominance of the ASP-104 site of fatty acid desaturase 3 promoted a linolenic acid (ALA) accumulation in <i>Plukenetia volubilis</i>

Yijun Fu; Zhaohui Wang; Lixuan Ye; Guofang Deng; Kexian Li; Yuan Zou; Jia Wang; Jun Niu; Yijun Fu; Zhaohui Wang; Lixuan Ye; Guofang Deng; Kexian Li; Yuan Zou; Jia Wang; Jun Niu

doi:10.48130/tp-0025-0028

Figures (5) Tables (2)

Figure 1.
Phenotypes of Plukenetia volubilis and its fruits.
Figure 2.
Model prediction and molecular docking. (a) PvFAD3 tertiary structure prediction. (b) The reliability of the predicted model was evaluated. The Ramachandran plot results showed that 84.1% of the amino acids in the PvFAD3 protein model were located in the most favored region (red region), 11.6% in other allowed regions (yellow region), 1.3% in generally allowed regions (light brown region), and 3% in disallowed regions (white region). If the proportion of amino acid residues falling within the allowed region of the protein model is higher than 90%, the conformation of the model can be considered to conform to the principles of stereochemistry. (c) The tertiary structure of ligand LA. (d) Substrate protein docking prediction. The dashed rectangular region represents the rigid docking active site. (e) Top view of ligand LA small molecule binding to PvFAD3 mimicking protein. (f) Side-view magnification of ligand LA small molecule binding to PvFAD3 mimicking protein. The LA ligand molecule forms hydrogen bonds with the side chain R group of ASP-104 in the simulated PvFAD3 protein. (g) The intramolecular structure of the PvFAD3 protein model was analyzed. The amino acid residues at positions 104 (ASP-104) and 109 (ASN-109) were connected by hydrogen bonds.
Figure 3.
Conserved domains are predicted by homologous sequence alignment. (a) ASP-104 and ASN-109 are located in the PvFAD3 protein conserved domain motif 2. The larger the letter font, the more conserved the site. (b) The comparison of FAD3 proteins in 27 different species. There were significant differences in ASP-104 among different species, while ASN-109 was absolutely conserved. Dark blue: amino acid homology = 100%, Pink: amino acid homology ≥ 75%, Light blue: amino acid homology ≥ 50%. RcFAD3, EEF36775.1; VfFAD3, AAC98967.2; JcFAD3, NP_001292929.1; MeFAD3, XP_021610352.1; MeFAD3, XP_021610352.1; HbFAD3, XP_021666224.1; TsFAD3, ABM68629.1; PfFAD3, AQZ42316.1; PsFAD3, AVZ47050.1; LuFAD3, BAG70950.1; BnFAD3, AAT09135.1; EuFAD3, ARQ20744.1; AtFAD3, NP_180559.1; ParFAD3, CAB4284757.1; GmFAD3, NP_001236114.1; ChFAD3, AEF80000.1; CmFAD3, KAF3952997.1; JrFAD3, XP_018835457.1; AhFAD3, QBI71562.1; CsFAD3, XP_028086292.1; TwFAD3, XP_038705526.1; CfFAD3, KAE8039281.1; DzFAD3, XP_022756628.1; AcFAD3, PSS09495.1; MdFAD3, XP_008384790.2; VvFAD3, XP_002277573.1; PavFAD3, XP_021816263.1.
Figure 4.
The topological model of PvFAD3. (a) PvFAD3 protein cross membrane structure prediction. The purple structure represents a cross-membrane or inlaid protein structure. (b) Topological model prediction of PvFAD3 proteins. TM1, TM2, and TM3: Protein transmembrane structure. AH1 and AH2: Protein mosaic structure. His-boxI, His-boxII, and His-boxIII: Histidine active region. Red Star: ASP-104.
Figure 5.
Homozygous screening and genetic transformation of AtFAD3m. (a) AtFAD3m homozygote, WT and AtFAD3m heterozygote. (b) AtFAD3m seeding. (c) The transgenic PvFAD3 positive seedlings of T₁ generation were screened for Kana⁺ resistance. (d) The transgenic mPvFAD3 positive seedlings of T₁ generation were screened for Kana⁺ resistance. (e) PCR screening of transgenic plants. (f) RT-qPCR validation of transgenic plants. (g) When T₃ positive seedlings were transplanted into soil for 5 weeks, the growth state of transformed PvFAD3 and mPvFAD3 was significantly better than that of AtFAD3m. (h) Plant height statistics of AtFAD3m, PvFAD3, and mPvFAD3.

