铁皮石斛14-3-3基因家族鉴定及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0839

摘要/Abstract

摘要：

【目的】 14-3-3蛋白，亦称通用调节因子（GRF），由多基因家族编码，在植物生长发育和逆境应答发挥关键的作用。鉴定铁皮石斛（Dendrobium officinale）GRF基因家族，为铁皮石斛GRF基因功能研究及遗传改良提供理论依据。【方法】 通过生物信息学的方法鉴定铁皮石斛14-3-3家族成员，分析其理化性质、染色体定位、系统进化发育、基因结构和启动子顺式作用元件等，同时通过荧光定量PCR技术检测它们在不同组织、低温处理及盐胁迫处理后的表达量。【结果】 铁皮石斛有17个GRF家族成员，分为ε类和非ε类亚族，不均匀地分布在7条染色体上，且存在7对串联复制基因。同一亚族成员基因结构、保守基序和蛋白质二级结构相类似。DoGRF家族基因的启动子区域存在大量激素和环境胁迫应答相关的调控元件。DoGRF家族基因在铁皮石斛各组织中均有表达，具有组织表达特异性，大多数基因在花器官中表达最高，其次是茎和根。同时，在低温处理、盐胁迫处理下呈现差异化表达，可能受到低温和盐胁迫的调控，特别是DoGRF2在铁皮石斛逆境应答过程中起着关键的作用。【结论】 在全基因组水平从铁皮石斛中鉴定出17个DoGRF家族成员，不同基因对非生物胁迫响应不一，并具有器官表达特异性。DoGRF2对低温和盐胁迫呈现正向响应。

关键词: 铁皮石斛, 14-3-3家族, 基因表达, 非生物胁迫

Abstract:

【Objective】 14-3-3 protein, also known as general regulatory factor（GRF）, encodes through multi-gene family, and plays a central role in encouraging plant growth and development and stress-related adversity responses. The identification of GRF gene family in Dendrobium officinale may provide theoretical basis for the study of D. officinale GRF gene function and genetic improvement.【Method】 Bioinformatics was used to identify the members of 14-3-3 gene family, and analyze their physicochemical properties, chromosomal localization, phylogenetic tree, gene structure, and cis-acting elements of each DoGRF member. Simultaneously, their expression profiles in different tissues and under the treatment of low-temperature or salt stress were detected using the real-time quantitative PCR.【Result】 Results demonstrated that seventeen DoGRF gene family members were presented in D. officinale, which harbored two subfamilies（ε group and non-ε group）. They were unevenly located on 7 chromosomes, and harbored seven tandem repeat gene pairs. The gene structure, conserved motifs, and secondary structure of DoGRF members within the same subfamily were similar. The cis-acting elements at the promoter regions of DoGRF family genes contained abundant hormone and stress response elements. DoGRF genes family were expressed in various tissues（flower buds, sepals, labellum（lip）, pollinia, gynostemium（column）, stems, leaves, roots, green root tips, and white part of roots）, and most of them were highly expressed in floral organs, followed by stems and roots. Besides, they were differentially expressed under treatment of low-temperature or salt-stress, suggesting that they might be involved in regulating the stresses of low-temperature and salinity. Especially, DoGRF2 might play a crucial role in the process of stress responses.【Conclusion】 17 members of DoGRF family were systematically identified from D. officinale at the genome level. Different genes possessed different responses to abiotic stresses, and exhibited tissue-specific expression profiles. DoGRF2 showed positively response to low-temperature and salt stress.

Key words: Dendrobium officinale, 14-3-3 family, gene expression, abiotic stress

江林琪, 赵佳莹, 郑飞雄, 姚馨怡, 李效贤, 俞振明. 铁皮石斛14-3-3基因家族鉴定及表达分析[J]. 生物技术通报, 2024, 40(3): 229-241.

JIANG Lin-qi, ZHAO Jia-ying, ZHENG Fei-xiong, YAO Xin-yi, LI Xiao-xian, YU Zhen-ming. Identification and Expression Analysis of 14-3-3 Gene Family in Dendrobium officinale[J]. Biotechnology Bulletin, 2024, 40(3): 229-241.

