蓝莓R2R3-MYB基因鉴定及类黄酮调控基因表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0103

摘要/Abstract

摘要：

目的解析蓝莓R2R3-MYB转录因子家族在类黄酮调控中的表达模式，为探究蓝莓参与类黄酮合成调控机制提供理论基础。方法运用生物信息学手段，对蓝莓R2R3-MYB基因的理化性质、亚细胞定位等进行分析；通过RT-qPCR检测VcMYBs基因在不同组织和果实发育期的表达。结果在蓝莓中鉴定出278个R2R3-MYB基因，其氨基酸序列长度为160-970 aa；多数成员亚细胞定位在细胞核，进化树分析将VcMYBs分为23个亚家族，同亚家族基因结构特性相似；顺式作用元件分析表明，VcMYBs可能参与生物和非生物胁迫、激素响应、植物生长发育以及光响应；从可能参与类黄酮合成调控的基因（SG4、SG5、SG6和SG7）中随机选择14个，RT-qPCR分析显示，多数VcMYBs基因在果实中表达水平较高，其中，VcMYB91（SG6）和VcMYB56（SG5）等基因在绿果期低表达，成熟期高表达，可能参与果实着色；然而VcMYB88（SG4）和VcMYB96（SG4）等基因在绿果期高表达，成熟期低表达，可能抑制果实着色；VcMYB236（SG7）和VcMYB44（SG7）基因可能参与黄酮醇合成。结论全基因组分析鉴定出278个蓝莓R2R3-MYB基因，解析发现SG5/6亚组4个基因（VcMYB56、VcMYB91等）调控花青素合成，SG4/5亚组6个基因（VcMYB88、VcMYB96等）参与原花青素合成，SG7亚组2个基因（VcMYB236、VcMYB44）可能调控黄酮醇代谢。

关键词: 蓝莓, R2R3-MYB, 生物信息学, 组织表达模式, 不同果实发育期, 类黄酮合成

Abstract:

Objective Elucidating the expression patterns of the R2R3-MYB transcription factor family in blueberry during flavonoid regulation, thereby providing a theoretical foundation for exploring the regulatory mechanisms underlying flavonoid biosynthesis in blueberry. Method Bioinformatics was used to analyze the characteristics of R2R3-MYB genes in blueberry, such as physicochemical properties and subcellular localization, and RT-qPCR was to detect the expression patterns of VcMYBs in different tissues and different fruit development stages. Result A total of 278 R2R3-MYB genes were identified in blueberry, with amino acid sequence lengths ranging from 160 to 970 aa, and most of them were located in the nucleus. The blueberry VcMYBs was divided into 23 subfamilies by phylogenetic tree analysis, and the genes belonging to the same subfamily had similar structural characteristics. Cis-acting element analysis showed that VcMYBs may be involved in biological stress, abiotic stress, hormone induction, plant growth and development, and light response. Fourteen genes (SG4, SG5, SG6 and SG7) that may be involved in the regulation of flavonoid synthesis were randomly selected for further analysis. RT-qPCR analysis showed that the most of the VcMYBs had high expressions in fruits. Among them, VcMYB91(SG6) and VcMYB56(SG5) genes were lowly expressed in the green fruit stage and highly expressed in the mature stage, suggesting that they may be involved in fruit coloring. However, VcMYB88(SG4) and VcMYB96(SG4) showed the opposite expression pattern, suggesting that they may inhibit fruit coloring. In addition, VcMYB236(SG7) and VcMYB44(SG7) genes may be involved in flavonoid synthesis. Conclusion The 278 blueberry R2R3-MYB genes were identified via genome-wide analysis. Functional dissection revealed that four genes (VcMYB56, VcMYB91, etc.) from the SG5/6 subgroups regulate anthocyanin biosynthesis, six genes (VcMYB88, VcMYB96, etc.) from the SG4/5 subgroups participate in proanthocyanidin synthesis, and two genes (VcMYB236, VcMYB44) from the SG7 subgroup may govern flavonol metabolism.

Key words: blueberry, R2R3-MYB, bioinformatics, tissue expression pattern, different fruit development stages, flavonoid synthesis

刘佳丽, 宋经荣, 赵文宇, 张馨元, 赵子洋, 曹一博, 张凌云. 蓝莓R2R3-MYB基因鉴定及类黄酮调控基因表达分析[J]. 生物技术通报, 2025, 41(9): 124-138.

LIU Jia-li, SONG Jing-rong, ZHAO Wen-yu, ZHANG Xin-yuan, ZHAO Zi-yang, CAO Yi-bo, ZHANG Ling-yun. Identification of the R2R3-MYB Gene and Expression Analysis of Flavonoid Regulatory Genes in Blueberry[J]. Biotechnology Bulletin, 2025, 41(9): 124-138.

