摘要: 目的探究枣类钙调蛋白（ZjCML）在抗寒性中的重要作用，旨在挖掘关键抗寒 ZjCML基因，为进一步利用其进行枣抗寒育种提供理论参考、�/sec> 方法本研究利用生物信息学技术，全面分析了枣 CML基因家族成员的数目及相关结构特征，并利用RNA-seq和实时荧光定量PCR技术分析其响应低温胁迫时的表达模式，为筛选关键抗寒基因奠定基础、�/sec> 结果在枣基因组数据中共鉴定到23�?i>ZjCML基因，不均匀地分布在12条染色体上。与拟南芥CMLs蛋白构建进化树分析发现ZjCMLs可被分为12个类群。蛋白网络互作预测发�?6个ZjCMLs存在于该网络中，且具有一定的互作关系。RNA-seq结果表明 ZjCML13咋�i>ZjCML6基因表达模式在‘冬枣’及其同源四倍体响应低温胁迫中具有显著差异，其中 ZjCML13在‘冬枣’响应低温胁�?�?4 h时极显著上调表达，而在‘冬枣’同源四倍体中表达差异不显著。外源CaCl ₂和褪黑素处理后， ZjCML13在‘冬枣’同源四倍体响应低温胁迫6�?4 h时极显著上调表达，说明其可能通过诱导 ZjCML13表达调控‘冬枣’同源四倍体抗寒性、�/sec> 结论枢�i>CML基因家族具有特定的结构特征及响应低温胁迫＋�i>ZjCML13可能在调控‘冬枣’及其同源四倍体抗寒性差异中发挥重要作用、�/sec>

关键诌�
• 枢�/a> /
• 类钙调蛋白（CML（�/a> /
• 基因家族/
• 抗寒�?/a> /
• 表达分析

Abstract: ObjectiveThis paper aims to investigate the important founction of CaM-like proteins in Chinese jujube in resistance to cold stress and identify the key ZjCML, which could provide theoretical basis for cold breeding in Chinese jujube by using them. MethodThrough using bioinformatics technology, the number and related structural information of CMLin Chinese jujube were analyzed comprehensively in the current study. And then, we used RNA-seq and real-time fluorescence quantitative PCR technology to analyze its expression pattern in response to low temperature stress, laying a foundation for screening key cold resistance genes. ResultA total of 23 ZjCMLgenes were identified in Chinese jujube genome. All of them distributed unevenly on 12 chromosomes. In addition, ZjCMLcan be divided into 12 subgroups, and protein interaction network prediction showed that 16 ZjCML existed in the network and they had mutual interaction relationships. Moreover, RNA-seq analysis showed that the expression patterns of ZjCML13 and ZjCML6 in response to cold stress between ‘Dongzao� and its autotetraploids were significantly different. The expression level of ZjCML13 in response to cold stress in ‘Dongzao� was significantly higher than that in its autotetraploid at 6 and 24 h. External CaCl ₂and melatonin treatments could significantly induce the expression of ZjCML13 in autotetraploids ‘Dongzao� under cold stress at 6 and 24 h, indicating that it may regulate cold resistance of autotetraploid ‘Dongzao� by inducing the expression of ZjCML13. ConclusionThe ZjCMLgene family have specific characteristics and expression patterns, ZjCML13 might play important role in regulation of cold difference between ‘Dongzao� and its autotetraploids.

Key words:
• Chinese jujube/
• CaM-like protein (CML)/
• gene family/
• cold resistance/
• expression analysis

�?nbsp; 1枢�i>CML基因在染色体上的定位

Figure 1.Position ofCMLgenes on the chromosomes of Chinese jujube

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�?nbsp; 2枣CML的保守基序分枏�/p>

Figure 2.Analysis of the conserved motifs of CMLs in Chinese jujube

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�?nbsp; 3枢�i>CML家族基因结构

Figure 3.Gene structure ofCMLfamily in Chinese jujube

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�?nbsp; 4枣和拟南芥CML蛋白系统进化栐�/p>

