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GUO Yuting, DU Changxia. Identification and bioinformatics analysis of R2R3-MYB subfamily in cucumber[J]. Journal of Zhejiang A&F University, 2024, 41(2): 286-296. doi: 10.11833/j.issn.2095-0756.20230278
Citation: GUO Yuting, DU Changxia. Identification and bioinformatics analysis of R2R3-MYB subfamily in cucumber[J]. Journal of Zhejiang A&F University, 2024, 41(2): 286-296. doi: 10.11833/j.issn.2095-0756.20230278
shu

Identification and bioinformatics analysis of R2R3-MYB subfamily in cucumber

doi: 10.11833/j.issn.2095-0756.20230278
  • 1.

    Collaborative Innovation Center for Efficient and Green Production of Agriculture in Mountainous Areas of Zhejiang Province, College of Horticulture Science, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China

  • 2.

    Key Laboratory of Quality and Safety Control for Subtropical Fruit and Vegetable, Ministry of Agriculture and Rural Affairs, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China

  • Received Date: 2023-05-05
  • Accepted Date: 2023-11-30
  • Rev Recd Date: 2023-11-08
    • Available Online: 2024-03-21
  • Publish Date: 2024-04-01
  •   Objective  This study is aimed to conduct an in-depth study of the functions associated with members of the R2R3-MYB subfamily in cucumber (Cucumis sativus).   Method  First, an analysis was conducted of the whole cucumber genome so as to identify R2R3-MYB subfamily members. Then, bioinformatics was employed to analyze their phylogenetic relationships, physicochemical properties of proteins, chromosomal localization, gene structure, conserved motifs, and cis-acting elements, as well as the protein interactions.   Result  The whole cucumber genome contained 99 R2R3-MYB transcription factors with typical structural domains, and the protein sequences contained amino acids ranging from 195 to 552, with conserved motifs and amino acid sites. They were unevenly distributed on chromosomes. They were unstable proteins with the instability index of most subfamily members being greater than 40. As was shown by the analysis of cis-acting regulatory elements, most of the elements contained in the promoter regions of genes were closely related to hormone regulation, MYB binding sites, and stress.   Conclusion  The R2R3-MYB family members were obtained through whole cucumber genome identification, which were divided into 30 subgroups and mapped on 7 chromosomes and the upstream promoter region of this family members contained stress-related action elements. [Ch, 7 fig. 1 tab. 36 ref.]
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Identification and bioinformatics analysis of R2R3-MYB subfamily in cucumber

doi: 10.11833/j.issn.2095-0756.20230278
  • 1. Collaborative Innovation Center for Efficient and Green Production of Agriculture in Mountainous Areas of Zhejiang Province, College of Horticulture Science, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China
  • 2. Key Laboratory of Quality and Safety Control for Subtropical Fruit and Vegetable, Ministry of Agriculture and Rural Affairs, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China
  • Received Date: 2023-05-05
  • Accepted Date: 2023-11-30
  • Rev Recd Date: 2023-11-08
  • Available Online: 2024-03-21
  • Publish Date: 2024-04-01

Abstract:   Objective  This study is aimed to conduct an in-depth study of the functions associated with members of the R2R3-MYB subfamily in cucumber (Cucumis sativus).   Method  First, an analysis was conducted of the whole cucumber genome so as to identify R2R3-MYB subfamily members. Then, bioinformatics was employed to analyze their phylogenetic relationships, physicochemical properties of proteins, chromosomal localization, gene structure, conserved motifs, and cis-acting elements, as well as the protein interactions.   Result  The whole cucumber genome contained 99 R2R3-MYB transcription factors with typical structural domains, and the protein sequences contained amino acids ranging from 195 to 552, with conserved motifs and amino acid sites. They were unevenly distributed on chromosomes. They were unstable proteins with the instability index of most subfamily members being greater than 40. As was shown by the analysis of cis-acting regulatory elements, most of the elements contained in the promoter regions of genes were closely related to hormone regulation, MYB binding sites, and stress.   Conclusion  The R2R3-MYB family members were obtained through whole cucumber genome identification, which were divided into 30 subgroups and mapped on 7 chromosomes and the upstream promoter region of this family members contained stress-related action elements. [Ch, 7 fig. 1 tab. 36 ref.]

