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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (10): 2494-2504.doi: 10.3724/SP.J.1006.2022.14150

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and functional study of lysophosphatidic acid acyltransferase gene in Perilla frutescens

XU Hua-Xiang(), LU Geng, GUO Xi, LI Yuan-Yuan, ZHANG Tao()   

  1. College of Life Sciences, Chongqing Normal University, Chongqing 401331, China
  • Received:2021-08-17 Accepted:2022-02-25 Online:2022-10-12 Published:2022-03-31
  • Contact: ZHANG Tao E-mail:xhx990123@163.com;zht2188@126.com
  • Supported by:
    National Natural Science Foundation of China(31171588);Chongqing Normal University Postgraduate Research and Innovation Project(YKC21033)

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Abstract:

Lysophosphatidic acid acyltransferase (LPAAT) is a key enzyme in plant triacylglycerol biosynthesis. Investigating the function of Perilla frutescens LPAAT gene (PfLPAAT) in oil biosynthesis can help to reveal the molecular mechanism of plant oil accumulation. In this study, RT-PCR was used to obtain PfLPAAT, and the basic physical and chemical properties, transmembrane domain, and subcellular localization of the putative protein of PfLPAAT was analyzed by bioinformatics methods, and phylogenetic analysis of homologous proteins was conducted. The qRT-PCR was used to analyze the relative expression level of PfLPAAT in different tissues and developmental stages of seeds in Perilla. The expression vector pCAMBIA1303-PfLPAAT was constructed and transformed into Arabidopsis thaliana by floral dip method, and the oil content and fatty acid composition of transgenic Arabidopsis seeds were analyzed. The results showed that the PfLPAAT sequence was 1149 bp in length and encoded 382 amino acids. The theoretical isoelectric point of the putative protein was 9.60, and the molecular mass was 43.02 kD. Bioinformatics indicated that PfLPAAT functioned in endoplasmic reticulum and belonged to the PLN02380 superfamily. The qRT-PCR revealed that PfLPAAT expressed in all tissues of Perilla, and the highest level was observed in leaves and seeds of 15 days after flowering. Compared with wild-type Arabidopsis, the overexpression of PfLPAAT in Arabidopsis improved seed oil content significantly, with a range of 2.04% to 13.40%. Fatty acid composition analysis demonstrated that the content of oleic acid, linoleic acid, linolenic acid, and arachidic acid increased significantly, while the content of palmitic acid, stearic acid, arachidic acid, arachidonic acid, and docosaenoic acid decreased. In short, the overexpression of the PfLPAAT in Arabidopsis could not only increase the oil content of transgenic Arabidopsis seeds, but also increase the content of unsaturated fatty acids with varying degrees. The results provide novel candidate genes for the mining and innovative utilization of germplasm resources of the oil crop Perilla.

Key words: Perilla frutescens, lysophosphatidic acid acyltransferase, oil content, unsaturated fatty acids

Table 1

Primer sequences used in this study"

引物代码
Primer name		 引物序列
Primer sequence (5'-3')
PfLPAAT-F GGTTTTACTCATTATGGCGAT
PfLPAAT-R TCACCTCACCGCTCTCTGT
PfLPAAT-Bgl II-F GGGACTCTTGACCATGGTAGATCTGGCGATCCCAGCCGCGATTGTTG
PfLPAAT-Bgl II-R ACAGGACGTAAACTAGTCAGATCTCGCTGTCTGTCTTGTCTCGTTGAG
PfLPAAT-q-F TACATGCCATACTGCACTCACAGTT
PfLPAAT-q-R TATTTCCGGCCTGGGATTCA
β-Actin-F AGACCTTCAATGTGCCAGCCA
β-Actin-R CACGACCAGCAAGATCCAACCA

Fig. 1

Cloning and analysis of PfLPAAT A: PCR amplification product of PfLPAAT, M: DL2000 marker, 1: PfLPAAT ORF amplification product; B, C: transmembrane structure and hydrophobicity analysis of PfLPAAT, respectively; D, E: prediction of secondary structure and conserved domains of PfLPAAT, respectively."

Fig. 2

Prediction of two motifs of LPAAT by MEME The logo was generated from an alignment of eight plant sequences of the LPAAT protein, the outlined boxes indicate the highly conserved motifs NHX4D (A, catalytic domain) and EGTR (B, binding domain)."

Fig. 3

Mutiple sequence alignment of LPAAT in different plants"

Fig. 4

Phylogenetic analysis of PfLPAAT and its homologous proteins The number on the branches represents the reliability percent of bootstraps values based on 1000 replications."

Fig. 5

Relative expression of PfLPAAT in different tissues (A) and seeds at different development stages (B)"

Fig. 6

Identification and acquisition of transgenic Arabidopsis A: T-DNA element of plasmid pCAMBIA1303 containing the PfLPAAT coding region; LB: left border; CaMV 35S: cauliflower mosaic virus 35S promoter; PfLPAAT: lysophosphatidic acid acyltransferase gene from Perilla; GUS: β-glucuronidase; GFP: green fluorescent protein; NOS: nopaline synthase terminator; RB: right border. B: identification of T3 generation transgenic Arabidopsis; M: DL2000 marker; 1-10: amplification products of transgenic Arabidopsis lines. C-E: GUS staining of transgenic Arabidopsis seedlings, rosette leaves, and fruit pods, respectively."

Fig. 7

Phenotypic observation of transgenic Arabidopsis seeds A, B: dry seeds of wild-type and transgenic Arabidopsis were viewed under a stereo microscope, respectively, scale bar in the lower right corner indicates 1mm; C, D: length and width of wild-type and transgenic Arabidopsis (lines 1-10) seeds, respectively. The data are means ± standard errors (n = 31)."

Table 2

Oil content and fatty acid composition of transgenic Arabidopsis seeds"

株系
Line		 含油量
Oil content (%)		 C16:0
(%)		 C18:0 (%) C18:1 (%) C18:2 (%) C18:3 (%) C20:0 (%) C20:1 (%) C20:2 (%) C22:1 (%)
Col 31.260 11.02 3.45 17.64 30.86 17.86 1.09 15.12 1.81 1.15
1 33.471* 5.32* 2.63* 20.71* 36.74* 17.52 0.45* 15.26 0.46* 0.91*
2 31.896 4.76* 2.47* 18.57 35.6* 15.6* 0.7* 21.07* 0.32* 0.91*
3 33.400* 7.57* 2.37* 16.53* 40.19* 20.08* 0.51* 11.98* 0.34* 0.43*
4 32.640* 3.32* 0.47* 20.83* 38.88* 19.5* 0.71* 14.99 0.37* 0.93
5 32.005 6.85* 3.44 19.92* 35.83* 16.39* 0.47* 16.05 0.53* 0.52*
6 32.150* 7.23* 2.87 19.5* 37.48* 14.9* 0.68* 16.22 0.6* 0.52*
7 33.117* 6.23* 2.44* 16.24* 40.08* 18.19* 0.55* 14.64 0.75* 0.88*
8 35.092* 6.17* 2.3* 15.96* 39.36* 20.41* 0.56* 14.01* 0.51* 0.72*
9 32.205* 10.02 2.34* 14.42* 33.00* 20.23* 0.71* 17.56* 0.67* 1.05
10 35.450* 6.67* 2.53* 16.7 32.46* 21.69* 0.66* 17.48* 0.53* 1.28
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