图/表 8

表1 RT-qPCR引物

Table 1 Primers used for RT-qPCR

基因 Gene	引物 Primer （5'-3'）	∆G/∆g （25 ℃）（kcal/mol）	∆H/∆h （kcal/mol）	3'∆G （kcal/mol）	Tm （℃）	GC （%）	Primer score	PCR score
VcMYB44	F： GACCAGACCTAAAACGAGGCAAC	-36.5/-35.0	-190.5/-181.6	-7.3	60.8	52.2	941	920
VcMYB44	R： GGCAATCTTCCTGCTATTAGTGACC	-37.9/-36.7	-201.5/-194.0	-7.1	60.4	48.0	963	920
VcMYB56	F： TCGGAATATCCTAACGACCAACCAC	-37.9/-37.2	-202.2/-194.5	-7.2	60.3	48.0	946	889
VcMYB56	R： GACTTTCTGCTTTCGGGCCTC	-34.0/-32.9	-173.0/-166.5	-7.7	60.8	57.1	856	889
VcMYB77	F： TTCTTCCGATCAGGACAAACCCTC	-36.9/-36.0	-193.2/-185.4	-7.3	60.9	50.0	940	877
VcMYB77	R： TGACACTGCCGTGGTAATACCC	-34.5/-34.0	-179.3/-171.5	-6.7	60.0	54.5	897	877
VcMYB88	F： TTGCCTTTCCCTCTCCTCTAATGC	-36.6/-35.9	-191.7/-184.0	-6.7	60.6	50.0	880	900
VcMYB88	R： TAGGAATCCCAAGACTTTCCGTTT	-34.5/-34.3	-177.7/-181.3	-6.8	60.5	41.7	920	900
VcMYB91	F： CCACCTGACCGACAAATGCAA	-33.3/-32.7	-168.9/-164.8	-7.3	60.6	52.4	930	926
VcMYB91	R： AACCGTGCATATCCCTTACGAC	-34.5/-33.2	-177.3/-172.0	-7.5	60.5	50.0	941	926
VcMYB96	F： CCTTATCAAAACCAATCTGAACCGT	-37.0/-35.6	-194.0/-192.1	-8.0	60.9	40.0	839	867
VcMYB96	R： TTTGCTACTGCCACTATTCCCAC	-35.1/-34.2	-182.7/-177.3	-7.6	60.3	47.8	901	867
VcMYB103	F： TCACGCGCTGAATCCTAACCC	-34.2/-33.5	-174.1/-166.6	-7.1	61.0	57.1	936	883
VcMYB103	R： AACACTGCCGTGGTAATACCC	-32.9/-32.0	-167.4/-162.7	-6.7	60.1	52.4	889	883
VcMYB139	F： GTTGAGAGATCCCTCCGACCA	-32.9/-32.1	-167.1/-161.7	-7.1	60.3	57.1	920	888
VcMYB139	R： CTTTTCGGTCAGTAGGACTTCGTT	-36.0/-35.4	-187.2/-187.6	-7.1	60.8	45.8	933	888
VcMYB172	F： CCTCGCTCTCAAAATCCACGTC	-35.4/-34.2	-183.7/-175.9	-7.5	60.5	54.5	928	879
VcMYB172	R： TCTAGACAACTTGCAGCATCGG	-33.7/-33.4	-171.9/-172.6	-7.3	60.5	50.0	954	879
VcMYB214	F： GAAAGTCCATAACCCAAAGCCCAA	-36.1/-35.4	-188.9/-183.5	-7.1	60.6	45.8	918	893
VcMYB214	R： TCTTTAGGAGGGCAATCAAAGCTAC	-37.0/-36.1	-195.5/-191.3	-6.6	60.3	44.0	867	893
VcMYB229	F： TGAATTACCTCCGTCCGAACGTC	-36.3/-35.5	-191.1/-182.4	-7.5	60.3	52.2	920	793
VcMYB229	R： TCTCCCAGCTATCAGCGACCA	-32.8/-32.9	-166.6/-161.9	-7.1	60.2	57.1	919	793
VcMYB236	F： AGTTGTAATGCCGTCAACACCGA	-35.7/-35.5	-186.5/-180.7	-7.9	60.5	47.8	887	857
VcMYB236	R： ACTAGTGCCGTGATCATTTATTCCG	-38.1/-36.7	-201.0/-195.2	-7.4	61.0	44.0	904	857
VcMYB256	F： ACTACTGGAACACCCATATCCGAA	-35.4/-35.0	-183.1/-181.9	-7.4	60.8	45.8	937	861
VcMYB256	R： ATTAGCAGTCGCAAAAGATATGGT	-34.7/-34.0	-180.2/-183.3	-6.6	60.1	37.5	879	861
VcMYB259	F： TCCTTAATTGCGGGTAGACTTCCA	-35.1/-35.3	-182.2/-182.8	-6.7	60.3	45.8	887	866
VcMYB259	R： TCCTTTCAGTCGTATTCAAGGTCG	-36.1/-35.3	-189.0/-187.4	-7.9	60.4	45.8	866	866

