Genome-wide Identification and Expression Analysis of CBL Gene Family in Glycyrrhiza uralensis

doi:10.13560/j.cnki.biotech.bull.1985.2020-1128

Abstract

Abstract:

CBL（calcineurin B-like proteins）,as the typical Ca²⁺ sensor protein in plants,plays important role in plant stress response and development. In order to discover the characteristics of the CBL family members of Glycyrrhiza uralensis and to investigate their functions,10 CBL genes were identified from the G. uralensis genome using bioinformatics method,designated as GuCBL1-GuCBL10,with the locations on 10 different scaffolds. Amino acid sequence alignment indicated that all CBL family members contained 4 EF-motifs that bound to calcium ion at the C-terminus,and some CBL members included myristoylation sites and palmitoylation sites at the N-terminus,which might be related to the subcellular localization of CBL protein. The phylogenetic tree analysis showed that the CBL gene family can be divided into 4 subfamilies,and each of the subfamilies contained Arabidopsis and soybean CBL gene family members,indicating that they had high homology. In the upstream promoter sequences of the G. uralensis CBL genes,there existed some cis-elements that responded to abiotic stresses and plant hormones,implying that the CBL family genes might have different biological functions. Tissue-specific expression analysis showed that GuCBL was diversely expressed in different tissues,with the highest accumulation in roots. qRT-PCR detection results indicated that,the CBL family genes showed different induced expressions under diverse stress treatments of salt,oxidation,drought,high temperature,and low temperature,respectively,suggesting the close associations between the expressions of CBL family genes and the responses to abiotic stress. All these results provide a reference for in-depth understanding the regulatory mechanisms of CBL genes in response to abiotic stress and for elucidating their functions in secondary metabolite accumulation.

Key words: Glycyrrhiza uralensis, CBL gene family, genome-wide identification, expression analysis, abiotic stress

GAO Ling, WANG Fei, XIE Shuang-quan, CHEN Xi-feng, SHEN Hai-tao, LI Hong-bin. Genome-wide Identification and Expression Analysis of CBL Gene Family in Glycyrrhiza uralensis[J]. Biotechnology Bulletin, 2021, 37(4): 18-27.

Figures/Tables 12

References 34

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引物名称Primer name	引物序列Primer sequence（5'-3'）	引物功能 Primer function
GuCBL1-F	CGAGCTTGACGTTGGGAGAG	GuCBL的RT-PCR检测 qRT-PCR detection of GuCBL
GuCBL1-R	CTGGCGAGGGCGACAATATC
GuCBL2-F	TTTGTTTGCTGATCGGGTGT
GuCBL2-R	ATTCCGCAAGCGTAGCAACT
GuCBL3-F	CCGATTCGTCAAAGCAACCT
GuCBL3-R	AGGATCCCATTGTGCTTTGTG
GuCBL4-F	GAGAGGCGGTTTGCGTATTG
GuCBL4-R	GCCTTTTTACGGTTCCTGGC
GuCBL5-F	TGTCAGTGAGGTTGAGGCATT
GuCBL5-R	CGCATTTGGGTGGAAGACAT
GuCBL6-F	GCAGTGCAGTTATTGACGATGG
GuCBL6-R	TGATGCACTGGGATGGAAGAC
GuCBL7-F	GCTGTGTGCGGTGTTCATTC
GuCBL7-R	GCGCAACGCCTCAATTTCAT
GuCBL8-F	ATGAGGATCCCACCGTTCTTG
GuCBL8-R	ACCCCATTGCGCTTGACATC
GuCBL9-F	TCCTTGCTTCTGAAACACCCT
GuCBL9-R	TGGCCATTGCGATTGACATC
GuCBL10-F	CAGCAACAGCACAGGCATTT
GuCBL10-R	CAGCAAGGCGAGCGAAATC
GuActin-F	CCTCAACCCAAAGGTCAACAG	内参基因 Internal reference gene
GuActin-R	GACCAGCGAGATCCAAACGAA	内参基因 Internal reference gene
GuCBL4-1	GAAATTGAAGCATTATATGATCTGT	全长基因克隆 Full-length gene cloning
GuCBL4-2	GGAGCCTATGGAAAAACGCCAGCAA
GuCBL7-1	ACTTCGTAGCTAGCACTCCG
GuCBL7-2	GACTGCATGATGGCGGAAAA
GuCBL10-1	AGCAACAGCACAGGCATTTCC
GuCBL10-2	CAGCCTCCGATTTGAAAACAAAACT

