葡萄AKR基因家族的鉴定和组织特异性表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0068

摘要/Abstract

摘要：

醛酮还原酶（aldo-keto reductase,AKR）是一类NADP-依赖型氧化还原酶,通过对葡萄AKR基因鉴定分析,旨在揭示葡萄AKR基因分子生物学功能和遗传调控机制。基于2020年最新组装的葡萄参考基因组数据,利用生物信息学对葡萄AKR基因进行理化性质、基因结构、Motif分析、顺式作用元件、亚细胞定位、二级结构预测以及组织特异性表达分析,最后用荧光定量分析了葡萄AKR基因在盐胁迫下表达水平变化。从全基因组水平上共鉴定出13个葡萄AKR基因家族成员,分为2个亚族;基因结构分析显示,不同成员编码的氨基酸序列外显子数目及位置存在差异;motif分析表明,AKR蛋白保守结构域高度相似;顺式作用元件分析显示,该基因上游启动子元件不仅存在核心元件和增强元件,同时还存在各种激素及胁迫响应元件,器官特异性元件和光调控元件;二级结构和亚细胞定位预测表明,该基因家族主要在细胞核中表达,并以α-螺旋和不规则卷曲为主。基因芯片表达谱分析显示,AKR家族成员间表达水平差异大且存在组织特异性。荧光定量结果显示,盐胁迫条件下VvAKR02、VvAKR05、VvAKR07、VvAKR11、VvAKR12和VvAKR13表达量显著上调。从葡萄中鉴定了13个AKR家族基因,并分析其盐胁迫下的表达水平,对于挖掘葡萄AKR家族中耐盐基因提供了一定的理论依据和实验基础。

关键词: 葡萄, AKR基因家族, 生物信息学, 基因芯片表达谱, 盐胁迫

Abstract:

Aldo-Keto Reductase（AKR）is a kind of NADP-dependent oxidoreductase. Through the identification and analysis of grape AKR genes,the aim is to reveal the molecular biological function and genetic regulation mechanism of AKR genes. Based on the latest assembly reference genome data of grape in 2020,bioinformatics was used to analyze the physicochemical properties,gene structure,Motif analysis,cis-acting elements,subcellular localization,secondary structure prediction,and tissue-specific expression analysis of grape AKR genes. Finally,fluorescence quantitative analysis was used to analyze the change of grape AKR gene expressions under salt stress. Thirteen members of AKR gene family were identified at the genome-wide level and divided into 2 subfamilies. Genetic structure analysis demonstrated that the number and location of exons among different members varied. Motif analysis showed that the conserved domains of AKR protein were highly similar. The cis-acting element analysis showed that the upstream promoter elements of this gene not only had core elements and enhancement elements,but also had various hormone and stress response elements,organ specific elements and light regulatory elements. The prediction of secondary structure and subcellular localization indicated that the gene family was mainly expressed in the nucleus,and was dominated by α-helix and irregular coil. Analysis via gene chip expression profile showed that the expression levels of AKR family members were significantly different and had tissue specificity. Fluorescence quantitative results showed that the expression levels of VvAKR02,VvAKR05,VvAKR07,VvAKR11,VvAKR12 and VvAKR13 were up-regulated under salt stress. In conclusion,13 AKR family genes were identified from grape and their expression levels under salt stress were analyzed,which may provide a theoretical and experimental basis for mining salt-tolerant genes in AKR family of grape.

Key words: grape, the AKR gene family, bioinformatics, gene chip expression profile, salt stress

马亚男, 卢旭, 魏云春, 李康, 魏若男, 李胜, 马绍英. 葡萄AKR基因家族的鉴定和组织特异性表达分析[J]. 生物技术通报, 2021, 37(8): 141-151.

MA Ya-nan, LU Xu, WEI Yun-chun, LI Kang, WEI Ruo-nan, LI Sheng, MA Shao-ying. Identification and Tissue Specific Expression Analysis of AKR Gene Family in Grape[J]. Biotechnology Bulletin, 2021, 37(8): 141-151.

图/表 9

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基因Gene	上游引物Forward primer（5'-3'）	下游引物Reverse primer（5'-3'）
VvAKR01	AGGAGGCAGCAGTGTCAAAATGG	CCAGCCACCGCATCATCATCTC
VvAKR02	ACCATTGAAAGCCTCAGCCAGTG	GGCGTTGTCAGAGGGTTATCGTC
VvAKR03	TTGTGCTGAGGAAGCTGGGTAAAC	GCCGCAGAGTGGGAACAAGAAG
VvAKR04	AGCGGTGATTTGAATGCCTTTTGC	GGCGGTCAATACGGTGGAGATG
VvAKR05	AGCGGGTTCATCTCCACTTGATTG	GGAAGGCATGGAAGAGTGTCAGAG
VvAKR06	TCTGCTCTGTCTTGTGCGAAACC	GAACCAACCCCATACCCTAAAGCC
VvAKR07	TGCTGCCAAAGTGGGTTCATCTC	GTCAGAAGCTCGGTCTCACCAAA
VvAKR08	AAAGCTTTGGTGCACCGACG	TCCCAGGCTTCAAGCTCACC
VvAKR09	TAGCCGCCTTAAGAGCCTGTCC	ACGCTGTCTTCACCGGCAATCG
VvAKR10	GACACTGTGACCGCTCTGAATACC	CTCTCGGCGTACTCTCCTCTTGG
VvAKR11	CTTGGATGACAGCAGGAGGAACAC	AGCACATGGAGAGCAATGGAAGC
VvAKR12	AGTGCTTCCATTGCTCTCCATGTG	CTTATCCATTGGCCGGTCCGTATG
VvAKR13	CTGCCTGCTCTTGCCAGATGTG	CCGTGTTCCTCCTGCTGTCAATC
UBI	GCTCGCTGTTTTTGCAGTTCTAC	AACATAGGTGAGGCCGCACTT

