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Acta Agronomica Sinica ›› 2019, Vol. 45 ›› Issue (7): 993-1001.doi: 10.3724/SP.J.1006.2019.84122

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

Transcriptome analysis of the peanut transgenic offspring with depressing AhPEPC1 gene

PAN Li-Juan1,CHEN Na1,Ming-CHEN Na1,WANG Tong1,WANG Mian1,CHEN Jing1,YANG Zhen1,WAN Yong-Shan2,YU Shan-Lin1,CHI Xiao-Yuan1,*(),LIU Feng-Zhen2,*()   

  1. 1 Shandong Peanut Research Institute, Qingdao 266100, Shandong, China
    2 College of Agronomy, Shandong Agricultural University / National Key Laboratory of Crop Biology, Tai’an 271018, Shandong, China;
  • Received:2018-09-20 Accepted:2019-01-19 Online:2019-07-12 Published:2019-03-22
  • Contact: Xiao-Yuan CHI,Feng-Zhen LIU E-mail:chi000@126.com;liufz@sdau.edu.cn
  • Supported by:
    This study was supported by the Grants from the National Ten Thousand Youth Talents Plan of 2014(W02070268);the China Agriculture Research System(CARS-13);the National Natural Science Foundation of China(31701464);the Natural Science Foundation of Shandong Province(ZR2017YL017);the Natural Science Foundation of Shandong Province(ZR2016CM07);the Youth Scientific Research Foundation of Shandong Academy of Agricultural Sciences(2016YQN14);Young Talents Training Program of Shandong Academy of Agricultural Sciences;Agricultural Scientific and Technological Innovation Project of Shandong Academy of Agricultural Sciences(CXGC2016B02);Agricultural Scientific and Technological Innovation Project of Shandong Academy of Agricultural Sciences(CXGC2018E21);the Basic Research Project of Qingdao(17-1-1-51-jch);Improved Variety Engineering Project of Shandong Province(2017LZGC003);Taishan Scholars Project.

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Abstract:

Phosphoenolpyruvate carboxylase is considered as a key enzyme to control the ratio of protein to lipid of oilseeds. In this study, the antisense expression of the peanut PEPC isoform 1 (AhPEPC1) gene increased the lipid content by 5.7%-10.3% compared with the non-transgenic control. The high-throughput sequencing technology — RNA-Seq was used to analyze whether the inhibitory expression of AhPEPC1 gene in peanut affected the function of other genes. The results showed that 110 genes were differentially expressed, of which 25 genes were up-regulated and 85 genes were down-regulated. KEGG enrichment analysis was performed on 110 differentially expressed genes, among which 34 genes were successfully obtained KEGG annotation, and two genes (Aradu.M0JX8 and Aradu.FE0Z7) were down-regulated in the biosynthesis pathway of amino acids. Fifteen DEGs between non-transgenic control and transgenic peanut seeds were selected to analyze the gene expression levels using qRT-PCR. The results of qRT-PCR agreed well with most findings from RNA-seq analysis. This research might resolve to some extent the molecular mechanisms of AhPEPC1 gene regulating oil content of peanut seeds.

Key words: peanut, AhPEPC1 gene, transgenic, transcriptome analysis

Table 1

Primers used for real time PCR analysis"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 引物名称
Primer name		 引物序列
Primer sequence (5°-3°)
Aradu.2N2A3-F TTCATTCATCCCCCTTTTCAG Aradu.CZ64T-F GGAGTAAACGACCCGATAAGC
Aradu.2N2A3-R GGCTGGTTTTGGTTGCTCC Aradu.CZ64T-R GTCCTACGATGCCCGAGAACG
Aradu.32P5Q-F TTGTTTTTGGGTCGGTTGA Aradu.FE0Z7-F TTTTCTCCGCACTCACCTAA
Aradu.32P5Q-R TTAGGTGTTGTGTAGGGTG Aradu.FE0Z7-R TGCCGACTCTTCTTCACCTT
Aradu.38MP3-F TGATTGGTGTGGTGTGTTGC Aradu.G395P-F CGTTCTTGGTGTCCCTGTG
Aradu.38MP3-R CTCCTTATGTGTTCGTGTTTTG Aradu.G395P-R TTCCTTGTGCTTGAGCCTT
Aradu.3UA04-F AGTGAAAATAAGGGAAAGAC Aradu.JE7IW-F GCAATTTGTCCTGATGACCCTA
Aradu.3UA04-R CAACTGAAACTAAACGGGAT Aradu.JE7IW-R CCTCCACTGTGCTCCTCCCT
Aradu.8XK1K-F CTCAAGCGTTCTGCATTGCC Aradu.M0JX8-F CCATTCATGTCTTCGTCGT
Aradu.8XK1K-R CCCTGAAGAGTTTTCCCCCA Aradu.M0JX8-R CTCTTCTTCACCTTCTGCG
Aradu.91JGK-F ATTCAACGAGTTATCCACGG Aradu.PU7RJ-F AGAGGAAGGGTTATGCATT
Aradu.91JGK-R GGATTCCACAACATCAGCAG Aradu.PU7RJ-R AACTTTCGATTTGGGGCTG
Aradu.94LL9-F ATTGAAAGTGGTGGCGAAGT Aradu.Y0LYZ-F GGTGGTGTCACTGCTGGTA
Aradu.94LL9-R TTGACGGAATGGGTGAGG Aradu.Y0LYZ-R GAACTCGTGGCATTGTCC
Aradu.AAS86-F TGGGCTTCCAGGAGTTACAGG β-actin-F TTGGAAT GGGTCAGAAGGATGC
Aradu.AAS86-R GTTCTTCCAGTGTCCACAGTGT β-actin-R AGTGGTGCCTCAGTAAGAAGC

Fig. 1

Contents of lipid (A), protein (B), and fatty acid (C) in transgenic and control plants WT: control plants; L6, L11, L21: transgenic plants. Bars superscripted by different letters are significant different at P = 0.05."