Figure 4.Phylogenetic tree of CML protein in Chinese jujube andArabidopsis thaliana

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�?nbsp; 5枣CML蛋白网络互作预测分析

Figure 5.Putative interaction network of CML protein in Chinese jujube

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�?nbsp; 6ZjCML基因在‘冬枣’及其同源四倍体响应低温胁迫时的RNA-seq热图

Figure 6.Heat map ofZjCMLs expressing profiles in response to cold stress between ‘Dongzao� and its autotetraploids from RNA-seq data

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�?nbsp; 7ZjCML6咋�i>ZjCML13基因在‘冬枣’及其同源四倍体中响应低温胁迫时qRT-PCR表达模式分析

*表示�?i>P< 0.05水平上有显著性差异；**表示�?i>P< 0.01水平上有极显著性差异。下同�? represents significant difference atP< 0.05 level; ** represents extremely significant difference atP< 0.01 level. The same below.

Figure 7.Expression profiles in response to cold stress between ‘Dongzao� and its autotetraploids from qRT-PCR test ofZjCML6 andZjCML13

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�?nbsp; 8ZjCML家族基因在CaCl₂和褪黑素处理冬枣四倍体后响应低温胁迫时的相对表达量分析

Figure 8.Relative expression levels of some CML genes in tetraploid of ‘Dongzao� in response to cold stress under CaCl₂and melatonin treatments

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�?nbsp; 1实时荧光定量PCR引物�?/p>

Table 1.Primers used in qRT-PCR

基因名称 Gene name	引物序列�?′�?′） Primer sequence �?′−3′）
ZjCML1	F: AGCACATGAAGCCCGAACC R: CGGACCCAACATCCACCTC
ZjCML2	F: GGGGTGTAGTAGGAAAGGCT R: TTCCTTGAGATGACCTCCGC
ZjCML3	F: TAGCCTTGGGACTATCCGCT R: CGCCAGTGACCAAGACAACT
ZjCML4	F: TGCTGAAGAGGAGCTTCGAG R: CCATCACCATCCAAATCAGCTT
ZjCML5	F: CGAAGGTTCGGTCCGTGAAA R: TTGCTTTCTGGGCTGCAAAA
ZjCML6	F: AACGGCAAGTCTGTAGACGA R: CAATGTGTCCGTCGCCATTC
ZjCML7	F: CGTGACCGCATTTTCAAGCG R: GGCCATCATACGACTCACCT
ZjCML8	F: TTGCAGGTTCAATGGCGAAG R: GCTGACTTTGCCATCACAGA
ZjCML9	F: TCTGTGGGAAAACCATGCCT R: ATGAAACCGTCGCCGTTGAT
ZjCML10	F: AGGAGGACATGAGAGAGGCTT R: GTTGACCATCCCATCACCGT
ZjCML11	F: AGCTGAAAGAGGCATTCCGA R: GGTTAGCTTCTCACCGAGGTT
ZjCML12	F: ACAAGGATGGAAGTGGTGCAA R: TCTCACCAAGATCCTTTGCCA
ZjCML13	F: ACCAAGAATCAACAGCGGCA R: TACTCACCGACCGATCCGAA
ZjCML14	F: GATTCAGACGGCTTCATT R: TCTATTGTCCCATTTCCA
ZjCML15	F: ATCCCAAGACAGGCAAAGCTC R: GATGCCCTTGTATTCGTGCTG
ZjCML16	F: ATGGAGACGGGAAGATATCGC R: CCAAACCAAGCAACCCATCTC
ZjCML17	F: GCCAGCGAAAACGACAACAA R: CGGTGGCATGTTGACCTAGA
ZjCML18	F: TCATGGAGGTTTTCCGGTCG R: CGACAGCTCACGGTATGTCA
ZjCML19	F: CTCCATACACAGAGGACCAGC R: GCCTCCTTGATCATCCCTGTC
ZjCML20	F: TCGACAAGAACAGAGACGGC R: GTCCCTCATTTCCGGGTCTT
ZjCML21	F: CGCCATGATGTTGAGCGAAG R: GCCGTGATTTTCCCATCGTG
ZjCML22	F: AGAGGTTTGACCGTGACAGG R: ACTTGGACACCAGCAAATCCA
ZjCML23	F: TCGGGACCATAACGGACTCA R: ACAGATCGGATCATGCGTCG
ZjActin	F: AGCCTTCCTGCCAACGAGT R: TTGCTTCTCACCCTTGATGC

