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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (8): 1283-1290.doi: 10.3724/SP.J.1006.2020.94192

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Cloning and function analysis of a type 2 diacylglycerol acyltransferase (DGAT2) from Perilla frutescens

LU Geng,TANG Xin,LU Jun-Xing,LI Dan,HU Qiu-Yun,HU Tian,ZHANG Tao()   

  1. College of Life Sciences, Chongqing Normal University, Chongqing 401331, China
  • Received:2019-12-11 Accepted:2020-03-24 Online:2020-08-12 Published:2020-04-26
  • Contact: Tao ZHANG E-mail:zht2188@126.com
  • Supported by:
    National Natural Science Foundation of China(31171588);Chongqing Technology Innovation and Application Development Project(cstc2019jscx-msxm0612)

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Abstract:

Diacylglycerol acyltransferase (DGAT) is a key enzyme in the final step of triacylglycerol (TAG) synthesis in plant. In seed oil of certain plants, DGAT2 can selectively accumulate more unsaturated fatty acids. In this paper, we successful cloned PfDGAT2 from Perilla frutescens and performed bioinformatics analysis. Real-time fluorescence quantitative analysis showed that PfDGAT2 was expressed in different organs, with the highest in seeds at 10 d after anthesis, the medium in roots, and gradual decrement in the middle and late stages of seed. Compared with the wild-type, the oil content of seeds in PfDGAT2 transgenic Arabidopsis was increased by 21.68%-77.89%. The fatty acid components of the four strains with the largest increase in seed oil content were analyzed. Compared with the control, linolenic acid (C18:3), arachidonic acid (C20:1), arachidonic acid (C20:2), docosaenoic acid (C22:1) increased significantly by 4.57%, 7.44%, 5.40%, and 0.37%, respectively. Palmitic acid (C16:0), stearic acid (C18:0), and linoleic acid (C18:2) were obviously reduced by 3.47%, 6.64%, and 4.83%, respectively. Oleic acid (C18:1) only decreased by 0.18% and arachidic acid (C20:0) by 1.91%. In conclusion that PfDGAT2 gene can not only increase the oil content, but promote the accumulation of unsaturated fatty acids such as linolenic acid and arachidonic acid, which provides a reference and theoretical basis for studying the synthesis and accumulation of unsaturated fatty acid in plants.

Key words: diacylglycerol acyltransferase, seed oil content, unsaturated fatty acid, Perilla frutescens

Table 1

Primers used for PCR"

引物名称
Primer name		 引物序列
Sequences (5'-3')
PfDGAT2 F CTCGCTTACTGCTACTTCAATG
PfDGAT2 R CGACAATTAGAGAATCCTGAGC
PfDGAT2-DL F AGTCCGAGCCCAACGGCGATGTCAG
PfDGAT2-DL R GGATGCCCAACCCTTGCTTTGTGCC
Actin F AGACCTTCAATGTGCCAGCCA
Actin R CACGACCAGCAAGATCCAACC
18S RNAF CGGCGACGCATCATTCAAA
DGAT2-BglII F ACTCTTGACCATGGTAGATCTGGAGTCCGAGCCCAACGGCG
DGAT2-BglII R GGACGTAAACTAGTCAGATCTAGAATCCTGAGCTCTAAGTCG
JD-F TTTCATTTGGAGAGAACACGGGGGA
JD-R CGCTGATCAATTCCACAGTTTTCGC

Fig. 1

Cloning and analysis of the PfDGAT2 gene A: PCR amplification product of PfDGAT2, M: DL2000 DNA marker. B: conserved domains analysis of PfDGAT2 protein."

Fig. 2

Phylogenetic tree of different plants based on amino acid of DGAT2 The number on the branches represents the reliability percent of bootstraps values based on 1000 replications."

Fig. 3

Multiple sequence alignment analysis of DGAT2 in different plants PfDGAT2: Perilla frutescens; SiDGAT2: Sesamum indicum (XP_011098009.1); StDGAT2: Solanum tuberosum (XP_006365015.1); AtDGAT2: Arabidopsis thaliana (OAP06431.1); GmDGAT2: Glycine max (NP_001299586.1); NtDGAT2: Nicotiana tabacum (AGL46984.1); CsDGAT2: Camelina sativa (XP_010426724). In the black box there are the YFP, EPHS, GGVQE, RXGFX(K/R)XAXXXGXX(L/V) VPXXXFG(E/Q), and VVGRPI conserved domains in turn."

Fig. 4

Analysis of relative expression of PfDGAT2 gene in different tissues and different growth stages of seeds Bars superscripted by different lowercase letters are significantly different at P < 0.05. Data points are means±SE (n = 3)."

Fig. 5

Seeds oil content of transgenic PfDGAT2 Arabidopsis thaliana Bars superscripted by different lowercase letters are significantly different at P < 0.05. Data are means±SE (n = 3)."

Fig. 6

Relative fatty acid content in PfDGAT2 transgenic Arabidopsis thaliana seeds In the figure, * shows significant difference of each component of PD2-1, PD2-3, PD2-5 with wild-type Col-0. * represents the significant difference at P < 0.05, ** represents significant difference at P < 0.01, *** represents significant difference at P < 0.001. Data are means±SE (n = 3)."

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