Genome-wide Identification and Expressions under Stresses of RLCK VI Family in Gossypium barbadense
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摘要:
目的 对海岛棉（Gossypium barbadense）类受体胞质激酶RLCK VI（GbRLCK VI）家族基因进行全基因组分析，为深入研究 RLCK VI家族基因参与棉花生长发育和抗逆的调控机制提供参考。 方法 基于最新发布的海岛棉基因组数据，利用生物信息学手段对GbRLCK VI家族基因进行全基因组鉴定，并系统分析该家族基因成员的理化性质、序列特征、基因复制、系统进化和表达特征。 结果 海岛棉中共鉴定出39个GbRLCK VI家族基因，经聚类分析将其分为A、B两组，其中A组22个，B组17个，均含有1个激酶结构域，分布于16条染色体，多数位于质膜。基因复制分析表明，该家族在进化过程中发生染色体片段复制事件；Ka/Ks分析显示，所有基因对Ka/Ks均小于1，表明GbRLCK VI家族基因在进化过程中可能经历了严格的纯化选择作用。转录组分析表明，GbRLCK VI家族基因在10个不同组织中的表达模式不同，11个基因在花器官中优势表达，9个基因在根茎叶中优势表达；逆境胁迫下的表达分析显示，8个基因在干旱、盐、黄萎病胁迫下优势表达，4个基因只在黄萎病胁迫下优势表达，说明GbRLCK VI基因能快速地参与抗逆反应，挑选4个基因GB_A12G0061、GB_A11G2234、GB_D01G2010、GB_D03G0730进行qRT-PCR验证，表达分析结果显示，4个基因在干旱、盐或黄萎病菌胁迫下的表达趋势与转录组数据一致，表明它们参与了棉花对逆境胁迫（干旱、盐和黄萎病菌）的响应过程。 结论 明确了GbRLCK VI家族基因在基因组中的分布特征、结构特征以及系统进化特征，根据转录组数据初步揭示了该家族基因在棉花生长发育和抗逆胁迫中的功能。 Abstract:Objective Genomes and expressions under stresses of the RLCK VI family genes in Gossypium barbadense were determined to study the regulatory mechanisms of the growth, development, and stress resistance of cotton plants. Method Based on the latest released data on G. barbadense genome, bioinformatics of GbRLCK VI was analyzed to understand the associated physiochemical properties, sequence characteristics, gene replication, phylogenetic evolution, and expression. Result Thirty-nine RLCK VI were identified in G. barbadense which were clustered into two categories of 22 in Group A and 17 in Group B. Both groups contained a kinase domain distributed in 16 chromosomes with most of them located in plasma membrane. The gene family had undergone chromosome fragment duplication events during evolution. Since all Ka/Ks of the gene pairs were less than 1, strict purification and selection might have taken place in the process. The expressions of GbRLCK VI as shown by the transcriptome analysis varied in 10 different tissues with 11 predominantly expressed in the floral organs, while 9 in the roots, stems, and leaves. Under different stresses, 8 genes were significantly expressed by the imposed drought, salt, and verticillium wilt, and 4 only by verticillium wilt. On 4 selected genes, i.e., GB_ A12G0061, GB_ A11G2234, GB_ D01G2010, and GB_ D03G0730, qRT-PCR showed their expressions under drought, salt, or verticillium wilt stress to agree with what the transcriptome data did. Their involvement in the stress response of the cotton plant was confirmed. Conclusion The genome, structure, and phylogenetic characteristics of GbRLCK VI family in G. barbadense were determined. Their roles in the growth, development, and stress responses of the cotton plant were clarified. -
图 1 拟南芥、水稻与海岛棉RLCK VI家族基因系统进行分析
红色方块为RLCK VI_A亚家族；绿色方块为RLCK VI_B亚家族；At：拟南芥，LOC_Os：水稻，GB：海岛棉。
Figure 1. Phylogenetic evolution of RLCK VI family genes in Arabidopsis thaliana, Oryza sativa, and G. barbadense
Red square indicates RLCK VI_A subfamily group; green square, RLCK VI_B subfamily group; At: Arabidopsis thaliana; LOC_Os: Oryza sativa; GB: G. barbadense.
图 2 海岛棉RLCK VI家族蛋白序列的保守基序和基因结构分析
Figure 2. Conserved motifs and structures of RLCK VI protein sequences in G. barbadense
图 4 GbRLCK VI基因在海岛棉不同组织器官的表达分析
Figure 4. Expressions of GbRLCK VI in tissues and organs of G. barbadense
图 5 GbRLCK VI基因在海岛棉不同逆境胁迫下聚类表达分析
Figure 5. Clustered GbRLCK_VI expressions under stresses
图 6 GbRLCK VI家族基因在海岛棉不同逆境胁迫下qRT-PCR分析
A～D：PEG处理；E～H：NaCl处理；I～L：黄萎病菌处理。
Figure 6. qRT-PCR of GbRLCK_VI under stresses
A–D: PEG treatments; E–H: NaCl treatments; I–L: verticillium wilt pathogen treatments.
