葡萄NCED基因家族进化及表达分析

doi:10.11983/CBB18200

植物学报 ›› 2019, Vol. 54 ›› Issue (4): 474-485.DOI: 10.11983/CBB18200

葡萄NCED基因家族进化及表达分析

王小龙,刘凤之,史祥宾,王孝娣,冀晓昊,王志强,王宝亮,郑晓翠,王海波()

中国农业科学院果树研究所, 农业部园艺作物种质资源利用重点实验室, 辽宁省落叶果树矿质营养与肥料高效利用重点实验室, 兴城 125100

收稿日期:2018-09-19 接受日期:2019-01-15 出版日期:2019-07-01 发布日期:2020-01-08
通讯作者: 王海波
基金资助:
国家现代农业产业技术体系建设专项(nycytx-29-zp);国家科技支撑项目(2014BAD16B05-2);中国农业科学院创新工程(2014BAD16B05-2);中国农业科学院创新工程(CAAS-ASTIP-2017-RIP-04);农业部948重点项目(2011-G28)

Evolution and Expression of NCED Family Genes in Vitis vinifera

Xiaolong Wang,Fengzhi Liu,Xiangbin Shi,Xiaodi Wang,Xiaohao Ji,Zhiqiang Wang,Baoliang Wang,Xiaocui Zheng,Haibo Wang()

Key Laboratory of Mineral Nutrition and Fertilizers Efficient Utilization of Deciduous Fruit Tree, Liaoning Province, Key Laboratory of Germplasm Resources Utilization of Horticultural Crops, Ministry of Agriculture, Fruit Research Institute, Chinese Academy of Agricultural Sciences, Xingcheng 125100, China

Received:2018-09-19 Accepted:2019-01-15 Online:2019-07-01 Published:2020-01-08
Contact: Haibo Wang

摘要/Abstract

摘要： 9-顺式-环氧类胡萝卜素双加氧酶(NCED)是植物体内ABA生物合成的关键限速酶, 参与植物对干旱、外源ABA和高盐的响应过程, 降低环境胁迫对植株的危害。基于全基因组鉴定分析葡萄(Vitis vinifera) NCED基因家族成员, 探讨各成员的物种进化关系及各个基因成员在不同组织中的时空表达模式及对干旱、ABA和高盐(NaCl)胁迫的响应, 为进一步揭示该基因家族成员的生物学功能奠定基础。在葡萄基因组中共发现12个NCED基因。其推测的编码蛋白质长度在510 (VvNCED2)-625 aa (VvNCED10)之间。VvNCED蛋白的分子量最大值是70.53 kDa (VvNCED10), 最小值是57.85 kDa (VvNCED2)。在从祖先基因分化之后, 葡萄NCED基因发生了5次复制事件, 同时有2次丢失事件。NCED1/2、NCED3/4、NCED6/7和NCED9/10基因对被认为是通过片段复制产生。上述4对复制基因复制时间分布在3.08-120.0百万年前, 晚于单双子叶植物分化的时间。与对照相比, VvNCED1在ABA处理48小时后显著上调(72.1%), 而VvNCED2显著下调(84.0%)。VvNCED6只在干旱处理14、21和28天的根系中表达量高于对照, 分别为对照的2.49、1.05和1.09倍。VvNCED7只在干旱处理14天的根系中表达量高于对照, 为对照的1.07倍。在ABA处理72小时后, VvNCED3表达量较对照显著下调(59.5%), 而VvNCED4较对照显著上调(169.9%)。VvNCED3/VvNCED4分别在NaCl处理24和48小时出现显著性峰值, 较对照分别上调219.2%和114.4%。保守结构域不同组成和不同胁迫处理下差异表达模式是NCED蛋白发生功能分化的基础。推测NCED在进化过程中发生的功能分化有利于复制事件的发生。

