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作物杂志,2020, 第5期: 23–32 doi: 10.16035/j.issn.1001-7283.2020.05.004

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

亚麻荠油脂相关转录因子CsLEC2基因家族的鉴定及表达分析

王志龙1(), 薛应红1, 郝月茹1, 刘宝玲1, 苑丽霞2, 薛金爱1(), 李润植1   

  1. 1山西农业大学分子农业与生物能源研究所,030801,山西太谷
    2晋中学院生物科学与技术学院,030600,山西榆次
  • 收稿日期:2020-05-11 修回日期:2020-07-21 出版日期:2020-10-15 发布日期:2020-10-12
  • 通讯作者: 薛金爱
  • 作者简介:王志龙,研究方向为生物技术与组学工程,E-mail: wangzhilong7661@163.com
  • 基金资助:
    国家自然科学基金(31801400);山西省重点研发项目(201703D221002-3);203省1331成果转化培育项目;晋中市科技重点研究项目(Y182009);晋中市科技重点研究项目(31401430);晋中市重点创新团队的“1331项目”(jzxycxtd2019009)

Identification and Expression Analysis of Oil-Related Transcription actor CsLEC2 Gene Family in Camelina sativa (L.) Crantz

Wang Zhilong1(), Xue Yinghong1, Hao Yueru1, Liu Baoling1, Yuan Lixia2, Xue Jin'ai1(), Li Runzhi1   

  1. 1Institute of Molecular Agriculture and Bioenergy, Shanxi Agricultural University, Taigu 030801, Shanxi, China
    2College of Biological Science and Technology, Jinzhong University, Yuci 030600, Shanxi, China
  • Received:2020-05-11 Revised:2020-07-21 Online:2020-10-15 Published:2020-10-12
  • Contact: Xue Jin'ai

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摘要：

对亚麻荠CsLEC2家族进行全基因组鉴定、半定量RT-PCR、qRT-PCR,检测CsLEC2基因的时空表达特性并通过转录组数据预测CsLEC2下游靶基因,以解析CsLEC2调控亚麻荠种子油脂合成和积累等生物学功能。结果表明,在亚麻荠基因组中共鉴定到3个亚麻荠CsLEC2基因（CsLEC2.1、CsLEC2.2和CsLEC2.3）。蛋白理化性质和高级结构分析显示,亚麻荠CsLEC2蛋白具有与拟南芥AtLEC2相似的理化性质,且其二级结构的主体也相似。系统进化分析表明,亚麻荠CsLEC2蛋白与模式植物拟南芥AtLEC2蛋白及拟南芥琴亚种AlLEC2蛋白亲缘关系最近。qRT-PCR结果显示亚麻荠CsLEC2在未成熟的种子中高表达。转录组数据分析显示下游基因CsWRI1和CsOLE3高表达,推测CsLEC2可能直接上调CsWRI1和CsOLE3的转录表达,参与油脂代谢调控。

关键词: 亚麻荠, LEC2基因家族, 表达分析, 油脂生物合成

Abstract:

We conducted the genome-wide identification of CsLEC2 gene family and examined spatiotemporal expression patterns of CsLEC2 genes and their downstream target genes by semi-quantitative RT-PCR and qRT-PCR, predicted the downstream target genes of CsLEC2 using transcriptomic data, aiming to analyze the biological functions of CsLEC2 in regulating oil synthesis and accumulation in Camelina sativa seeds. The results showed that CsLEC2.1, CsLEC2.2 and CsLEC2.3 were identified in the genome of C.sativa. Protein physicochemical properties and advanced structural analysis indicated that the C.sativa CsLEC2 protein had similar physicochemical properties and similar secondary structure to Arabidopsis thaliana AtLEC2. Phylogenetic analysis revealed that CsLEC2 protein had the closest genetic relationship with AtLEC2 protein and AlLEC2 protein. qRT-PCR revealed that CsLEC2 only expressed in seeds and had a high expression in immature seeds. The downstream genes CsWRI1 and CsOLE3 were highly expressed, showed CsLEC2 might directly up-regulate the transcriptional expression of CsWRI1 and CsOLE3 involving in oil metabolism regulation.

Key words: Camelina sativa, LEC2 gene family, Expression analysis, Oil biosynthesis

表1

各物种的LEC2蛋白序列基本信息

物种Species 名称
Name		 NCBI编号
NCBI number
木薯Manihot esculenta MeLEC2 XP_021598372.1
麻风树Jatropha curcas JcLEC2 XP_012092409.1
蓖麻Ricinus communis RcLEC2 NP_001310615.1
毛果杨Populus trichocarpa PtLEC2 XP_024454872.1
银中杨Populus alba PaLEC2 TKR58082.1
可可Theobroma cacao TcLEC2 XP_017978733.1
克莱门柚Citrus clementina CcLEC2 XP_024046749.1
杨梅Morella rubra MrLEC2 KAB1221959.1
欧洲栓皮栎Quercus suber QsLEC2 XP_023881426.1
橡树Quercus lobata QlLEC2 XP_030973220.1
山嵛菜Eutrema salsugineum EsLEC2 XP_006415675.1
拟南芥琴亚种Arabidopsis lyrata subsp. AlLEC2 XP_020866568.1
拟南芥Arabidopsis thaliana AtLEC2 NP_564304.1

