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作物学报 ›› 2019, Vol. 45 ›› Issue (7): 993-1001.doi: 10.3724/SP.J.1006.2019.84122

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

花生AhPEPC1基因抑制表达的转基因后代转录组分析

潘丽娟1,陈娜1,陈明娜1,王通1,王冕1,陈静1,杨珍1,万勇善2,禹山林1,迟晓元1,*(),刘风珍2,*()   

  1. 1 山东省花生研究所, 山东青岛 266100
    2 山东农业大学农学院/作物生物学国家重点实验室, 山东泰安 271018
  • 收稿日期:2018-09-20 接受日期:2019-01-19 出版日期:2019-07-12 网络出版日期:2019-03-22
  • 通讯作者: 迟晓元,刘风珍
  • 作者简介:E-mail: panlijuan_2008@aliyun.com
  • 基金资助:
    本研究由2014年国家“万人计划”青年拔尖人才(W02070268);国家现代农业产业技术体系建设专项(CARS-13);国家自然科学基金项目(31701464);山东省自然科学基金项目(ZR2017YL017);山东省自然科学基金项目(ZR2016CM07);山东省农业科学院青年科研基金项目(2016YQN14);山东省农业科学院青年英才培养计划;山东省农业科学院农业科技创新工程(CXGC2016B02);山东省农业科学院农业科技创新工程(CXGC2018E21);青岛市应用研究专项青年专项(17-1-1-51-jch);山东省良种工程(2017LZGC003);泰山学者工程专项经费资助

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 Published:2019-07-12 Published online:2019-03-22
  • Contact: Xiao-Yuan CHI,Feng-Zhen LIU
  • 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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摘要：

磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvate carboxylase, PEPC)是控制油料作物种子中蛋白质/油脂含量比率的一个关键酶。本研究检测了花生AhPEPC1基因抑制表达的转基因株系种子含油量, 与非转基因花生相比, 转基因花生种子含油量提高了5.7%~10.3%。利用转录组测序(RNA-Seq)技术分析花生中AhPEPC1基因的抑制表达是否影响其他基因的功能。结果表明, 转录组分析筛选到110个基因差异表达, 其中25个基因上调表达, 85个基因表达下调。对110个差异表达基因进行了KEGG富集分析, 其中有34个基因成功获得了KEGG注释, 发现氨基酸的生物合成途径中有2个基因(Aradu.M0JX8, Aradu.FE0Z7)下调表达。利用荧光定量PCR分析了15个DEG (differential expressed gene)在非转基因对照和转基因花生种子中的表达情况, 发现其趋势与转录组测序结果基本一致。研究结果可在一定程度上解析AhPEPC1基因调控花生种子含油量的分子机制。

关键词: 花生, AhPEPC1基因, 转基因, 转录组分析

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

表1

荧光定量PCR的引物序列"

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

图1

转基因株系和对照花生种子含油量(A)、蛋白含量(B)和脂肪酸含量(C) WT: 对照; L6、L11、L21: 转基因株系。a、b、c、d的柱值在处理间差异显著(P = 0.05)。"

图2

基因表达维恩图 WT: 对照; TL: 转基因株系。每个大圆圈中的数字之和代表该group表达了的基因总个数, 圆圈交叠的部分表示group之间共有的表达基因。"

图3

差异基因火山图 Padj < 0.05为差异基因筛选标准。有显著性差异表达的基因用红色点(上调)和绿色点(下调)表示, 无显著性差异表达的基因用蓝色点表示。"

图4

GO富集分类图 纵坐标为富集的GO term, 横坐标为该term中差异基因个数。绿色表示生物过程, 橙色表示细胞组分, 紫色表示分子功能。"

表 2

差异基因KEGG富集情况"

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

图5

氨基酸生物合成途径的转录变化"

图6

氨基酸合成途径中基因相对表达量"

表3

RNA-seq与RT-PCR验证倍数差别"

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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