GUO Yuting, DU Changxia. Identification and bioinformatics analysis of R2R3-MYB subfamily in cucumber[J]. Journal of Zhejiang A&F University, 2024, 41(2): 286-296. doi: 10.11833/j.issn.2095-0756.20230278
Citation: GUO Yuting, DU Changxia. Identification and bioinformatics analysis of R2R3-MYB subfamily in cucumber[J]. Journal of Zhejiang A&F University, 2024, 41(2): 286-296. doi: 10.11833/j.issn.2095-0756.20230278
shu

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  • MYB蛋白质家族成员数量庞大，功能多样，存在于绝大多数真核生物中。大多数MYB蛋白作为转录因子发挥作用，MYB结合域有结合DNA的能力[1−2]，在植物生长发育、次生代谢及抗逆胁迫等方面具有重要作用[3]。R2R3-MYB作为家族中成员最多的一类，对植物生长发育意义重大[4−7]。

    MYB基因家族根据MYB结构域的数量和类型分为4类：1R-MYB (或MYB相关)、2R-MYB (R2R3-MYB)、3R-MYB (R1R2R3-MYB)和4R-MYB[8]。2R-MYB类是植物特有的，并且是最大的亚家族，已在许多植物中进行了全基因组注释。R2R3-MYB家族成员数量各不相同，拟南芥Arabidopsis thaliana[9]有126个，水稻Oryza sativa[10]有102个，葡萄Vitis vinifera[11]有108个，甜橙Citrus sinensis[12]有100个，苹果Malus domestica[13]有222个，毛果杨Populus trichocarpa[14]有192个，玉米Zea mays[15]有200多个。LI等[16]在黄瓜Cucumis sativus中仅鉴定出55个R2R3-MYB基因，而黄瓜基因组V2版本中鉴定出69个R2R3-MYB基因[17]。通过最新版黄瓜基因组数据库可知：黄瓜基因组V3版本基因组数量为24 317个，较V2版本(23 248个)增多。鉴于此，本研究对黄瓜基因组数据库V3版本中的R2R3-MYB亚家族进行鉴定，分析黄瓜CsMYB基因家族成员，为研究黄瓜CsMYB家族功能提供参考。

    • 1.   材料与方法

      1.1.   数据来源

    • 全基因组序列来源于黄瓜基因组数据库(http://www.cucurbitgenomics.org/organism/20)。序列中的MYB结合域(PF00249)使用PFam数据库(http://pfam.xfam.org/)的隐马尔可夫模型(HMM)配置文件进行识别和确认，该配置文件用作对先前注释的黄瓜MYB序列的查询，通过HMMER SEARCH进一步识别并使用默认参数手动筛选(目标序列的期望值E≤1.2×10−8)[17]。应用HMMER SEARCH建立黄瓜MYB (CsMYB)基因家族特异性模型[18]，使用黄瓜MYB特异性HMM选择E低于0.001的所有黄瓜MYB蛋白。CsMYB家族的候选基因根据黄瓜特异性MYB HMM确定。以上最初获得的所有候选CsMYB基因均通过保守域数据库(conserved domain database，CDD)[19]和简单模块化架构研究工具(simplemodular architecture research tool，SMART)使用默认参数E≤0.010[20]。根据MYB序列内的特征性保守结构域，分离黄瓜R2R3-MYB基因。

      • 1.2.   黄瓜R2R3-MYB基因家族系统进化分析

    • 通过ClustalW对模式植物拟南芥、木本植物杨梅Morella rubra[21]和草本植物黄瓜[1]的R2R3-MYB序列进行比对，采用MEGA-11软件的邻接法(neighbor-joining，NJ)，设置自展法系数(bootstrap)为1 000次，其余参数按系统默认构建进化树[22]。