图/表 11

参考文献 32

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引物名称Primer name	上游引物Forward primer（5'-3'）	下游引物Reverse primer（5'-3'）
EF-1α	GCTTGAGAAGGAGCCCAAGT	CCAACAGCCACAGTTTGTCG
DoGRF1	GAAGCTGCTGAACAGTCATTG	CCCAGTCTAATTGGGTGAGTAG
DoGRF2	AAAGAAGAGAGTCGTGGGAATG	GTAGCAGAAGGAATGAGATGGG
DoGRF3	GCTGGAATCCTTCGTTTGTTG	TACCGATGGTAATCGCCTTTC
DoGRF4	CGGTGGTATCCTCTCTTTGTTAG	AGCAAGATAGCGGTGGTAATC
DoGRF5	AGCATCACGTGAAGAGGATTAG	CAGCAGACGACGAAGGAATAA
DoGRF6	GAGGATCGTGTGGCGATTAT	GTAGCAGAGGGAATCAGATGAG
DoGRF7	CCATAGGTACCTTGCTGAGTTT	AGTGCCAATCCCAACCTTATAG
DoGRF8	CGGAGATGAAGGTATCCAACAG	TTGCAGTGTCCTCTACCAATC
DoGRF9	GAGATGCTGCAATCGCTAATTC	CCGCTACACTAAACGCAAATG
DoGRF10	ATCCGGCTCTTGAAGGAATG	GGAGCCAGAACAGACAAAGT
DoGRF11	GGATGCGGAAGTCGCTAAT	ATGCTCGGCCAGACTAAAC
DoGRF12	CCTCATTTCCGTCGCATACA	GCCTTCTTCCTTCTGCTCAA
DoGRF13	AAAGTATATGGCGGAGGTGAAG	AATTGCTCTTCGTCCTCCTTAG
DoGRF14	CATCGAGCAGAAGGAGGAAA	GGTGTGAGTCGAGTAACCTAAG
DoGRF15	GGCTGAGAGGTATGAGGAAATG	AGGCTACCGAGAGGAGATTT
DoGRF16	GCGGAGGATATTGCTCTTGT	GGCGAGCGTTCTCTGATAAA
DoGRF17	TGAGAGAGAGCAGCAAGTTTAC	CTACTGTCAGCTCCACATCTAATC

引物名称Primer name	上游引物Forward primer（5'-3'）	下游引物Reverse primer（5'-3'）
EF-1α	GCTTGAGAAGGAGCCCAAGT	CCAACAGCCACAGTTTGTCG
DoGRF1	GAAGCTGCTGAACAGTCATTG	CCCAGTCTAATTGGGTGAGTAG
DoGRF2	AAAGAAGAGAGTCGTGGGAATG	GTAGCAGAAGGAATGAGATGGG
DoGRF3	GCTGGAATCCTTCGTTTGTTG	TACCGATGGTAATCGCCTTTC
DoGRF4	CGGTGGTATCCTCTCTTTGTTAG	AGCAAGATAGCGGTGGTAATC
DoGRF5	AGCATCACGTGAAGAGGATTAG	CAGCAGACGACGAAGGAATAA
DoGRF6	GAGGATCGTGTGGCGATTAT	GTAGCAGAGGGAATCAGATGAG
DoGRF7	CCATAGGTACCTTGCTGAGTTT	AGTGCCAATCCCAACCTTATAG
DoGRF8	CGGAGATGAAGGTATCCAACAG	TTGCAGTGTCCTCTACCAATC
DoGRF9	GAGATGCTGCAATCGCTAATTC	CCGCTACACTAAACGCAAATG
DoGRF10	ATCCGGCTCTTGAAGGAATG	GGAGCCAGAACAGACAAAGT
DoGRF11	GGATGCGGAAGTCGCTAAT	ATGCTCGGCCAGACTAAAC
DoGRF12	CCTCATTTCCGTCGCATACA	GCCTTCTTCCTTCTGCTCAA
DoGRF13	AAAGTATATGGCGGAGGTGAAG	AATTGCTCTTCGTCCTCCTTAG
DoGRF14	CATCGAGCAGAAGGAGGAAA	GGTGTGAGTCGAGTAACCTAAG
DoGRF15	GGCTGAGAGGTATGAGGAAATG	AGGCTACCGAGAGGAGATTT
DoGRF16	GCGGAGGATATTGCTCTTGT	GGCGAGCGTTCTCTGATAAA
DoGRF17	TGAGAGAGAGCAGCAAGTTTAC	CTACTGTCAGCTCCACATCTAATC