表2

CsLEC2基因的特异性引物

基因名称Gene name 引物(5′-3′) Primer (5′-3′)
CsActin F: TTGGAAGGATCTGTACGGTAAC
R: TGTGAACGATTCCTGGACC
CsLEC2.1 F: CCTCTTCTAACGCAAACTCTGTCCA
R: GTTGACGAAATGAGTAGGCTACGAA
CsLEC2.2 F: ATGGATGCTAACAACAATCTCTCGC
R: TGTTTGGCCTTCACTCAAGACAAGA
CsLEC2.3 F: AGTGAACGAGAGGAACCA
R: CGGCTTGATAATGCTGATG

表3

预测基因在亚麻荠数据库中的编号

基因名称Gene name 亚麻荠数据库编号C. sativa database number
CsWRI1 Csa06g028810.1
CsFUS3 Csa04g012470.1
CsABI3-1 Csa15g050420.1
CsABI3-2 Csa19g036630.1
CsABI3-3 Csa01g030760.1
CsOLE1-1 Csa12g028090.1
CsOLE1-2 Csa11g019460.1
CsOLE2-1 Csa11g057650.1
CsOLE2-2 Csa10g047190.1
CsOLE2-3 Csa09048s010.1
CsOLE3-1 Csa02g041750.1
CsOLE3-2 Csa18g022020.1
CsOLE3-3 Csa11g082710.1

表4

亚麻荠CsLEC2家族蛋白理化性质

分析内容Analysis content AtLEC2 CsLEC2.1 CsLEC2.2 CsLEC2.3
数据库编号Database code At1g28300.1 Csa03g031590.1 Csa14g035910.1 Csa17g039140.1
NCBI编号NCBI code NP_564304.1 XP_010499344.1 XP_010460616.1 XP_010478197.1
开放阅读框Open read frame (bp) 1092 1092 1092 1044
染色体位置Location on chromosome
 Chr1:9896566-
9900177		 Chr3:13175576-
13178955		 Chr14:14188382-
14191779		 Chr17:14245237-
14248478
蛋白长度The number of amino acids coding protein 363 363 363 347
相对分子量Relative molecular weight (kDa) 41.71 41.22 41.21 39.34
理论等电点Theoretical (pI) 5.21 5.54 5.60 5.46
碱性氨基酸数Basic amino acid number 36 34 35 36
酸性氨基酸数Acid amino acid number 50 46 45 45
亲水性指数Hydropathy index -0.818 -0.556 -0.654 -0.600
氨基酸Amino acid (%) Ser (10.2) Ser (9.6) Ser (9.6) Ser (10.4)
Asn (9.1) Asn (8.5) Asn (8.5) Asn (8.9)
Leu (7.4) Leu (8.5) Leu (8.5) Leu (8.4)

图1

亚麻荠CsLEC2家族的基因结构

图2

亚麻荠CsLEC2家族蛋白功能结构域的分析

图3

亚麻荠CsLEC2蛋白二级结构

图4

亚麻荠CsLEC2家族蛋白三级结构预测

图5

亚麻荠CsLEC2家族蛋白多序列比对

图6

不同物种LEC2蛋白的系统进化树和保守基序

表5

保守基序信息

Motif编号Motif number 氨基酸残基Amino acid residue
motif 1 QSWSFKYKFWSNNKSRMYVLENTGEFVKQNGAEIGDFLTIYEDESKNLYF
motif 2 YKFCTPDNKRLRVLLRKELKNSDVGSLGRIVLPKREAEGNLPTLSDKEGI
motif 3 RRALDAYKTKVARCKRKLARQRSL
motif 4 RDEEEASLALLIEQLRHKEQQ
motif 5 FFPFSSSNANSVQEFAMDANNNLSHLTTMPTYDHHQAEPHH
motif 6 YIDDCYSGLDVLPDVNRYNF
motif 7 YSSDAYPQIPVSQTGSEFCSLVSNPNPCL
motif 8 SSPNSSPDEVVDSKRQVMMLNMKNNVQIP
motif 9 PNDLMGLTIDHQHHQ
motif 10 SVDYAHVGSLDDQVSFDDIVW

表6

CsLEC2基因上游启动子顺式元件

顺式作用元件Cis-acting element 核心序列Core sequence 功能Function
CAAT-box CAAT 启动子和增强子区域常见的顺式作用元件
TATA-box TATA 转录起始位点上游-30bp附近核心启动子元件
ARE AAACCA 厌氧诱导必需的调节元素
Box 4 ATTAAT 涉及光响应的保守DNA模块
CGTCA-motif CGTCA MeJa响应的相关顺式作用调控元件
TGACG-motif TGACG MeJa响应的相关顺式作用调控元件
MBSI aaaAaaC(G/C)GTTA MYB结合位点,参与类黄酮生物合成基因的调控
MYB CAACCA MYB结合元件
TGA-element AACGAC 生长素响应元件
ERE ATTTCATA 雌激素应答元件
as-1 TGACG 根特异表达元件

图7

亚麻荠CsLEC2在不同组织和种子不同发育时期的RT-PCR结果

图8

CsLEC2基因在种子不同发育时期的表达分析 不同小写字母表示在P<0.05水平上差异显著

图9

基因在亚麻荠种子不同发育时期的FPKM变化趋势

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