Culture condition	Group	FA components (%)
Culture condition	Group	C14:0	C16:0	C16:1	C18:0	C18:1	C18:2	C18:3
30 °C	pYES2	1.28 ± 0.34^c	12.16 ± 0.76^e	43.12 ± 0.61^a	2.82 ± 0.06^c	21.36 ± 0.65^a	19.26 ± 0.40^c	0.00 ± 0.00^e
	pYES2-PvFAD3	12.63 ± 0.45^a	44.03 ± 0.54^b	6.24 ± 0.08^c	1.59 ± 0.04^d	9.87 ± 0.55^c	17.14 ± 0.64^d	8.49 ± 0.09^b
	pYES2-mPvFAD3	1.67 ± 0.07^c	15.27 ± 0.53^d	42.23 ± 0.49^a	4.15 ± 0.06^b	22.77 ± 0.55^a	7.35 ± 0.39^e	6.56 ± 0.17^c
20 °C	pYES2	0.00 ± 0.00^d	15.98 ± 0.30^d	29.01 ± 0.42^b	4.53 ± 0.22^a	14.09 ± 0.76^b	36.39 ± 0.71^a	0.00 ± 0.00^e
	pYES2-PvFAD3	10.99 ± 0.23^b	62.04 ± 0.34^a	1.42 ± 0.15^d	1.33 ± 0.07^d	6.78 ± 0.43^d	1.92 ± 0.28^f	15.52 ± 0.32^a
	pYES2-mPvFAD3	1.94 ± 0.22^c	18.50 ± 0.54^c	28.09 ± 0.43^b	4.66 ± 0.05^a	19.64 ± 0.68^a	22.10 ± 0.44^b	5.06 ± 0.47^d
Different letters indicate significant differences (p < 0.05).

Table 1.

Fatty acid compositions in different yeasts.

Group	FA components (%)
Group	C16:0	C18:0	C18:1	C18:2	C18:3	C20:0	C20:1	Total
AtFAD3m
AtFAD3m - line1	1.62 ± 0.02^d	0.78 ± 0.04^c	2.79 ± 0.06^de	6.94 ± 0.06^g	0.21 ± 0.02^b	0.42 ± 0.02^d	3.03 ± 0.05^f	15.78 ± 0.08^f
AtFAD3m - line2	1.65 ± 0.06^d	0.80 ± 0.04^c	2.93 ± 0.06^d	7.51 ± 0.08^e	0.20 ± 0.03^b	0.44 ± 0.03^cd	3.15 ± 0.04^e	16.68 ± 0.16^e
AtFAD3m - line3	1.64 ± 0.03^d	0.80 ± 0.06^c	2.74 ± 0.04^e	7.25 ± 0.15^f	0.19 ± 0.01^b	0.40 ± 0.02^d	3.07 ± 0.05^ef	16.09 ± 0.26^f
PvFAD3 transfer
PvFAD3 - line3	1.77 ± 0.06^c	0.79 ± 0.04^c	3.59 ± 0.19^c	9.78 ± 0.03^b	0.29 ± 0.02^a	0.55 ± 0.04^b	4.13 ± 0.07^c	20.90 ± 0.33^c
PvFAD3 - line4	1.75 ± 0.02^c	0.83 ± 0.06^c	3.62 ± 0.06^c	9.53 ± 0.06^c	0.30 ± 0.02^a	0.57 ± 0.02^b	4.12 ± 0.05^c	21.73 ± 0.12^c
PvFAD3 - line5	2.35 ± 0.06^a	1.10 ± 0.03^a	4.67 ± 0.13^a	10.96 ± 0.05^a	0.29 ± 0.03^a	0.65 ± 0.02^a	5.14 ± 0.05^a	25.17 ± 0.06^a
mPvFAD3 transfer
mPvFAD3 - line2	1.76 ± 0.08^c	0.85 ± 0.03^bc	3.68 ± 0.09^c	8.49 ± 0.08^d	0.19 ± 0.01^b	0.47 ± 0.02^c	3.94 ± 0.08^d	19.40 ± 0.16^d
mPvFAD3 - line3	1.92 ± 0.07^b	0.91 ± 0.02^b	4.08 ± 0.05^b	9.67 ± 0.10^bc	0.19 ± 0.02^b	0.53 ± 0.03^b	4.36 ± 0.06^b	21.66 ± 0.04^b
Different letters indicate significant differences (p < 0.05).

Table 2.

Fatty acid compositions in Arabidopsis seeds.