表 1 荧光定量 PCR引物序列
Table 1. Sequences of primers for quantitative PCR
基因名称
Gene name基因序列（5′-3′）
Primer sequence (5′-3′)GB_A11G2234-F AATGAAGAATGAGAAACAA GB_A11G2234-R GAGGTGAAAACTGAAGTAC GB_A12G0061-F AAACTGGACTCACCACAAC GB_A12G0061-R AGTACACCAAAGGCAAACA GB_D01G2010-F GCAATATGGGGACCAACTG GB_D01G2010-R AGAACAACACCGAAAGCGT GB_D03G0730-F CATAAACGAAATAGCTTGC GB_D03G0730-R CCTTGGTCTCATAGGAAAC GhUBQ7-F GAAGGCATTCCACCTGACCAAC GhUBQ7-R CTTGACCTTCTTCTTCTTGTGCTTG 
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表 2 GbRLCK VI亚族成员蛋白理化性质及亚细胞定位分析
Table 2. Physicochemical properties and subcellular localizations of proteins in GbRLCK VI subfamily members
基因编号
Gene ID分类
Classification理论等电点
pI相对分子质量
Relative molecular
mass/Da染色体
Chromosome位置
Location/bp开放阅读
框长度
ORF/bp氨基酸
Amino
acids/aa亚细胞定位
Subcellular
localizationGB_A01G0260 RLCK VI_A 5.30 62875.09 A 2206175～2210244 1683 561 细胞质 Cytoplasmic GB_A01G1081 RLCK VI_B 8.18 83349.28 A 20600106～20603458 2214 738 胞外 Extracellular GB_A01G1914 RLCK VI_B 5.97 75989.42 A 103561645～103564486 2046 682 胞外 Extracellular GB_A02G1246 RLCK VI_B 6.94 84611.20 A 63519857～63524024 2325 775 胞外 Extracellular GB_A02G1740 RLCK VI_A 5.94 50565.48 A 97801526～97803417 1362 454 质膜 Plasma membrane GB_A03G0311 RLCK VI_A 5.95 44313.24 A 3766673～3768994 1167 389 质膜 Plasma membrane GB_A05G3702 RLCK VI_A 8.52 67781.48 A 84140713～84143610 1815 605 细胞质 Cytoplasmic GB_A08G1010 RLCK VI_A 9.08 65230.47 A 33774846～33777243 1749 583 细胞质 Cytoplasmic GB_A09G2643 RLCK VI_A 5.6 58175.80 A 77782790～77786063 1554 518 细胞质 Cytoplasmic GB_A10G1115 RLCK VI_A 6.01 54570.32 A 22829603～22832184 1470 490 质膜 Plasma membrane GB_A11G0540 RLCK VI_A 6.03 50910.97 A 4885422～4887763 1362 454 质膜 Plasma membrane GB_A11G1251 RLCK VI_A 9.25 47356.59 A 12738544～12740333 1269 423 质膜 Plasma membrane GB_A11G2234 RLCK VI_A 6.09 52234.38 A 51930366～51932808 1395 465 质膜 Plasma membrane GB_A11G2998 RLCK VI_A 6.98 54619.72 A 102734569～102739375 1455 485 质膜 Plasma membrane GB_A11G3462 RLCK VI_A 8.39 54509.86 A 111382556～111386884 1455 485 细胞质 Cytoplasmic GB_A12G0061 RLCK VI_A 9.54 44738.44 A 770437～772212 1197 399 质膜 Plasma membrane GB_A12G0366 RLCK VI_B 5.53 51025.69 A 6110842～6112604 1365 455 质膜 Plasma membrane GB_A12G0580 RLCK VI_B 8.94 57833.28 A 12489405～12491904 1566 522 胞外 Extracellular GB_A12G0837 RLCK VI_B 6.28 72794.02 A 35264457～35267552 1959 653 胞外 Extracellular GB_A12G1016 RLCK VI_A 6.08 55330.70 A 58620348～58623557 1470 490 细胞质 Cytoplasmic GB_D01G0255 RLCK VI_A 5.61 54753.77 D 2136137～2138668 1461 487 质膜 Plasma membrane GB_D01G1159 RLCK VI_B 8.62 82638.79 D 16772231～16775594 2193 731 胞外 Extracellular GB_D01G2010 RLCK VI_B 5.87 75966.19 D 54299320～54302161 2049 683 胞外 Extracellular GB_D03G0345 RLCK VI_A 5.94 50468.36 D 3912795～3914684 1359 453 质膜 Plasma membrane GB_D03G0730 RLCK VI_B 6.52 84629.22 D 19203868～19208089 2328 776 胞外 Extracellular GB_D03G1685 RLCK VI_A 5.86 44338.23 D 49960463～49962648 1167 389 质膜 Plasma membrane GB_D04G0916 RLCK VI_A 8.82 67857.57 D 17923478～17926392 1824 608 细胞质 Cytoplasmic GB_D09G2477 