乌拉尔甘草CBL基因家族的鉴定与表达分析

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

摘要/Abstract

摘要：

钙依赖磷酸酶B类似蛋白（Calcineurin B-like proteins,CBL）是植物特有的一类Ca²⁺传感蛋白,在植物逆境应答及发育过程中发挥重要作用。为挖掘乌拉尔甘草CBL家族成员特征并预测CBL功能,利用生物信息学方法从乌拉尔甘草基因组中鉴定出10个CBL基因,命名为GuCBL1-GuCBL10,分别定位在10个不同的支架上。氨基酸序列比对发现,CBL家族成员在C-末端都含有4个与钙离子结合的EF-motif,部分CBL成员N端含有肉豆蔻酰化位点及棕榈酰化位点,预测可能与CBL蛋白的亚细胞定位有关。进化树分析显示,乌拉尔甘草CBL基因家族可分为4个亚家族,且各亚族中都包含拟南芥和大豆的CBL基因家族成员,说明它们具有高同源性。在乌拉尔甘草CBL基因的上游序列中,存在一些应答多种激素和逆境的顺式元件,预测CBL基因家族可能具有不同的生物学功能。组织表达分析表明,GuCBL在乌拉尔甘草根、茎和叶片中均有表达,在根中的表达量最高。荧光定量分析显示,在盐、氧化、干旱、高温和低温胁迫下,CBL家族基因受到不同程度的诱导,说明乌拉尔甘草CBL家族基因的表达与逆境胁迫响应之间的密切联系。该研究结果为深入理解CBL基因响应非生物胁迫的调控机制和解析其参与次生代谢物质累积的功能奠定基础。

关键词: 乌拉尔甘草, CBL, 基因家族鉴定, 表达分析, 非生物胁迫

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

高玲, 王斐, 谢双全, 陈喜凤, 沈海涛, 李鸿彬. 乌拉尔甘草CBL基因家族的鉴定与表达分析[J]. 生物技术通报, 2021, 37(4): 18-27.

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.

图/表 12

表1 所使用的引物

Table 1 Primers used in this study

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

表2 乌拉尔甘草CBL基因家族特征分析

Table 2 Characteristic analysis of G. uralensis CBL family genes

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

图1 乌拉尔甘草GuCBL在染色体支架上的分布 GuCBL位于10个单独的支架上,黑线条表示片段重复基因对,各染色体支架以不同颜色的圆形显示

Fig. 1 Chromosomal scaffold distribution of GuCBL genes in G. uralensis GuCBL genes are located on 10 individual scaffolds. Pairs of segmentally duplicated genes are indicated by black lines. Chromosomal scaffolds are showed with different colored circles

图2 CBL家族的系统发育关系分析红色、绿色和蓝色分别表示乌拉尔甘草、拟南芥和大豆的 CBL 蛋白

Fig. 2 Phylogenetic relationship analysis of the CBL family The CBL proteins of G. uralensis, A. thaliana, and G. max were indicated in red, green, and blue, respectively

图3 乌拉尔甘草CBL家族基因基因结构和保守基序分析 a：GuCBL 基因的外显子/内含子结构分析。绿框和黑线分别代表外显子和内含子,基因组长度在底部标示。b ：GuCBL 蛋白的保守基序分析

Fig. 3 Conserved motif and gene structure analyses of CBL family genes in G. uralensis a: Exon/intron structure analysis of GuCBL genes. Green boxes and black lines represent the exons and introns respectively, and the genomic length is indicated at the bottom. b: Conserved motif analysis of GuCBL proteins

图4 乌拉尔甘草 CBL的多序列比对和保守结构域分析 EF-hand、PFPF-motif、豆蔻酰化位点和 PFPF结构域内的丝氨酸残基分别用红色线框标注

Fig. 4 Multiple alignment and conserved motif analysis of CBL in G. uralensis EF-hand, PFPF-motif, myristoylation site, and serine residue of PFPF-motif are showed in red boxes

图5 乌拉尔甘草、大豆和拟南芥CBL基因的共线性分析蓝色、红色、紫色线条分别表示乌拉尔甘草重复 CBL 基因对、乌拉尔甘草CBL 基因与拟南芥和大豆 CBL 基因之间的共线关系、及大豆和拟南芥之间或内部的 CBL 基因对

Fig. 5 Syntenic analysis of CBL genes in G. uralensis, G. max, and A. thaliana Blue, red, and purple lines represent duplicated CBL pairs in G. uralensis, syntenic relationships of CBL genes between G. uralensis and the other two species of G. max and A. thaliana, and CBL pairs between or inside G. max and A. thaliana

表3 乌拉尔甘草同源CBL基因的Ka/Ks比值

Table 3 Ka/Ks ratios for homologous CBL genes in G. uralensis

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

表4 乌拉尔甘草中同源CBL基因对的田岛相对进化速率分析a

Table 4 Tajima relative rate tests of homologous CBL gene pairs in G. uralensisa

测试组 Testing group	测试值Testing value
测试组 Testing group	Mt^b	M1^c	M2^d	Χ²	P^e
GuCBL1/GuCBL7 with GmCBL7	174	16	39	9.62	0.00193
GuCBL3/GuCBL2 with GmCBL6	236	19	5	8.27	0.00026

图6 GuCBL启动子中的顺式作用元件分布

Fig. 6 Distribution of cis-elements in GuCBL promoter regions

图7 乌拉尔甘草GuCBL的组织特异性表达分析

Fig. 7 Tissue-specific expression analysis of G. uralensis GuCBL genes

图8 乌拉尔甘 GuCBL响应非生物胁迫的表达分析

Fig. 8 Expression analysis of G. uralensis GuCBL genes in response to abiotic stress

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