      • 1.3.   黄瓜R2R3-MYB家族成员的基本性质及定位分析

    • 通过筛选结果得到的黄瓜R2R3-MYB基因的序列信息，利用TBtools将其定位于染色体上。采用ExPASy-ProtParam (https://www.expasy.org/resources/protparam)在线预测黄瓜R2R3-MYB蛋白的氨基酸数量、分子量、理论等电点、不稳定指数及脂肪系数；使用Plant-mPLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/)预测亚细胞定位。

      • 1.4.   黄瓜R2R3-MYB家族成员的染色体定位及共线性分析

    • 利用TBtools中的Gene Location Visualize功能绘制黄瓜R2R3-MYB家族成员的染色体定位图，并通过MCscanX将黄瓜R2R3-MYB亚家族成员基因与拟南芥同源基因进行共线性分析。

      • 1.5.   黄瓜R2R3-MYB亚家族成员的基因结构及基序分析

    • 利用TBtools对黄瓜R2R3-MYB亚家族成员的基因结构进行可视化分析，并对其蛋白的保守基序进行分析，限值为15，绘制结果图。

      • 1.6.   黄瓜R2R3-MYB亚家族上游的顺式元件预测

    • 利用TBtools提取黄瓜R2R3-MYB亚家族成员上游1 500 bp的序列，通过PlantCARE 数据库(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)获得启动子顺式作用元件，利用Excel筛选结果，再利用TBtools对其最终结果进行可视化。

      • 1.7.   黄瓜R2R3-MYB亚家族蛋白成员的互作预测

    • 通过网站STRING (https://www.string-db.org/)对黄瓜R2R3-MYB蛋白成员之间进行互作预测分析，研究黄瓜R2R3-MYB蛋白成员之间的作用关系。

    2.   结果与分析

      2.1.   黄瓜R2R3-MYB亚家族全基因组鉴定及进化树构建

    • 将黄瓜基因组数据与拟南芥进行比对，根据HMM模型进行搜索，取其交集，筛选鉴定其具有特殊结构域的序列，最终获得黄瓜99个R2R3-MYB基因家族成员。参照DUBOS等[23]的分类方法，将鉴定出的成员进一步分为30个亚组。为了探索系统发育关系，通过ClustalW进行序列比对，使用黄瓜99个CsMYB蛋白、拟南芥125个AtMYB蛋白和杨梅122个MrMYB蛋白构建了系统发育树(图1)。按照染色体定位结果，分别命名为Cs2RMYB1~Cs2RMYB99 (表1)。根据蛋白质序列的相似性和聚类结果的高自举性，将CsMYB进一步分为30个进化枝(指定为C1~C30)，黄瓜R2R3-MYB基因家族成员被分成30个亚组。其中，C16、C20和C23亚组的家族成员数量最多，有21个成员；C18亚组成员数为16个；C14、C24和C27亚组家族成员数均为15个；C10和C28亚组家族成员数量等同，为4个；C9亚组成员数最少，仅3个。黄瓜R2R3-MYB亚家族的进化树聚类方式与拟南芥有一定的相似性。

      Figure 1.  Phylogenetic tree construction of C. sativus, A. thaliana and M. rubra R2R3-MYB subfamily members