基因Gene	上游引物Forward primer（5'-3'）	下游引物Reverse primer（5'-3'）
VvAKR01	AGGAGGCAGCAGTGTCAAAATGG	CCAGCCACCGCATCATCATCTC
VvAKR02	ACCATTGAAAGCCTCAGCCAGTG	GGCGTTGTCAGAGGGTTATCGTC
VvAKR03	TTGTGCTGAGGAAGCTGGGTAAAC	GCCGCAGAGTGGGAACAAGAAG
VvAKR04	AGCGGTGATTTGAATGCCTTTTGC	GGCGGTCAATACGGTGGAGATG
VvAKR05	AGCGGGTTCATCTCCACTTGATTG	GGAAGGCATGGAAGAGTGTCAGAG
VvAKR06	TCTGCTCTGTCTTGTGCGAAACC	GAACCAACCCCATACCCTAAAGCC
VvAKR07	TGCTGCCAAAGTGGGTTCATCTC	GTCAGAAGCTCGGTCTCACCAAA
VvAKR08	AAAGCTTTGGTGCACCGACG	TCCCAGGCTTCAAGCTCACC
VvAKR09	TAGCCGCCTTAAGAGCCTGTCC	ACGCTGTCTTCACCGGCAATCG
VvAKR10	GACACTGTGACCGCTCTGAATACC	CTCTCGGCGTACTCTCCTCTTGG
VvAKR11	CTTGGATGACAGCAGGAGGAACAC	AGCACATGGAGAGCAATGGAAGC
VvAKR12	AGTGCTTCCATTGCTCTCCATGTG	CTTATCCATTGGCCGGTCCGTATG
VvAKR13	CTGCCTGCTCTTGCCAGATGTG	CCGTGTTCCTCCTGCTGTCAATC
UBI	GCTCGCTGTTTTTGCAGTTCTAC	AACATAGGTGAGGCCGCACTT

基因Gene	登录号 Gene accession No.	染色体定位 Chromosomal location	全长Full length/bp	CDS/bp	氨基酸 Amino acid	分子量 Molecular weight/bp	等电点 pI	不稳定系数 Instability index	脂肪系数Aliphatic index	平均亲水性Hydropathicity
VvAKR01	GSVIVT01009396001	chr18:7983438..7986889	3452	969	322	36192.49	5.9	38.98	89.66	-0.238
VvAKR02	GSVIVT01010238001	chr1:18082562..18106134	23573	1002	333	36674.09	7.07	34.21	90.75	-0.213
VvAKR03	GSVIVT01011576001	chr1:6306239..6307845	1607	867	288	32083.49	9.01	42.94	96.88	-0.086
VvAKR04	GSVIVT01011577001	chr1:6298552..6305704	7153	912	303	34017.51	6.54	47.47	89.8	-0.164
VvAKR05	GSVIVT01011579001	chr1:6289243..6290820	1578	951	316	35455.01	5.85	51.55	95.73	-0.206
VvAKR06	GSVIVT01011582001	chr1:6274423..6285766	11344	1293	430	48655.51	7.1	43.48	94.12	-0.184
VvAKR07	GSVIVT01011583001	chr1:6264420..6272529	8110	909	302	34322.71	6.34	27.01	92.02	-0.274
VvAKR08	GSVIVT01011585001	chr1:6241008..6248273	7266	1092	363	40730.95	5.98	49.92	95.92	-0.228
VvAKR09	GSVIVT01030263001	chr8:9745143..9747339	2197	969	322	35975.24	6.17	45.07	83.57	-0.319
VvAKR10	GSVIVT01034098001	chr8:15157629..15161115	3487	948	315	35068.99	5.83	50.57	89.46	-0.243
VvAKR11	GSVIVT01034099001	chr8:15153012..15157096	4085	828	275	30440	6.45	46.14	87.56	-0.204
VvAKR12	GSVIVT01034101001	chr8:15144714..15149849	5136	903	300	33663.65	6.76	42.71	88.37	-0.317