表1 RT-qPCR引物

Table 1 Primers used for RT-qPCR

基因 Gene	引物 Primer （5'-3'）	∆G/∆g （25 ℃）（kcal/mol）	∆H/∆h （kcal/mol）	3'∆G （kcal/mol）	Tm （℃）	GC （%）	Primer score	PCR score
VcMYB44	F： GACCAGACCTAAAACGAGGCAAC	-36.5/-35.0	-190.5/-181.6	-7.3	60.8	52.2	941	920
VcMYB44	R： GGCAATCTTCCTGCTATTAGTGACC	-37.9/-36.7	-201.5/-194.0	-7.1	60.4	48.0	963	920
VcMYB56	F： TCGGAATATCCTAACGACCAACCAC	-37.9/-37.2	-202.2/-194.5	-7.2	60.3	48.0	946	889
VcMYB56	R： GACTTTCTGCTTTCGGGCCTC	-34.0/-32.9	-173.0/-166.5	-7.7	60.8	57.1	856	889
VcMYB77	F： TTCTTCCGATCAGGACAAACCCTC	-36.9/-36.0	-193.2/-185.4	-7.3	60.9	50.0	940	877
VcMYB77	R： TGACACTGCCGTGGTAATACCC	-34.5/-34.0	-179.3/-171.5	-6.7	60.0	54.5	897	877
VcMYB88	F： TTGCCTTTCCCTCTCCTCTAATGC	-36.6/-35.9	-191.7/-184.0	-6.7	60.6	50.0	880	900
VcMYB88	R： TAGGAATCCCAAGACTTTCCGTTT	-34.5/-34.3	-177.7/-181.3	-6.8	60.5	41.7	920	900
VcMYB91	F： CCACCTGACCGACAAATGCAA	-33.3/-32.7	-168.9/-164.8	-7.3	60.6	52.4	930	926
VcMYB91	R： AACCGTGCATATCCCTTACGAC	-34.5/-33.2	-177.3/-172.0	-7.5	60.5	50.0	941	926
VcMYB96	F： CCTTATCAAAACCAATCTGAACCGT	-37.0/-35.6	-194.0/-192.1	-8.0	60.9	40.0	839	867
VcMYB96	R： TTTGCTACTGCCACTATTCCCAC	-35.1/-34.2	-182.7/-177.3	-7.6	60.3	47.8	901	867
VcMYB103	F： TCACGCGCTGAATCCTAACCC	-34.2/-33.5	-174.1/-166.6	-7.1	61.0	57.1	936	883
VcMYB103	R： AACACTGCCGTGGTAATACCC	-32.9/-32.0	-167.4/-162.7	-6.7	60.1	52.4	889	883
VcMYB139	F： GTTGAGAGATCCCTCCGACCA	-32.9/-32.1	-167.1/-161.7	-7.1	60.3	57.1	920	888
VcMYB139	R： CTTTTCGGTCAGTAGGACTTCGTT	-36.0/-35.4	-187.2/-187.6	-7.1	60.8	45.8	933	888
VcMYB172	F： CCTCGCTCTCAAAATCCACGTC	-35.4/-34.2	-183.7/-175.9	-7.5	60.5	54.5	928	879
VcMYB172	R： TCTAGACAACTTGCAGCATCGG	-33.7/-33.4	-171.9/-172.6	-7.3	60.5	50.0	954	879
VcMYB214	F： GAAAGTCCATAACCCAAAGCCCAA	-36.1/-35.4	-188.9/-183.5	-7.1	60.6	45.8	918	893
VcMYB214	R： TCTTTAGGAGGGCAATCAAAGCTAC	-37.0/-36.1	-195.5/-191.3	-6.6	60.3	44.0	867	893
VcMYB229	F： TGAATTACCTCCGTCCGAACGTC	-36.3/-35.5	-191.1/-182.4	-7.5	60.3	52.2	920	793
VcMYB229	R： TCTCCCAGCTATCAGCGACCA	-32.8/-32.9	-166.6/-161.9	-7.1	60.2	57.1	919	793
VcMYB236	F： AGTTGTAATGCCGTCAACACCGA	-35.7/-35.5	-186.5/-180.7	-7.9	60.5	47.8	887	857
VcMYB236	R： ACTAGTGCCGTGATCATTTATTCCG	-38.1/-36.7	-201.0/-195.2	-7.4	61.0	44.0	904	857
VcMYB256	F： ACTACTGGAACACCCATATCCGAA	-35.4/-35.0	-183.1/-181.9	-7.4	60.8	45.8	937	861
VcMYB256	R： ATTAGCAGTCGCAAAAGATATGGT	-34.7/-34.0	-180.2/-183.3	-6.6	60.1	37.5	879	861
VcMYB259	F： TCCTTAATTGCGGGTAGACTTCCA	-35.1/-35.3	-182.2/-182.8	-6.7	60.3	45.8	887	866
VcMYB259	R： TCCTTTCAGTCGTATTCAAGGTCG	-36.1/-35.3	-189.0/-187.4	-7.9	60.4	45.8	866	866