      基因命名氨基酸
      数量/个      		分子量/kDa等电点不稳定
      指数      		脂肪指数染色体
      位置      		基因命名氨基酸
      数量/个      		分子量/
      kDa      		等电点不稳定
      指数      		脂肪指数染色体
      位置
      Cs2RMYB136040.916.4452.4157.17Chr 1Cs2RMYB5126830.018.5954.6564.40Chr 3
      Cs2RMYB247154.885.9142.8466.43Chr 1Cs2RMYB5221725.137.2155.7565.21Chr 3
      Cs2RMYB336741.945.8359.2275.50Chr 1Cs2RMYB5319522.678.3772.5973.54Chr 3
      Cs2RMYB433137.609.0265.1561.90Chr 1Cs2RMYB5435539.216.2554.4682.96Chr 3
      Cs2RMYB527731.195.9078.8866.21Chr 1Cs2RMYB5530534.196.1945.7974.52Chr 3
      Cs2RMYB623226.598.8357.2666.51Chr 1Cs2RMYB5625328.529.1049.0965.53Chr 3
      Cs2RMYB732636.766.4253.9167.94Chr 1Cs2RMYB5731335.054.9768.2475.97Chr 4
      Cs2RMYB828832.506.3171.3064.03Chr 1Cs2RMYB5831436.066.1062.9858.06Chr 4
      Cs2RMYB937742.096.4650.2366.71Chr 1Cs2RMYB5932235.686.1454.4667.55Chr 4
      Cs2RMYB1033737.925.7445.2471.19Chr 1Cs2RMYB6037740.505.4139.9073.24Chr 4
      Cs2RMYB1129933.999.1758.1761.64Chr 1Cs2RMYB6135339.816.2260.2859.92Chr 4
      Cs2RMYB1237242.336.1254.3267.63Chr 1Cs2RMYB6222925.199.2958.9061.70Chr 4
      Cs2RMYB1327732.115.8363.4679.96Chr 1Cs2RMYB6326329.148.3461.1467.15Chr 4
      Cs2RMYB1433738.248.6652.3859.08Chr 1Cs2RMYB6428031.518.9259.5372.11Chr 4
      Cs2RMYB1537041.156.1351.9761.95Chr 1Cs2RMYB6526028.845.0038.8676.54Chr 5
      Cs2RMYB1636441.759.5455.5869.81Chr 1Cs2RMYB6632336.416.0053.2472.45Chr 5
      Cs2RMYB1732737.067.6862.3169.48Chr 2Cs2RMYB6725829.465.8445.2867.67Chr 5
      Cs2RMYB1829532.999.0745.4170.10Chr 2Cs2RMYB6823626.986.7153.4874.83Chr 5
      Cs2RMYB1922225.746.0053.8672.43Chr 2Cs2RMYB6928431.236.4156.1872.46Chr 5
      Cs2RMYB2026429.988.9758.2463.14Chr 2Cs2RMYB7030033.775.2955.2371.50Chr 5
      Cs2RMYB2126531.429.6454.5267.02Chr 2Cs2RMYB7133738.257.6955.1066.59Chr 5
      Cs2RMYB2228632.595.6058.7259.69Chr 2Cs2RMYB7221024.435.1951.2560.38Chr 5
      Cs2RMYB2322826.8710.2758.0865.44Chr 2Cs2RMYB7346452.296.8266.0966.85Chr 5
      Cs2RMYB2425629.158.2448.2579.22Chr 2Cs2RMYB7436941.406.6150.6856.34Chr 5
      Cs2RMYB2521023.5410.3165.3259.48Chr 2Cs2RMYB7530734.257.5148.6464.59Chr 5
      Cs2RMYB2623026.328.8357.6463.65Chr 2Cs2RMYB7639844.526.4953.5662.29Chr 5
      Cs2RMYB2720423.048.4443.7867.94Chr 2Cs2RMYB7720223.938.2543.6158.37Chr 5
      Cs2RMYB2833838.468.5658.0068.99Chr 2Cs2RMYB7829433.138.7252.0770.41Chr 5
      Cs2RMYB2929031.809.2359.3365.93Chr 2Cs2RMYB7928632.776.4064.0365.17Chr 6
      Cs2RMYB3029733.709.9755.6569.97Chr 2Cs2RMYB8055261.135.5052.5161.54Chr 6
      Cs2RMYB3130132.308.1955.9064.15Chr 2Cs2RMYB8130134.405.5670.9765.08Chr 6
      Cs2RMYB3230834.865.9348.4784.90Chr 2Cs2RMYB8226931.336.4545.9563.42Chr 6
      Cs2RMYB3351957.037.4659.7057.53Chr 3Cs2RMYB8333438.244.9646.9559.85Chr 6
      Cs2RMYB3430534.645.7057.5870.72Chr 3Cs2RMYB8428632.925.8056.5054.58Chr 6
      Cs2RMYB3524829.149.1056.6166.49Chr 3Cs2RMYB8529133.536.6361.7046.94Chr 6
      Cs2RMYB3625929.986.5048.7459.54Chr 3Cs2RMYB8627732.269.3551.4559.49Chr 6
      Cs2RMYB3729633.705.3556.2767.20Chr 3Cs2RMYB8730132.459.3250.9164.82Chr 6
      Cs2RMYB3821925.256.3259.7970.32Chr 3Cs2RMYB8831935.856.5348.5165.49Chr 6
      Cs2RMYB3935440.509.2058.2277.40Chr 3Cs2RMYB8934438.446.1257.7280.00Chr 6
      Cs2RMYB4026730.105.5955.7069.33Chr 3Cs2RMYB9038744.365.8154.9361.01Chr 6
      Cs2RMYB4124528.135.7548.7062.57Chr 3Cs2RMYB9128933.196.4553.1570.17Chr 6
      Cs2RMYB4222425.607.6465.4374.87Chr 3Cs2RMYB9224827.749.1146.2476.73Chr 7
      Cs2RMYB4327531.618.7139.6779.75Chr 3Cs2RMYB9350054.625.8955.4657.82Chr 7
      Cs2RMYB4431636.225.6358.8756.52Chr 3Cs2RMYB9425429.455.3674.1375.63Chr 7
      Cs2RMYB4531234.716.9955.7062.79Chr 3Cs2RMYB9527131.905.5653.1762.66Chr 7
      Cs2RMYB4624829.708.4652.7369.60Chr 3Cs2RMYB9625429.405.0243.8679.49Chr 7
      Cs2RMYB4734839.138.8646.8264.77Chr 3Cs2RMYB9728032.365.2059.2168.86Chr 7
      Cs2RMYB4829432.666.1757.4064.42Chr 3Cs2RMYB9828532.825.3258.4764.70Chr 7
      Cs2RMYB4923326.139.3351.3365.41Chr 3Cs2RMYB9924827.786.2444.8065.28Scaffold 72
      Cs2RMYB5031335.135.9554.6365.50Chr 3
      　　说明：Cs2RMYB48基因定位于叶绿体，其他基因定位于细胞核。