基因Gene	登录号 Gene accession No.	染色体定位 Chromosomal location	全长Full length/bp	CDS/bp	氨基酸 Amino acid	分子量 Molecular weight/bp	等电点 pI	不稳定系数 Instability index	脂肪系数Aliphatic index	平均亲水性Hydropathicity
VvAKR01	GSVIVT01009396001	chr18:7983438..7986889	3452	969	322	36192.49	5.9	38.98	89.66	-0.238
VvAKR02	GSVIVT01010238001	chr1:18082562..18106134	23573	1002	333	36674.09	7.07	34.21	90.75	-0.213
VvAKR03	GSVIVT01011576001	chr1:6306239..6307845	1607	867	288	32083.49	9.01	42.94	96.88	-0.086
VvAKR04	GSVIVT01011577001	chr1:6298552..6305704	7153	912	303	34017.51	6.54	47.47	89.8	-0.164
VvAKR05	GSVIVT01011579001	chr1:6289243..6290820	1578	951	316	35455.01	5.85	51.55	95.73	-0.206
VvAKR06	GSVIVT01011582001	chr1:6274423..6285766	11344	1293	430	48655.51	7.1	43.48	94.12	-0.184
VvAKR07	GSVIVT01011583001	chr1:6264420..6272529	8110	909	302	34322.71	6.34	27.01	92.02	-0.274
VvAKR08	GSVIVT01011585001	chr1:6241008..6248273	7266	1092	363	40730.95	5.98	49.92	95.92	-0.228
VvAKR09	GSVIVT01030263001	chr8:9745143..9747339	2197	969	322	35975.24	6.17	45.07	83.57	-0.319
VvAKR10	GSVIVT01034098001	chr8:15157629..15161115	3487	948	315	35068.99	5.83	50.57	89.46	-0.243
VvAKR11	GSVIVT01034099001	chr8:15153012..15157096	4085	828	275	30440	6.45	46.14	87.56	-0.204
VvAKR12	GSVIVT01034101001	chr8:15144714..15149849	5136	903	300	33663.65	6.76	42.71	88.37	-0.317

基因 Gene	分子式 Formula	α-螺旋 Alpha helix/%	β-转角 Beta turn/%	扩展链结构 Hydropathicity/%	不规则卷曲 Random coil/%	亚细胞定位 Subcellular localization
VvAKR01	C₁₆₂₁H₂₅₃₃N₄₃₇O₄₇₃S₁₅	42.55	4.04	14.29	39.13	Nucleus,cytosol,cytoskeleton and chloroplast
VvAKR02	C₁₆₃₀H₂₅₈₄N₄₅₂O₄₈₀S₁₅	37.84	6.01	15.32	40.84	Nucleus,cytosol,cytoskeleton and chloroplast
VvAKR03	C₁₄₃₇H₂₃₁₄N₃₈₈O₄₀₉S₁₆	41.32	4.51	18.06	36.11	Nucleus,membrane,cytosol,chloroplast and mitochondrion,peroxisome
VvAKR04	C₁₅₃₃H₂₄₁₈N₄₀₂O₄₃₇S₁₇	42.57	4.62	13.86	38.94	Nucleus,cytosol,cytoskeleton and chloroplast
VvAKR05	C₁₅₈₄H₂₅₃₄N₄₂₄O₄₆₆S₁₅	43.04	3.80	15.51	37.66	Extracellular,cytosol,chloroplast,mitochondrion and endoplasmic reticulum
VvAKR06	C₂₂₀₇H₃₄₈₁N₅₇₁O₆₂₇S₁₉	34.42	3.95	17.44	44.19	Nucleus,membrane,cytosol,cytoskeleton,chloroplast and mitochondrion
VvAKR07	C₁₅₆₀H₂₄₄₅N₄₀₁O₄₄₅S₁₂	41.39	5.30	13.58	39.74	Nucleus,extracellular,cytosol and cytoskeleton
VvAKR08	C₁₈₂₉H₂₉₀₀N₄₉₀O₅₃₃S₁₄	42.15	4.41	13.50	39.94	Nucleus,membrane,cytosol and cytoskeleton
VvAKR09	C₁₆₀₁H₂₅₁₈N₄₄₂O₄₆₉S₁₆	40.37	8.39	14.60	36.65	Nucleus,membrane,cytosol,cytoskeleton and chloroplast
VvAKR10	C₁₅₈₂H₂₄₅₁N₄₁₉O₄₆₃S₁₀	42.54	6.67	13.97	36.83	Nucleus,cytosol and chloroplast
VvAKR11	C₁₃₇₈H₂₁₄₇N₃₆₃O₃₉₅S₁₀	43.27	5.09	14.18	37.45	Nucleus,cytosol,cytoskeleton,endoplasmic reticulum and vacuole
VvAKR12	C₁₅₁₈H₂₃₇₅N₄₁₁O₄₃₅S₁₀	43.00	6.67	12.00	38.33	Nucleus,membrane,cytosol,cytoskeleton and vacuole
VvAKR013	C₁₈₇₂H₂₉₁₆N₄₉₂O₅₃₁S₁₁	48.91	6.25	11.41	33.42	Nucleus,cytosol and chloroplast