芍药胼胝质合成酶基因家族鉴定及PlCalS5功能分析

doi:10.3864/j.issn.0578-1752.2023.16.011

摘要/Abstract

摘要：

【目的】CalS家族在调控植物胼胝质合成方面具有重要作用，鉴定芍药CalS家族成员，并进行生物信息学和表达模式分析，为芍药属远缘杂交不亲和研究提供依据。【方法】通过荧光显微镜观察自交、杂交柱头内花粉管生长过程及花粉萌发情况；测定柱头所含胼胝质、内源脱落酸含量（ABA）以及β-1,3-葡聚糖酶活性；分段克隆8个PlCalS并进行序列分析；使用Expasy、MEME、TBtools、MEGA 7.0等软件和在线工具预测PlCalS家族成员蛋白质基本理化性质、保守基序并构建系统发育进化树；利用qRT-PCR技术检测8个PlCalS在自交24 h、杂交24 h、杂交36 h的相对表达水平；对CalS5进行多序列比对并构建系统进化树，分析PlCalS5响应不同浓度ABA处理的表达特征。【结果】花粉管荧光显微观察发现杂交柱头内发生较为严重的胼胝质堵塞从而影响花粉管的正常生长及花粉的萌发。测定柱头内胼胝质含量发现多数时期自交柱头均低于同时期杂交柱头的含量，柱头内β-1,3-葡聚糖酶活性、ABA含量变化均具有一定的规律性。通过对结构域的完整性分析鉴定芍药PlCalS基因家族成员，后经同源性比对分析命名8个CalS，均含有15个保守基序且在PlCalS基因家族中的分布类似。多物种系统进化关系表明，CalS家族可分为3个分支，PlCalS家族仅分布在2个分支，其中PlCalS5与牡丹、拟南芥和番茄的CalS5亲缘关系较近。生物信息学分析结果表明8个家族成员编码1 745—1 951个氨基酸，原子总数为28 583—31 870个，等电点为7.99—9.13。对转录组FPKM值的分析表明，PlCalS家族成员在相同时期杂交处理中有较高表达，相同处理情况下在杂交36 h时高表达；荧光定量PCR表明，8个基因的相对表达量在自交24 h时均低于杂交24 h和杂交36 h。经两个浓度的ABA处理，发现PlCalS5对高浓度的ABA更为敏感。【结论】芍药CalS家族有8个基因成员且具有较高的保守性，8个家族成员对芍药胼胝质的生成起重要调控作用；大部分时期PlCalS在杂交柱头的表达量高于自交柱头，可能参与胼胝质异常沉积的过程；芍药杂交柱头异源花粉的刺激可能会增强某个ABA合成途径，ABA可能通过正调控胼胝质基因诱导胼胝质的积累，从而抑制花粉的萌发与花粉管的伸长，影响授粉亲和性。

关键词: 芍药属, 远缘杂交不亲和, CalS基因家族, 表达分析

Abstract:

【Objective】 The CalS family plays an important role in regulating callose synthesis in plants. In this study, the members of the CalS family were identified and their bioinformatics and expression patterns were analyzed, which provided an evidence for distant cross incompatibility of Paeonia lactiflora (P. lactiflora). 【Method】The pollen germination and tube growth in self- and cross-pollinated stigma were observed by fluorescence microscope. The callose content, endogenous Abscisic acid (ABA) content and β-1,3-glucanase activity in stigma were measured. Eight members of the CalS family were cloned, and Expasy, MEME, TBtools, MEGA 7.0 and so on were used to predict the basic physicochemical properties and conserved motifs of proteins of the PlCalS family members and to construct a phylogenetic evolutionary tree. The expression of eight PlCalS in stigma at 24 h of self-pollination, 24 h of cross-pollination and 36 h of cross-pollination were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Multiple sequence alignment of CalS5 was performed, a phylogenetic tree was constructed, and the expression characteristics of PlCalS5 in response to ABA treatment at different concentrations were analyzed. 【Result】Fluorescence microscopic observation of pollen tube showed that callose plugging occurred in the stigma of hybrid, which restricted the pollen germination and tube growth. Callose content in cross-pollinated stigma was found to be higher in most periods than that in self-pollinated stigma. Then, the callose activity of β-1,3-glucose and ABA content in stigma with different pollination affinity showed regular differences. The members of the PlCalS family of P. lactiflora were identified and named by integrity analysis of the structural domains and sequence matching, and 15 conserved motifs with stable distribution in the PlCalS family were encoded by every eight genes. Multi-species phylogenetic relationships showed that the CalS family could be divided into three branches, with the PlCalS family distributed in only two branches, of which PlCalS5 was more closely related to CalS5 in Paeonia suffruticosa, Arabidopsis thaliana and Solanum lycopersicum. The bioinformatics analysis showed that the eight family members encoded 1 745-1 951 amino acids, with a total number of 28 583-31 870 atoms and isoelectric points of 7.99-9.13. The analysis of the FPKM values in transcriptome showed that the PlCalS family members were highly expressed in the same period of hybridization and at 36 h of hybridization under the same treatment. qRT-PCR showed that the relative expression levels of 8 genes at 24 h of self-pollination were lower than those at 24 h and 36 h of cross-pollination. In addition, the PlCalS5 gene was found to be more sensitive to high ABA treatment. 【Conclusion】 There were eight gene members in the CalS family of P. lactiflora, which played an important role in regulating the callose formation of P. lactiflora. The expression level of the PlCalS gene in cross-pollinated stigma was higher than that in self-pollinated stigma in most periods, which might be involved in callose abnormal deposition. Heterologous pollen stimulation to the stigma of P. lactiflora could enhance a certain ABA synthesis pathway. ABA induced callose deposition by positively regulating the expression of callose synthesis genes, thus inhibiting pollen germination and tube elongation, and finally affecting pollination compatibility.

Key words: Paeonia lactiflora, distant hybridization incompatibility, CalS gene family, gene expression analyses

贺丹, 尤啸龙, 何松林, 张明星, 张佼蕊, 华超, 王政, 刘艺平. 芍药胼胝质合成酶基因家族鉴定及PlCalS5功能分析[J]. 中国农业科学, 2023, 56(16): 3183-3198.

HE Dan, YOU XiaoLong, HE SongLin, ZHANG MingXing, ZHANG JiaoRui, HUA Chao, WANG Zheng, LIU YiPing. Identification of Callose Synthetase Gene Family and Functional Analysis of PlCalS5 in Paeonia lactiflora[J]. Scientia Agricultura Sinica, 2023, 56(16): 3183-3198.

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图/表 15

表1

分段扩增引物"