图1 蓝莓和拟南芥R2R3-MYB家族系统进化树分析图中At代表拟南芥基因，Vc代表蓝莓基因，SG（Subgroup）是指R2R3-MYB转录因子家族中系统发育中的亚家族，下同

Fig. 1 Phylogenetic tree analysis of R2R3-MYB family in V. corymbosum and A. thalianaAt and Vc refer to genes from Arabidopsis thaliana and Vaccinium corymbosum, respectively; SG (Subgroup) refers to phylogenetically defined subgroups within the R2R3-MYB transcription factor family, the same below

图2 蓝莓R2R3-MYB家族成员基因结构、保守基序及保守结构域分析A：蓝莓R2R3-MYB家族成员内部系统进化树；B：蓝莓R2R3-MYB家族成员基因结构；C：蓝莓R2R3-MYB家族成员保守基序；D：蓝莓R2R3-MYB家族成员基因保守结构域；E：蓝莓R2R3-MYB家族成员保守基序的序列Logo

Fig. 2 Analysis of gene structures, conserved motifs, and conserved domains in R2R3-MYB family members from blueberryA: Phylogenetic tree of blueberry R2R3-MYB family members; B: Gene structure of blueberry R2R3-MYB family members. C: Conserved motifs of blueberry R2R3-MYB family members. D: Conserved domains of blueberry R2R3-MYB family members. E: Sequence logo of conserved motifs in R2R3-MYB family members from blueberry

图3 蓝莓R2R3-MYB家族成员的支架定位

Fig. 3 Scaffold localization of blueberry R2R3-MYB family members

图4 蓝莓R2R3-MYB家族成员的支架分布及其重复事件

Fig. 4 Scaffold distribution and repetitive events of blueberry R2R3-MYB family members

图5 蓝莓与不同物种的R2R3-MYB基因共线性分析A：蓝莓与不同物种（拟南芥、番茄、葡萄、桃和猕猴桃）的R2R3-MYB基因共线性分析；B：Venn图分析蓝莓和其他物种（拟南芥、番茄、葡萄、桃和猕猴桃）的相同和不同R2R3-MYB基因

Fig. 5 Colinearity analysis of R2R3-MYB genes between blueberry and different speciesA: Colinearity analysis of R2R3-MYB genes between blueberry and different species (A. thaliana, Solanum lycopersicum, Vitis vinifera, Prunus persica, and Actinidia chinensis); B: The Venn diagram analyzed the same and different R2R3-MYB genes in blueberry and different species (A. thaliana, Solanum lycopersicum, Vitis vinifera, Prunus persica, and Actinidia chinensis)

图6 蓝莓R2R3-MYB家族成员启动子顺式作用元件

Fig. 6 Promoter cis-acting elements of blueberry R2R3-MYB family members

图7 蓝莓14个R2R3-MYB家族成员在不同组织和不同果实发育期中的RT-qPCR检测A：不同组织（根、茎、叶和花）和不同果实发育期（小绿果、大绿果、转色果和成熟果的表型图，标尺=0.25 cm；B：蓝莓14个不同R2R3-MYB基因在不同组织和不同果实发育期的相对表达量，数据采用单因素方差分析和邓肯多范围检验进行分析，不同字母表示显著差异（P<0.05）

Fig. 7 RT-qPCR detection of 14 R2R3-MYB family members in different tissues and different fruit development stages of blueberryA: Phenotypic maps of different tissues (roots, stems, leaves and flowers) and different fruit development stages (small green fruit, large green fruit, color-changing fruit and mature fruit), scale=0.25 cm. B: Relative expressions of 14 different R2R3-MYB genes in different tissues and different fruit development stages of blueberry. Data were analyzed by one-way ANOVA with Duncan's multiple range test, where different superscript letters denote significant differences (P<0.05)

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