引物名称Primer name	引物序列Primer sequence（5'-3'）	引物功能 Primer function
GuCBL1-F	CGAGCTTGACGTTGGGAGAG	GuCBL的RT-PCR检测 qRT-PCR detection of GuCBL
GuCBL1-R	CTGGCGAGGGCGACAATATC
GuCBL2-F	TTTGTTTGCTGATCGGGTGT
GuCBL2-R	ATTCCGCAAGCGTAGCAACT
GuCBL3-F	CCGATTCGTCAAAGCAACCT
GuCBL3-R	AGGATCCCATTGTGCTTTGTG
GuCBL4-F	GAGAGGCGGTTTGCGTATTG
GuCBL4-R	GCCTTTTTACGGTTCCTGGC
GuCBL5-F	TGTCAGTGAGGTTGAGGCATT
GuCBL5-R	CGCATTTGGGTGGAAGACAT
GuCBL6-F	GCAGTGCAGTTATTGACGATGG
GuCBL6-R	TGATGCACTGGGATGGAAGAC
GuCBL7-F	GCTGTGTGCGGTGTTCATTC
GuCBL7-R	GCGCAACGCCTCAATTTCAT
GuCBL8-F	ATGAGGATCCCACCGTTCTTG
GuCBL8-R	ACCCCATTGCGCTTGACATC
GuCBL9-F	TCCTTGCTTCTGAAACACCCT
GuCBL9-R	TGGCCATTGCGATTGACATC
GuCBL10-F	CAGCAACAGCACAGGCATTT
GuCBL10-R	CAGCAAGGCGAGCGAAATC
GuActin-F	CCTCAACCCAAAGGTCAACAG	内参基因 Internal reference gene
GuActin-R	GACCAGCGAGATCCAAACGAA	内参基因 Internal reference gene
GuCBL4-1	GAAATTGAAGCATTATATGATCTGT	全长基因克隆 Full-length gene cloning
GuCBL4-2	GGAGCCTATGGAAAAACGCCAGCAA
GuCBL7-1	ACTTCGTAGCTAGCACTCCG
GuCBL7-2	GACTGCATGATGGCGGAAAA
GuCBL10-1	AGCAACAGCACAGGCATTTCC
GuCBL10-2	CAGCCTCCGATTTGAAAACAAAACT

基因名称 Gene name	染色体位置 Chromosome location	开放阅读框长度 Open reading frame sequence length/bp	氨基酸数目Amino acid number/aa	分子量Molecular weight/kD	等电点Isoelectric point	N-端氨基酸 N-terminal amino acid	棕榈酰化位点 Palmitoylation site	豆蔻酰化位点 Myristoylation site	亚细胞定位 Subcellular localization
GuCBL1	Scaffold00021（+）：357697-361866	828	275	31.68	4.99	MMIDMDFS	√	×	质膜 Plasma membrane
GuCBL2	Scaffold00027（+）：170105-174365	699	232	26.76	4.77	MVKRDIML	√	×	质膜 Plasma membrane
GuCBL3	Scaffold00030（-）：499662-505101	750	249	28.71	4.93	MPDSDSLF	√	×	质膜 Plasma membrane
GuCBL4	Scaffold00036（-）：366361-373219	1 008	331	37.63	9.31	WLIFQKLK	√	×	质膜 Plasma membrane
GuCBL5	Scaffold00067（+）：227402-231449	642	213	24.36	4.67	MGCFNSKA	√	√	质膜 Plasma membrane
GuCBL6	Scaffold00088（-）：140793-143426	681	226	25.72	4.57	MVQFLDEL	√	×	质膜 Plasma membrane
GuCBL7	Scaffold00219（-）：429-5651	861	364	41.46	5.33	MDFVASTP	√	×	质膜 Plasma membrane
GuCBL8	Scaffold00461（-）：93781-104251	651	216	24.87	4.76	MGCYCSTS	√	√	质膜 Plasma membrane
GuCBL9	Scaffold00669（-）：36499-39615	717	238	27.41	5.38	MGCRSSKV	√	√	质膜 Plasma membrane
GuCBL10	Scaffold01818（+）：19453-23753	735	269	31.13	5.35	MLQQQHRH	√	×	质膜 Plasma membrane

基因名称 Gene name	染色体位置 Chromosome location	开放阅读框长度 Open reading frame sequence length/bp	氨基酸数目Amino acid number/aa	分子量Molecular weight/kD	等电点Isoelectric point	N-端氨基酸 N-terminal amino acid	棕榈酰化位点 Palmitoylation site	豆蔻酰化位点 Myristoylation site	亚细胞定位 Subcellular localization
GuCBL1	Scaffold00021（+）：357697-361866	828	275	31.68	4.99	MMIDMDFS	√	×	质膜 Plasma membrane
GuCBL2	Scaffold00027（+）：170105-174365	699	232	26.76	4.77	MVKRDIML	√	×	质膜 Plasma membrane
GuCBL3	Scaffold00030（-）：499662-505101	750	249	28.71	4.93	MPDSDSLF	√	×	质膜 Plasma membrane
GuCBL4	Scaffold00036（-）：366361-373219	1 008	331	37.63	9.31	WLIFQKLK	√	×	质膜 Plasma membrane
GuCBL5	Scaffold00067（+）：227402-231449	642	213	24.36	4.67	MGCFNSKA	√	√	质膜 Plasma membrane
GuCBL6	Scaffold00088（-）：140793-143426	681	226	25.72	4.57	MVQFLDEL	√	×	质膜 Plasma membrane
GuCBL7	Scaffold00219（-）：429-5651	861	364	41.46	5.33	MDFVASTP	√	×	质膜 Plasma membrane
GuCBL8	Scaffold00461（-）：93781-104251	651	216	24.87	4.76	MGCYCSTS	√	√	质膜 Plasma membrane
GuCBL9	Scaffold00669（-）：36499-39615	717	238	27.41	5.38	MGCRSSKV	√	√	质膜 Plasma membrane
GuCBL10	Scaffold01818（+）：19453-23753	735	269	31.13	5.35	MLQQQHRH	√	×	质膜 Plasma membrane

同源基因 Paralogous genes	Ka	Ks	Ka/Ks	选择压 Selective pressure
GuCBL1-GuCBL7	0.2127	0.1996	1.0656	Positive selection
GuCBL3-GuCBL2	0.1968	0.1674	1.1756	Positive selection