RLCK VI_A 5.54 58118.73 D 52528068～52531290 1554 518 细胞质 Cytoplasmic GB_D10G1818 RLCK VI_A 6.1 58069.37 D 46511912～46514475 1569 523 细胞质 Cytoplasmic GB_D11G0554 RLCK VI_A 5.96 51742.65 D 4472367～4474758 1386 462 质膜 Plasma membrane GB_D11G1285 RLCK VI_A 9.37 50810.57 D 11385965～11387753 1347 449 细胞质 Cytoplasmic GB_D11G2293 RLCK VI_A 6.17 52301.37 D 30637528～30639969 1395 465 质膜 Plasma membrane GB_D11G2991 RLCK VI_A 7.67 54466.53 D 60438857～60443672 1455 485 质膜 Plasma membrane GB_D11G3433 RLCK VI_A 8.39 54454.82 D 67010248～67014591 1455 485 细胞质 Cytoplasmic GB_D12G0064 RLCK VI_A 9.51 44758.54 D 777708～781902 1197 399 质膜 Plasma membrane GB_D12G0350 RLCK VI_B 5.54 51097.86 D 4562988～4564741 1365 455 质膜 Plasma membrane GB_D12G0573 RLCK VI_B 9.04 57727.16 D 9309237～9311733 1563 521 胞外 Extracellular GB_D12G0968 RLCK VI_A 5.73 53551.68 D 20078236～20080755 1425 475 细胞质 Cytoplasmic GB_D12G0998 RLCK VI_B 6.62 79099.37 D 16414879～16417975 2133 711 胞外 Extracellular
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表 3 串联重复基因Ka/Ks计算
Table 3. Calculation of Ka/Ks for tandem repeat gene
基因编号
Gene ID基因编号
Gene ID非同义替换
Ka同义替换
Ks非同义替换/同义替换
Ka/KsGB_A01G1914 GB_A12G0366 0.104312114 0.45263137 0.230457101 GB_A01G0260 GB_D01G0255 0.028943225 0.046322927 0.624814254 GB_A01G1081 GB_D01G1159 0.020977245 0.054445666 0.385287692 GB_A01G1914 GB_D01G2010 0.013406752 0.02845499 0.471156428 GB_A01G1914 GB_D12G0350 0.109277637 0.473782321 0.230649461 GB_A02G1740 GB_D03G0345 0.005691277 0.03053857 0.186363574 GB_A02G1246 GB_D03G0730 0.006776517 0.037543027 0.18050001 GB_A02G1740 GB_D11G2293 0.197554085 1.040010514 0.18995393 GB_A05G3702 GB_D04G0916 0.023308504 0.056770511 0.410574135 GB_A09G2643 GB_D09G2477 0.012551719 0.050376868 0.249156396 GB_A10G1115 GB_D09G2477 0.250706504 0.744111803 0.336920478 GB_A10G1115 GB_D10G1818 0.010688224 0.02700967 0.395718402 GB_A11G1251 GB_A12G0061 0.103086568 0.88836622 0.116040621 GB_A11G0540 GB_D11G0554 0.01383297 0.035350161 0.391312788 GB_A11G1251 GB_D11G1285 0.012356552 0.038946803 0.31726743 GB_A11G2998 GB_D11G2991 0.01397591 0.054049622 0.258575533 GB_A11G3462 GB_D11G3433 0.013455287 0.069780882 0.192821966 GB_A11G0540 GB_D11G2293 0.239191447 0.679802163 0.351854496 GB_A11G2234 GB_D11G2293 0.009270106 0.04321328 0.214519851 GB_A11G1251 GB_D12G0064 0.108697399 0.818974304 0.132723821 GB_A12G0366 GB_D01G2010 0.110134899 0.497372712 0.221433336 GB_A12G0061 GB_D11G1285 0.102473606 0.89384392 0.114643735 GB_A12G0061 GB_D12G0064 0.017583223 0.05245583 0.335200547 GB_A12G0580 GB_D12G0573 0.006683419 0.045728447 0.146154523 GB_A12G0366 GB_D12G0350 0.01232937 0.026976777 0.457036417 GB_A12G1016 GB_D12G0968 0.014602332 0.065561783 0.222726282 GB_D01G2010 GB_D12G0350 0.116240086 0.513096743 0.22654614 GB_D03G0345 GB_D11G2293 0.19515733 1.062230892 0.18372402 GB_D09G2477 GB_D10G1818 0.254053518 0.74467519 0.341160175 GB_D11G0554 GB_D11G2293 0.210367409 0.660727812 0.318387398 GB_D11G1285 GB_D12G0064 0.106823583 0.823959217 0.129646687
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