      Table 1.  Cucumber R2R3-MYB subfamily member information

      • 2.2.   黄瓜R2R3-MYB亚家族的理化性质分析

    • 表1表明：黄瓜R2R3-MYB亚家族成员编码195~552个氨基酸，分子量为22.67~61.13 kDa，理论等电点为4.96~10.31，59个R2R3-MYB蛋白的等电点小于7，40个R2R3-MYB蛋白的等电点大于7，超过半数的蛋白为酸性。除Cs2RMYB43、Cs2RMYB60、Cs2RMYB65外，其余CsR2R3-MYB亚家族成员蛋白质不稳定指数为42.84~78.88，大于40.00，为不稳定蛋白。所有成员的脂肪系数相对较高，说明黄瓜R2R3-MYB亚家族成员适应环境的多样性。Plant-mPLoc 2.0网站预测结果显示：除Cs2RMYB48定位于叶绿体，其他成员均定位于细胞核。

      • 2.3.   黄瓜R2R3-MYB亚家族染色体定位及共线性分析

    • 黄瓜R2R3-MYB亚家族成员映射于1~7号染色体上，Cs2RMYB99的染色体定位结果未知(图2)。每条染色体上的基因分布相对不均匀，1、4、5、7号染色体上基因大多分布在上下两端，而剩下的染色体上基因分布较为均匀。3号染色体上数量最多，可以定位到24个R2R3-MYB亚家族成员；1、2号染色体各有16个R2R3-MYB亚家族成员；5、6号染色体分别为14和13个R2R3-MYB亚家族成员；7号染色体上最少，为7个R2R3-MYB亚家族成员。此外，2号染色体上的Cs2RMYB27和Cs2RMYB28、3号染色体上的Cs2RMYB45和Cs2RMYB46、5号染色体上的Cs2RMYB71和Cs2RMYB72、7号染色体上的Cs2RMYB94和Cs2RMYB95均在各自染色体上形成基因簇。根据进化树分析可知：形成基因簇的成员之间同源性较高，推测成员间功能相对保守。