Fig. 2

Venn diagram of the gene expression WT: control plants; TL: transgenic plants. The sum of the numbers in each large circle represents the total number of genes expressed in the group, the overlapping circles represent common genes between groups."

Fig. 3

Volcano plot of differentially expressed gene distribution The screening standard of differential genes is Padj < 0.05. Red dots indicate the up-regulated genes with significant differences, green dots indicate the down regulated genes with significant differences, no significant difference in the expression of genes with blue dots."

Fig. 4

GO annotation of differentially expressed genes The ordinate is the enriched GO term, the abscissa is the number of differential genes in term. The green represents the biological process, the orange represents the cell component, and the purple represents the molecular function."

Table 2

KEGG enrichment of the differentially expressed genes"

条目
Term		 数据库
Database		 编号
ID		 差异基因个数
Sample number		 调控模式Regulation 注释基因个数
Background number		 显著水平
P-value		 矫正显著水平
Corrected P-value
Photosynthesis - antenna proteins KEGG PATHWAY adu00196 2 Up 18 0.000093 0.000560
Purine metabolism KEGG PATHWAY adu00230 2 Up 194 0.008769 0.026306
DNA replication KEGG PATHWAY adu03030 1 Up 90 0.065891 0.116645
Peroxisome KEGG PATHWAY adu04146 1 Up 107 0.077763 0.116645
Pyrimidine metabolism KEGG PATHWAY adu00240 1 Up 140 0.100427 0.120512
Metabolic pathways KEGG PATHWAY adu01100 2 Up 2197 0.508576 0.508576
Selenocompound metabolism KEGG PATHWAY adu00450 2 Down 20 0.000926 0.015741
Cysteine and methionine metabolism KEGG PATHWAY adu00270 2 Down 108 0.021504 0.182780
Stilbenoid, diarylheptanoid and gingerol biosynthesis KEGG PATHWAY adu00945 1 Down 18 0.038622 0.218860
Base excision repair KEGG PATHWAY adu03410 1 Down 46 0.092940 0.284623
Biosynthesis of amino acids KEGG PATHWAY adu01230 2 Down 250 0.095036 0.284623
Phenylalanine metabolism KEGG PATHWAY adu00360 1 Down 50 0.100455 0.284623
Flavonoid biosynthesis KEGG PATHWAY adu00941 1 Down 62 0.122643 0.297848
RNA degradation KEGG PATHWAY adu03018 1 Down 111 0.207912 0.441813
Pyrimidine metabolism KEGG PATHWAY adu00240 1 Down 140 0.254567 0.471897
RNA transport KEGG PATHWAY adu03013 1 Down 166 0.294144 0.471897
Plant-pathogen interaction KEGG PATHWAY adu04626 1 Down 182 0.317487 0.471897
Phenylpropanoid biosynthesis KEGG PATHWAY adu00940 1 Down 193 0.333104 0.471897
Endocytosis KEGG PATHWAY adu04144 1 Down 217 0.365995 0.478609
Plant hormone signal transduction KEGG PATHWAY adu04075 1 Down 314 0.483750 0.543526
Biosynthesis of secondary metabolites KEGG PATHWAY adu01110 3 Down 1282 0.505639 0.543526
Ribosome KEGG PATHWAY adu03010 1 Down 340 0.511554 0.543526
Metabolic pathways KEGG PATHWAY adu01100 4 Down 2197 0.693074 0.693074

Fig. 5

Transcriptional changes in biosynthesis of amino acids pathways"

Fig. 6

Relative expression of genes (MOJX8, FEO27) in the biosynthesis of amino acids"

Table 3

Differental fold-change between RNA-seq and RT-PCR"

基因编号
Gene ID		 基因描述
Gene description		 RNA-seq变化倍数
RNA-seq fold change		 调控模式
Regulation		 RT-PCR变化倍数
RT-PCR fold change
Aradu.2N2A3 Protein IQ-DOMAIN 14 53.7 Up 2.2
Aradu.32P5Q Transketolase, chloroplastic 164.3 Down 2.1
Aradu.38MP3 — 61.8 Up 1.7
Aradu.3UA04 Pollen-specific protein SF21 11.2 Down 16.8
Aradu.8XK1K DNA polymerase alpha catalytic subunit 2.3 Up 1.1
Aradu.91JGK Probable leucine-rich repeat receptor-like serine/threonine-protein kinase 10.5 Down 1.6
Aradu.94LL9 F-box/FBD/LRR-repeat protein 4.7 Up 1.9
Aradu.AAS86 ABC transporter G family member 36 2.4 Up 3.0
Aradu.CZ64T Protein FAR1-RELATED SEQUENCE 12 5.2 Down 2.4
Aradu.FE0Z7 Cystathionine beta-lyase, chloroplastic 137.4 Down 1.4
Aradu.G395P Copper-transporting ATPase PAA1, chloroplastic 30.4 Down 1.5
Aradu.JE7IW Pollen-specific protein SF21 25.1 Down 2.9
Aradu.M0JX8 Cystathionine beta-lyase, chloroplastic 201.1 Down 2.0
Aradu.PU7RJ MADS-box transcription factor 6 2.9 Down 3.4
Aradu.Y0LYZ Myb-like protein 2.0 Up 2.4
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