基因名称 Gene name	片段名称 Fragment name	序列 Primer sequence (5′-3′)
PlCalS1	PlCalS1-1	F: 5′-ATGTCGTCGAGAGGCGGGTCT-3′ R: 5′-ATGGTCTGAGCAAATCCAGGAGG-3′
	PlCalS1-2	F: 5′-AGATACATTTTGAAGGTCGTTTCAGCTGC-3′ R: 5′-TTCATCTGGAAAGATTTTTTGCAAGTAAAAAAG-3′
	PlCalS1-3	F: 5′-GCTGTTCTTTTCTCCATAAAGGCCCTTG-3′ R: 5′-TATTCTGCACCCCCATGAAGTAATGTT-3′
	PlCalS1-4	F: 5′-GCAGTTACAACTGGCCCCTGTATTTTTC-3′ R: 5′-TTACTCTTTATTGCTAGTGGTGCGGT-3′
PlCalS2	PlCalS2-1	F: 5′-ATGAGTTACCCTGAGATCCGGGCT-3′ R: 5′-AGGAACAAGCAAGAGGTTCATTTCCCT-3′
	PlCalS2-2	F: 5′-GGCAGCAAAGTTTGCCCAGTTGT-3′ R: 5′-ATTTTGAACGCTTCTTCCATGTAGTTATCC-3′
	PlCalS2-3	F: 5′-AGCCTGAAAATCAAAACCATGCCATT-3′ R: 5′-TTACTCTTTATTGCTAGTGGTGCGGTC-3′
PlCalS3	PlCalS3-1	F: 5′-ATGTCGTCGAGAGGCGGGTCT-3′ R: 5′-AACGACCTTCAAAATGTATCTTAACTTAACATG-3′
	PlCalS3-2	F: 5′-AGCTGTGCTTGATGTTATCCTTAGTTGGA-3′ R: 5′-CCTCCGTGCAGTAAGGAGTCAAAA-3′
	PlCalS3-3	F: 5′-CTTCTCAAATTCATTGTTTATGGACATGCCT-3′ R: 5′-TGAAGTAAGGTCCTTCCAAAATAGTGGG-3′
	PlCalS3-4	F: 5′-ATACTCATGCAACTACAATTGGCTCCAGTATT-3′ R: 5′-TTACTCTTTGCTCCGTGACGAACG-3′
PlCalS5	PlCalS5-1	F: 5′-ATGTCGACCCTCGAGTCGGGCCCA-3′ R: 5′-CCCCTCCCAACATATATTCTTGGCTGT-3′
	PlCalS5-2	F: 5′-TGCGACGTTGGATTGAAAACTCGGACTGG-3′ R: 5′-AGTAAAGATATGCTCACGGACACCTAA-3′
	PlCalS5-3	F: 5′-TTGAAGATGCGCAATCTTCTGGAA-3′ R: 5′-TTACTTCTGCTTCTTTCCACCAGCCAG-3′
PlCalS9	PlCalS9-1	F: 5′-ATGGCTCGAGTAGAGAAGCTATGG-3′ R: 5′-TTTATATAAAGGAAGGATATGAAGACTGAG-3′
	PlCalS9-2	F: 5′-CGCATATTCTACAACAAGGCGTGTT-3′ R: 5′-CTGGCTAATGTTTGTCCCCGGTAA-3′
	PlCalS9-3	F: 5′-GATGAAAGTGCACTTGATTCGGAAC-3′ R: 5′-GCACAGTTAGCATGGTACAGAAATAGT-3′
	PlCalS9-4	F: 5′-GGCTTGGGCAACTTTTTGATTTC-3′ R: 5′-TTACATCCCAGTATTGGGATTGTTTCC-3′
PlCalS10	PlCalS10-1	F: 5′-ATGTCTGGGGTTTACGATAATTGGGA-3′ R: 5′-TAGCCTTGAAAGAGCCATGCCTCT-3′
	PlCalS10-2	F: 5′-GTAGAAAGTTTTCTGGACGTCCTGC-3′ R: 5′-ATCATACCACGAACTGTCCTCGCC-3′
	PlCalS10-3	F: 5′-TCAAGATAGCTCTCGGGATGCCC-3′ R: 5′-CAGTGCTGTGTTACCTAACAGTTTAG-3′
	PlCalS10-4	F: 5′-CTTATATGGAAGAGCTTACCTGGCTTTCT-3′ R: 5′-TTAGGTTTGCTGGTTAGCTTTGTTTCCAG-3′
PlCalS11	PlCalS11-1	F: 5′-ATGAGTACTATGAGACAACGACCTCC-3′ R: 5′-TACTTGACAGCTGCTCCTCCG-3′
	PlCalS11-2	F: 5′-CAACAGCTAAGGCTTAGGTTTCAGTTC-3′ R: 5′-CACCCCTAGTAAAAATAATGGCATGG-3′
	PlCalS11-3	F: 5′-AGATATTCCGAATCAGGTTGCCC-3′ R: 5′-TTAATTTGATCGTTTGCCAGTGAGAATGC-3′
PlCalS12	PlCalS12-1	F: 5′-ATGAGCCTACGCCAGCGTCC-3′ R: 5′-CATTTAACAGCTGCTCCTCCGG-3′
	PlCalS12-2	F: 5′-AGTTGAGGCTGAGATTCCAGTTCTTTG-3′ R: 5′-GCAGACATAAACCAGGCAAGTGAAGA-3′
	PlCalS12-3	F: 5′-AATATAAAACCTACTATGGTATCCGGAAGC-3′ R: 5′-TTACAACTCCACCTTGGATTTTTTTCC-3′