关键词: NCED基因家族, 葡萄, 非生物胁迫, 基因表达

Abstract: 9-cis-epoxycarotenoid dioxygenase (NCED), a key rate-limiting enzyme in ABA biosynthesis in plants, is involved in plant drought, exogenous abscisic acid (ABA) and high salt response, and can reduce the damage of environmental stress on plants. With genome-wide identification and analysis of the grape NCED gene family, we aimed to understand the species evolution relationship and study the expression patterns of various genes in different tissues and under drought, ABA and high salt (NaCl) stress treatment, to lay the foundation for further study of the biological functions of NCED genes. A total of 12 NCED genes were found in the grape genome. The amino acid residues encoded by the genes are distributed between 510 aa (VvNCED2) and 625 aa (VvNCED10). The maximum molecular weight of the VvNCED protein was 70.53 kDa (VvNCED10) and the minimum was 57.85 kDa (VvNCED2). After differentiation from the ancestral gene, the grape NCED genes had five replication events with two loss events. The NCED1/2, NCED3/4, NCED6/7 and NCED9/10 gene pairs are thought to be produced by segmental duplication. The replication time of segmental duplication ranged from 3.08 to 120.0 million years ago, which is later than the differentiation of monocotyledons. As compared with the control, VvNCED1 was significantly upregulated by 72.1% after 48 h of ABA treatment, whereas VvNCED2 was significantly downregulated by 84.0%. The expression of VvNCED6 was higher in only roots under drought treatment for 14, 21 and 28 days than in the control: 2.49, 1.05 and 1.09 times of control values, respectively. The expression of VvNCED7 was only 1.07 times higher than the control value in roots under drought treatment for 14 days. After 72 h of ABA treatment, the expression of VvNCED3 was significantly downregulated by 59.5% as compared with the control, whereas VvNCED4 was significantly upregulated by 169.9% as compared with the control. The significant peaks in expression of VvNCED3/VvNCED4 after NaCl treatment were 24 and 48 h, respectively, up by 219.2% and 114.4%. The differential conserved-domain expression patterns with different stress treatments are the basis for the functional differentiation of NCED proteins. The functional differentiation of NCED during evolution may be conducive to the occurrence of replication events.

Key words: NCED gene family, grapevine, abiotic stress, gene expression

王小龙,刘凤之,史祥宾,王孝娣,冀晓昊,王志强,王宝亮,郑晓翠,王海波. 葡萄NCED基因家族进化及表达分析. 植物学报, 2019, 54(4): 474-485.

Xiaolong Wang,Fengzhi Liu,Xiangbin Shi,Xiaodi Wang,Xiaohao Ji,Zhiqiang Wang,Baoliang Wang,Xiaocui Zheng,Haibo Wang. Evolution and Expression of NCED Family Genes in Vitis vinifera. Chinese Bulletin of Botany, 2019, 54(4): 474-485.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB18200

https://www.chinbullbotany.com/CN/Y2019/V54/I4/474

图/表 9

表1 RT-PCR分析所用引物序列

Table 1 The primer sequences used for quantitative RT-PCR

Gene name	Forward primer (5'-3')	Reverse primer (5'-3')
VvNCED1	GCTGGAGAAGCTGATAGTGAAG	GAAGATACCCAATGACCGGAAG
VvNCED2	GGCACTTTCGGAGGTTGATAA	TGGATGAGCAGTGAAGGAATG
VvNCED3	CGGTGGAGATGGTGAGAATAGA	CACTGCTGCGTACACGTATTT
VvNCED4	CTCAGCAGTAGGTGATCCTTTG	CAGGCTCGTACATTCTCTTAGC
VvNCED6	CTCGTGATTTGGGCTCTTTCT	GCTTGATGATGTGTGCTTTGG
VvNCED7	CGCTCTTCTTCTTCCTCACTAC	GGCGTTCCCTCTTCTACTATTG
VvNCED9	CCATGGACTTCCCGATGATAAA	ATCCCACAACTAGAGCTTGC
VvNCED10	CAGGGAGGTGTTGAAGAAGATG	CCCTTTGAGGCAGTGTGATT
VvActin	TACAATTCCATCATGAAGTGTGATG	TTAGAAGCACTTCCTGTGAACAATG