蛋白名称 Protein name	氨基酸数Number of amino acids/aa	分子量Molecular weight/kD	等电点Isoelectric point	不稳定系数 Instability index	脂肪系数 Aliphatic index	总平均亲水系数 Grand average of hydropathicity	亚细胞定位 Subcellular localization
DoGRF1	248	28.24	4.85	43.69	86.98	-0.444	细胞核Nucleus
DoGRF2	299	34.17	5.18	42.37	91.10	-0.341	细胞核Nucleus
DoGRF3	265	29.77	4.96	41.61	91.40	-0.283	细胞核Nucleus
DoGRF4	266	29.93	4.96	43.50	93.31	-0.293	细胞核Nucleus
DoGRF5	267	30.00	4.83	39.17	81.24	-0.537	细胞核Nucleus
DoGRF6	261	29.48	6.40	42.95	87.13	-0.357	细胞核Nucleus
DoGRF7	250	28.25	4.99	43.68	86.04	-0.358	细胞核Nucleus
DoGRF8	267	30.31	4.87	41.45	80.41	-0.552	细胞核Nucleus
DoGRF9	260	29.48	4.74	47.93	91.23	-0.483	细胞核Nucleus
DoGRF10	257	28.94	4.73	48.82	83.23	-0.472	细胞核Nucleus
DoGRF11	257	29.10	4.82	49.74	86.65	-0.503	细胞核Nucleus
DoGRF12	257	28.95	4.85	48.62	81.36	-0.508	细胞核Nucleus
DoGRF13	254	28.57	5.94	51.58	90.39	-0.259	细胞核Nucleus
DoGRF14	146	16.29	6.83	50.72	79.66	-0.125	叶绿体Chloroplast
DoGRF15	278	30.57	9.05	45.76	94.21	-0.042	细胞核Nucleus
DoGRF16	266	29.51	8.24	40.65	95.86	-0.021	细胞核Nucleus
DoGRF17	246	27.61	9.03	39.78	94.47	-0.128	叶绿体Chloroplast

蛋白名称 Protein name	氨基酸数Number of amino acids/aa	分子量Molecular weight/kD	等电点Isoelectric point	不稳定系数 Instability index	脂肪系数 Aliphatic index	总平均亲水系数 Grand average of hydropathicity	亚细胞定位 Subcellular localization
DoGRF1	248	28.24	4.85	43.69	86.98	-0.444	细胞核Nucleus
DoGRF2	299	34.17	5.18	42.37	91.10	-0.341	细胞核Nucleus
DoGRF3	265	29.77	4.96	41.61	91.40	-0.283	细胞核Nucleus
DoGRF4	266	29.93	4.96	43.50	93.31	-0.293	细胞核Nucleus
DoGRF5	267	30.00	4.83	39.17	81.24	-0.537	细胞核Nucleus
DoGRF6	261	29.48	6.40	42.95	87.13	-0.357	细胞核Nucleus
DoGRF7	250	28.25	4.99	43.68	86.04	-0.358	细胞核Nucleus
DoGRF8	267	30.31	4.87	41.45	80.41	-0.552	细胞核Nucleus
DoGRF9	260	29.48	4.74	47.93	91.23	-0.483	细胞核Nucleus
DoGRF10	257	28.94	4.73	48.82	83.23	-0.472	细胞核Nucleus
DoGRF11	257	29.10	4.82	49.74	86.65	-0.503	细胞核Nucleus
DoGRF12	257	28.95	4.85	48.62	81.36	-0.508	细胞核Nucleus
DoGRF13	254	28.57	5.94	51.58	90.39	-0.259	细胞核Nucleus
DoGRF14	146	16.29	6.83	50.72	79.66	-0.125	叶绿体Chloroplast
DoGRF15	278	30.57	9.05	45.76	94.21	-0.042	细胞核Nucleus
DoGRF16	266	29.51	8.24	40.65	95.86	-0.021	细胞核Nucleus
DoGRF17	246	27.61	9.03	39.78	94.47	-0.128	叶绿体Chloroplast

蛋白名称Protein name	α-螺旋 Alpha helix	延伸链 Extended strand	β-折叠 Beta turn	随机卷曲 Random coil
DoGRF1	52.42	12.54	5.41	29.63
DoGRF2	62.54	11.04	3.34	23.08
DoGRF3	49.75	14.39	5.30	30.56
DoGRF4	52.31	14.58	6.25	26.85
DoGRF5	59.20	11.66	3.37	25.77
DoGRF6	67.43	6.51	3.07	22.99
DoGRF7	52.96	12.81	5.17	29.06
DoGRF8	50.84	10.60	5.06	33.49
DoGRF9	51.75	11.05	4.85	32.35
DoGRF10	52.48	15.45	2.04	30.03
DoGRF11	52.06	11.60	4.38	31.96
DoGRF12	49.77	10.42	5.09	34.72
DoGRF13	44.19	16.28	5.04	34.50
DoGRF14	34.91	15.98	10.65	38.46
DoGRF15	46.38	13.62	8.09	31.91
DoGRF16	45.36	12.500	5.85	36.29
DoGRF17	43.03	13.75	6.97	36.25