表1

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基因 Gene name	引物序列 Primer sequence (5′-3′)	退火温度 Annealing temperature (℃)
PlCalS1	F: 5′-TTCAGCTGCAGCATGGGTTA-3′ R: 5′-CTCGAGGAATCGGCGAATGA-3′	52 54
PlCalS2	F: 5′-AGCTCTTGTGAAGGCTGCTT-3′ R: 5′-AAGCCTTCGCCACGAGTAAA-3′	52 52
PlCalS3	F: 5′-ATGAGCGTGCACTGACAGAA-3′ R: 5′-GCAGCTTCGCCCCAAATAAG-3′	52 54
PlCalS5	F: 5′-GCCGCTATAGTGTTCCCTCC-3′ R: 5′-AGGCTTTCCCTCTCCGATCT-3′	56 54
PlCalS9	F: 5′-CGGGGGCTTTTATGGACACT-3′ R: 5′-GAGGCCATTGAACCAAGGGA-3′	54 54
PlCalS10	F: 5′-TTGACGACTGGACGAGTTGG-3′ R: 5′-GTACAACCCACGAGAAGCCA-3′	54 54
PlCalS11	F: 5′-GCTACAGCTCGCGTCACTAT-3′ R: 5′-GCGAAGCTCTTATGCTGCAC-3′	54 54
PlCalS12	F: 5′-TCCCTTCGATTCCCTGAGGT-3′ R: 5′-GGACGTTGTCGCGTTGAAAA-3′	54 52
β-Tubulin	F: 5′-TGAGCACCAAAGAAGTGGACGAAC-3′ R: 5′-CACACGCCTGAACATCTCCTGAA-3′	57 57

基因名称 Gene names	预测分子式 Predicted molecular formula	编码氨基酸数目 Number of encoded amino acids	原子总数 Total number of atoms	理论等电点 Theoretical isoelectric point	不稳定系数 Instability coefficient	总平均亲水性 GRAVY	亚细胞定位预测 Prediction of subcellular localization
PlCalS1	C₁₀₂₈₆H₁₅₉₂₅N₂₆₉₅O₂₈₀₄S₇₈	1946	31788	9.10	39.61	-0.101	内质网 Endoplasmic reticulum
PlCalS2	C₉₂₇₈H₁₄₃₂₄N₂₄₀₈O₂₄₉₈S₇₅	1745	28583	9.10	40.71	-0.050	内质网 Endoplasmic reticulum
PlCalS3	C₁₀₃₃₄H₁₅₉₅₄N₂₇₀₂O₂₈₀₂S₇₈	1951	31870	9.12	42.01	-0.097	内质网 Endoplasmic reticulum
PlCalS5	C₁₀₀₈₉H₁₅₅₈₇N₂₆₄₇O₂₇₃₉S₇₉	1915	31141	8.95	42.27	-0.024	内质网 Endoplasmic reticulum
PlCalS9	C₁₀₀₄₈H₁₅₅₂₆N₂₆₁₂O₂₇₆₆S₆₈	1910	31020	7.99	36.40	-0.001	内质网 Endoplasmic reticulum
PlCalS10	C₉₉₇₃H₁₅₃₉₇N₂₆₀₇O₂₇₄₈S₆₈	1903	30793	8.45	38.86	0.009	内质网 Endoplasmic reticulum
PlCalS11	C₉₅₁₅H₁₄₆₂₅N₂₄₉₉O₂₅₄₉S₆₂	1782	29250	9.13	41.37	-0.007	内质网 Endoplasmic reticulum
PlCalS12	C₉₅₄₇H₁₄₆₆₀N₂₅₀₀O₂₅₆₄S₅₆	1780	29327	8.97	38.48	-0.030	内质网 Endoplasmic reticulum