      Figure 2.  Chromosome location map of C. sativus R2R3-MYB subfamily

      为揭示黄瓜R2R3-MYB亚家族成员在整个黄瓜基因组中的连锁关系，对黄瓜与拟南芥MYB基因进行共线性分析(图3)，结果表明：25对基因表现出共线性和保守的连锁关系，即表明2个物种之间MYB基因家族存在高同源性。

      Figure 3.  Analysis of the collinearity between C. sativus R2R3-MYB subfamily and A. thaliana

      • 2.4.   黄瓜R2R3-MYB亚家族基因结构及保守基序分析

    • 利用TBtools对黄瓜R2R3-MYB亚家族进行保守基序分析(图4)。结果显示：在99个R2R3-MYB蛋白中鉴定出10个不同的基序，其中Motif 3、Motif 7、Motif 2和Motif 4是高度保守的基序，所有成员均含有Motif 3，导致成员间功能相似。表明这些黄瓜R2R3-MYB亚家族成员间拥有特有或相同的基序，导致了它们之间具有特定的或者相似的功能。

      Figure 4.  Protein conservative motifs and gene structure of R2R3-MYB subfamily in C. sativus

      通过Jalview对黄瓜R2R3-MYB亚家族进行序列比对(图5)。结果显示：2R重复序列中有3个色氨酸残基(W)及3R重复序列中有3个色氨酸残基，形成疏水结构，此为鉴定MYB家族典型的结构域特征。除保守的W外，2R重复序列的末端还存在12个高度保守的氨基酸残基，如精氨酸(R)、甘氨酸(G)、赖氨酸(K)、丝氨酸(S)、半胱氨酸(C)、亮氨酸(L)等，3R重复序列中的谷氨酸(E)、G、丙氨酸(A)、R、苏氨酸(T)等。

      Figure 5.  Sequence comparison of C. sativus R2R3-MYB subfamily proteins

      • 2.5.   黄瓜R2R3-MYB亚家族顺式作用元件分析

    • 为了进一步研究黄瓜R2R3-MYB亚家族成员启动子区的顺式作用元件，通过PlantCARE在线网站分析了该亚家族上游1 500 bp区域(图6)。其启动子元件多与激素、抗逆及厌氧诱导相关，大部分黄瓜R2R3-MYB亚家族成员含雌激素响应(ERE)作用元件。从元件类型来看，ERE元件最多，分布于绝大多数成员的启动子区域上。从单个元件的具体分布来看，约68%家族成员启动子区含ERE元件，约60%成员启动子区含脱落酸应答元件(ABRE)。从单个基因家族成员来看，Cs2RMYB92启动子只有TCA-element激素调控相关元件；Cs2RMYB98有MYB结合位点，并富含激素响应与厌氧诱导元件，例如ABRE、ERE、茉莉酸甲酯相关元件(CGTCA)和MYB结合位点元件(MBS)。Cs2RMYB5、Cs2RMYB7和Cs2RMYB8等基因上有ABRE元件，说明这些基因可能与脱落酸响应有关；Cs2RMYB2、Cs2RMYB6和Cs2RMYB9等基因上含ERE元件，说明这些基因可能响应乙烯的调控；Cs2RMYB1、Cs2RMYB16和Cs2RMYB25等基因上有CGTCA元件，说明这些基因可能与茉莉酸甲酯响应有关；Cs2RMYB12、Cs2RMYB25、Cs2RMYB33和Cs2RMYB34等基因启动子元件上都含MBS元件，说明这些基因可能响应干旱胁迫的调控。

      Figure 6.  Prediction of upstream homeopathic elements of C. sativus R2R3-MYB subfamily