表2 葡萄候选NCED基因及其详细信息

Table 2 NCED genes identified in grapevine and their detailed information

Gene name	Accession No.	Chromosome location (start, end)	Length of amino acids (aa)	Molecular weight (kDa)	Theoretical pI	GRAVY
VvNCED1	VIT_213s0064g00840.1	Chr.13 (22672994, 22681910)	546	61.63	6.13	-0.271
VvNCED2	VIT_213s0064g00810.1	Chr.13 (22587965, 22596719)	510	57.85	6.23	-0.250
VvNCED3	VIT_202s0087g00910.1	Chr.2 (18560696, 18562591)	599	65.90	6.88	-0.236
VvNCED4	VIT_202s0087g00930.1	Chr.2 (18588853, 18590786)	589	65.61	6.63	-0.187
VvNCED5	VIT_216s0039g01370.1	Chr.16 (789473, 791221)	558	62.09	5.57	-0.197
VvNCED6	VIT_219s0093g00550.1	Chr.19 (17645348, 17647649)	609	67.13	6.38	-0.317
VvNCED7	VIT_210s0003g03750.1	Chr.10 (6374432, 6376728)	605	67.34	6.36	-0.365
VvNCED8	VIT_205s0051g00670.1	Chr.5 (11589343, 11591102)	575	63.14	8.24	-0.202
VvNCED9	VIT_204s0008g03510.1	Chr.4 (2883265, 2886523)	567	63.72	5.73	-0.335
VvNCED10	VIT_204s0008g03480.1	Chr.4 (2873553, 2878309)	625	70.53	6.43	-0.305
VvNCED11	VIT_204s0008g03380.1	Chr.4 (2784465, 2788790)	563	62.34	7.27	-0.339
VvNCED12	VIT_215s0021g02190.1	Chr.15 (13131078, 13135539)	610	68.46	7.31	-0.313

图1 葡萄、拟南芥和水稻NCED基因系统发育树(A)和扩增模式(B) 图中红色五角星代表物种分化前最近的共同祖先分化节点。红色和蓝色圆圈内的数字分别代表该物种在分化节点后发生的复制和丢失事件次数。绿色实心方框内的数字代表NCED基因的个数。

Figure 1 Joint phylogenetic tree (A) and amplification model (B) of NCED gene from grape, Arabidopsis and rice The red five-pointed stars in the figure represent the most recent common ancestral differentiation node before species differentiation. The numbers in the red and blue circles represent the number of duplication and loss events that occur after the species differentiated nodes, respectively. The numbers in the green solid box represent the number of NCED genes.

图2 葡萄NCED基因系统发育关系(A)和基因结构(B)

Figure 2 Phylogenetic relationship (A) and gene structure (B) of grape NCED genes

图3 葡萄NCED蛋白保守结构域示意图灰色条形框代表NCED蛋白全长, 其它彩色框中的数字是NCED蛋白保守结构域的随机编号。相同的数字代表相同的结构域。

Figure 3 Schematic structure of conserved motifs identified in grapevine NCED proteins The grey bars represent the full length of NCED proteins, and the numbers in the other colored boxes are random numbers for the conserved domains located at NCED protein. The same number represents the same conserved domain.

图4 NCED基因在葡萄不同发育阶段和组织中的表达模式 54种组织名称和基因名称分别位于热图的上方和右侧。热图上方比例尺表示基因表达量0.0-3.49。

Figure 4 Expression pattern of NCED genes at different developmental stages and in some specialized tissues of grapevine The 54 tissue names and gene names are located above and to the right of the heat map, respectively. The scale bar above the heat map indicates the geng expression level from 0.0 to 3.49.

图5 葡萄NCED基因在干旱条件下的表达模式干旱处理名称(DL1-DL5分别表示干旱处理0、7、14、21和28天叶组织; DR1-DR5分别表示干旱处理0、7、14、21和28天根组织)和基因名称分别位于热图的上方与右侧。热图上方比例尺表示基因表达量0.0-77.06。相同颜色的实心圆点代表复制基因对。

Figure 5 Expression pattern of grapevine NCED genes under drought treatments The drought treatments (DL1-DL5 indicate drought treatment for 0, 7, 14, 21 and 28 days of leaf tissue, respectively; DR1-DR5 indicate drought treatment for 0, 7, 14, 21 and 28 days of root tissue, respectively) and gene names are located above and to the right of the heat map, respectively. The scale bar above the heat map indicates the gene expression level from 0.0 to 77.06. Solid dots of the same color represent duplicated gene pairs.

图6 葡萄NCED基因在ABA处理下的表达模式不同小写字母表示不同处理时间点之间差异显著(P<0.05)。

Figure 6 Expression pattern of grapevine NCED genes under ABA treatments Different lowercase letters above the histogram indicate significant differences among the different time points of the treatment (P<0.05).

图7 葡萄NCED基因在NaCl处理下的表达模式不同小写字母表示不同处理时间点之间差异显著(P<0.05)。

Figure 7 Expression pattern of grapevine NCED genes under NaCl treatments Different lowercase letters above the histogram indicate significant differences among the different time points of the treatment (P<0.05).

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葡萄NCED基因家族进化及表达分析

Evolution and Expression of NCED Family Genes in Vitis vinifera

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