Crops ›› 2020, Vol. 36 ›› Issue (5): 23-32.doi: 10.16035/j.issn.1001-7283.2020.05.004

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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 E-mail:wangzhilong7661@163.com;306214803@qq.com

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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

Table 1

Basic information of LEC2 protein sequences from various species"

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

Table 2

Specific primers of CsLEC2 genes"

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

Table 3

Number of predicted genes in C. sativa database"

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

Table 4

Physicochemical properties of CsLEC2 family proteins from C. sativa"

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)

Fig.1

Gene structures of CsLEC2 family from C. sativa"

Fig.2

Analysis of functional domains of CsLEC2 family protein of C. sativa"

Fig.3

Comparison of secondary structure of CsLEC2 protein from C. sativa"

Fig.4

Prediction of the tertiary structure of CSLEC2 family protein in C. sativa"

Fig.5

Multiple sequence alignment of CsLEC2 family proteins of C. sativa"

Fig.6

Phylogenetic tree and conserved motifs of LEC2 proteins from different species"

Table 5

Conservative motif information"

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

Table 6

Cis-acting elements in the upstream promoter of CsLEC2"

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

Fig.7

RT-PCR results of CsLEC2 expressions in various tissues and different developmental stages seeds of C. sativa"

Fig.8

Expression analysis of CsLEC2 at different seed developmental stages of C. sativa Different lowercase letters indicate significantly difference at P < 0.05"

Fig.9

FPKM variation trend of genes at different seed developmental stages of C. sativa"

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