      • 2.6.   黄瓜R2R3-MYB亚家族成员蛋白的互作预测

    • 为深入了解黄瓜R2R3-MYB亚家族成员蛋白的互作情况，对99个成员进行蛋白质互作预测(图7)，结果表明：Cs2RMYB25、Cs2RMYB3、Cs2RMYB84、Cs2RMYB98、Cs2RMYB6、Cs2RMYB6均与Cs2RMYB44存在互作关系；Cs2RMYB96、Cs2RMYB22、Cs2RMYB31、Cs2RMYB41、Cs2RMYB56、Cs2RMYB75、Cs2RMYB7、Cs2RMYB4、Cs2RMYB50均与Cs2RMYB90存在互作关系，且有较强的关联性。此外，部分成员间虽存在互作相关性，但各成员之间的具体机制并不明晰，仍需进一步深入研究。

      Figure 7.  Prediction of protein interactions among R2R3-MYB subfamily members in C. sativus

    3.   讨论

    • 本研究共鉴定出99个黄瓜R2R3-MYB亚家族成员，约占黄瓜总MYB数的60%，数量多于西瓜(89个)[4]，少于拟南芥(126个)[9]、水稻(102个)[10]和玉米(200个)[15]。黄瓜的基因组大小为224.8~251.1 Mb[24]，而拟南芥、水稻、玉米和西瓜的基因组大小分别为125.0、430.0、2 300.0和365.1 Mb[25−28]，R2R3-MYB亚家族成员数量与植物的基因组大小并没有直接的相关性。

      作为MYB家族最大的亚家族，R2R3-MYB家族成员具有典型的模式结构，在植物发育、新陈代谢、信号转导及响应生物和非生物胁迫中起着非常重要的作用[29]。为了研究黄瓜基因组进化过程中功能保守的R2R3-MYB基因功能，构建其系统进化树并进行分析，结果表明：黄瓜Cs2RMYB 30个亚组中大多数包含来自拟南芥和杨梅R2R3-MYB成员，然而，C28亚组仅拥有黄瓜和拟南芥R2R3-MYB成员，表明它们可能来自共同的祖先。同时，外显子和内含子的结构分析有利于研究基因家族内部的进化关系，通过分组可以初步推测其功能，聚类较近的基因存在功能相似性，如在C30亚组中，拟南芥AtMYB42和AtMYB85被证明参与木质素生物合成代谢调控[30]，黄瓜Cs2RMYB72和Cs2RMYB73与其表现出较近的亲缘关系，可能与木质素生物合成代谢有关；在C15亚组中，AtMYB75、AtMYB90、AtMYB113和AtMYB114过量表达会促进植物花青素的积累[31]，与黄瓜Cs2RMYB19同属于C15亚组；在C22亚组中，AtMYB93是其分支上唯一一个影响拟南芥侧根发育的负调控因子[32]，而黄瓜Cs2RMYB89与AtMYB93进化关系最近；宫思宇等[33]研究表明：AtMYB94通过调节拟南芥表皮的蜡质含量提高植株耐旱性，而C22亚组中的Cs2RMYB48是其同源基因，可能具有类似功能，同一分支亚家族转录因子可能存在功能相似性[23, 34]。

      基序分析结果发现：所有的CsR2R3-MYB基因都有共同的保守基序，但也有独特的基序，这为CsR2R3-MYB亚家族成员的分类提供参考，且可能与CsR2R3-MYB基因的功能分化相关。此外，启动子序列上的顺式作用元件是转录因子识别并发挥转录调控作用的重要识别位点[35]。本研究发现：黄瓜CsR2R3-MYB亚家族成员启动子上的顺式作用元件种类繁多，绝大多数CsR2R3-MYB基因家族成员基因启动子区都含有ERE元件，暗示其可能参与黄瓜性别分化等生长发育过程；富含ABRE和CGTCA等激素响应顺式作用元件，表明CsR2R3-MYB基因家族在黄瓜应答逆境胁迫中发挥重要作用。由于CsR2R3-MYB基因调控元件的不同功能，启动子区域中这些高度多样化的顺式调控元件也可能反映了转录水平上的功能分歧[36]。本研究初步预测了MYB家族CsR2R3-MYB基因与植物生长发育及逆境胁迫响应相关，可为后续该亚家族成员基因功能深入研究提供参考。

Reference (36)

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