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�?nbsp; 2枢�i>CML基因家族基本信息

Table 2.Basic information ofCMLgene family in Chinese jujube

基因名称 Gene name	基因登录叶�br/>Gene ID	染色佒�br/>定位 Chromosome location	基因练�br/>全长 Genomic total length/bp	CDS长度 CDS length/bp	EF-hand 数目 Number of EF-hand	氨基酸数野�br/>Number of amino acid/aa	分子野�br/>Molecular mass/ kDa	等电炸�br/>Isoelectric point	亚细胞定佌�br/>Subcellular location
ZjCML1	LOC107420771	1	962	444	3	147	16.52	4.73	细胞膜、液泠�br/>Cell membrane and vacuole
ZjCML2	LOC107417331	1	1 325	582	3	193	21.71	5.17	细胞� Cell nucleus
ZjCML3	LOC107409684	2	1 250	777	3	258	28.86	6.16	细胞膜、细胞核 Cell membrane and nucleus
ZjCML4	LOC107413144	3	846	483	3	160	18.10	4.12	细胞膜、细胞质 Cell membrane and cytoplasm
ZjCML5	LOC107415541	4	1 014	642	2	213	24.62	4.72	细胞膛�br/>Cell membrane
ZjCML6	LOC107416249	4	562	459	4	152	16.68	4.80	液泡 Vacuole
ZjCML7	LOC107416258	4	667	255	2	84	93.44	4.45	细胞膛�br/>Cell membrane
ZjCML8	LOC107418957	5	782	486	4	161	17.45	4.14	液泡 Vacuole
ZjCML9	LOC107421107	6	1 158	507	3	168	19.01	4.33	细胞膛�br/>Cell membrane
ZjCML10	LOC107422102	7	1 214	729	5	242	27.62	4.93	细胞膛�br/>Cell membrane
ZjCML11	LOC107423403	7	952	450	4	149	16.84	4.11	细胞膜、细胞质 Cell membrane and cytoplasm
ZjCML12	LOC107424787	8	889	516	4	171	19.44	4.88	细胞膛�br/>Cell membrane
ZjCML13	LOC107424831	8	856	573	3	190	21.34	4.82	细胞膛�br/>Cell membrane
ZjCML14	LOC107425895	9	887	447	4	148	16.77	4.07	细胞膜、细胞质 Cell membrane and cytoplasm
ZjCML15	LOC107427067	9	1 294	561	4	186	21.34	6.62	细胞膛�br/>Cell membrane
ZjCML16	LOC107427847	9	580	423	4	140	15.56	4.30	细胞膛�br/>Cell membrane
ZjCML17	LOC107429242	10	1 224	606	4	201	22.19	4.50	细胞膛�br/>Cell membrane
ZjCML18	LOC107429541	10	721	480	4	159	17.65	4.34	液泡 Vacuole
ZjCML19	LOC107431195	11	1 286	492	4	163	18.02	4.57	液泡 Vacuole
ZjCML20	LOC107431392	11	1 219	645	3	214	23.89	4.34	细胞膛�br/>Cell membrane
ZjCML21	LOC107431644	12	1 192	693	3	230	25.22	4.74	细胞样�br/>Cell nucleus
ZjCML22	LOC107431634	12	1 166	807	3	268	29.24	6.59	细胞样�br/>Cell nucleus
ZjCML23	LOC107434111		930	507	4	168	18.60	4.37	液泡 Vacuole

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