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作物学报 ›› 2023, Vol. 49 ›› Issue (9): 2344-2361.doi: 10.3724/SP.J.1006.2023.24246

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

陆地棉漆酶基因家族成员表达模式分析

左春阳(), 李亚玮, 李焱龙, 金双侠, 朱龙付, 张献龙, 闵玲()   

  1. 华中农业大学作物遗传改良全国重点实验室 / 湖北洪山实验室, 湖北武汉 430070
  • 收稿日期:2022-11-02 接受日期:2023-02-21 出版日期:2023-09-12 网络出版日期:2023-03-13
  • 通讯作者: *闵玲, E-mail: lingmin@mail.hzau.edu.cn
  • 作者简介:左春阳, E-mail: 727202632@qq.com第一联系人:**同等贡献
  • 基金资助:
    湖北洪山实验室重大项目课题(2022hszd004);国家自然科学基金项目“GhHRK基因增强棉花花粉高温耐性的调控网络解析”(32072024)

Relative expression patterns of laccase gene family members in upland Gossypium hirsutum L.

ZUO Chun-Yang(), LI Ya-Wei, LI Yan-Long, JIN Shuang-Xia, ZHU Long-Fu, ZHANG Xian-Long, MIN Ling()   

  1. National Key Laboratory of Crop Genetic Improvement / Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan 430070, Hubei, China
  • Received:2022-11-02 Accepted:2023-02-21 Published:2023-09-12 Published online:2023-03-13
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    Major Project of Hubei Hongshan Laboratory(2022hszd004);National Natural Science Foundation of China “Regulatory Network Analysis of GhHRK Gene Enhancing Pollen High Temperature Tolerance in Cotton”(32072024)

摘要:

漆酶是铜蓝氧化酶蛋白家族的一员, 在植物木质素合成和提高植株抵御胁迫能力等方面发挥着重要作用。本研究从陆地棉基因组中鉴定到104个漆酶基因(GhLAC)家族成员, 进行了系统进化树和组织表达图谱的构建, 并随机选取了20个基因进行荧光定量PCR分析, 验证了表达热图的结果。为进一步探索漆酶在棉花中担当的角色, 本研究采用启动子-GUS融合载体转化拟南芥, 通过转基因拟南芥不同发育过程不同组织GUS染色结果, 分析了陆地棉漆酶基因家族6个成员(GhLAC12AGhLAC14AGhLAC20AGhLAC25DGhLAC59DGhLAC63D)的精细表达模式。为探究漆酶在逆境中发挥的作用, 对该6个漆酶基因进行了切割和刺洞2种创伤胁迫诱导表达分析, 并利用2个棉花品系‘84021’ (高温耐受型)和‘H05’ (高温敏感型)在常温和高温胁迫条件下不同时期的花药进行相应基因的荧光定量PCR分析。研究结果表明, 随机挑选的20个基因在根、茎、叶、花瓣、花药和柱头6个组织中差异表达, 大多数基因的表达与转录组结果一致。6个漆酶基因的启动子能够不同程度地驱动GUS基因在种子萌发时期、二叶期、四叶期表达; 创伤处理结果显示GhLAC12AGhLAC14A的启动子受创伤诱导后驱动GUS蛋白在叶片中表达的能力显著提高, 暗示着这2个基因可能参与创伤胁迫响应。6个GhLACs基因在棉花耐高温品系‘84021’的四分体时期和花药开裂期高温胁迫后都有表达量显著下调的趋势, 推测GhLACs基因可能负调控陆地棉花药高温耐受性。本研究结果为进一步探索漆酶家族基因功能提供了参考。

关键词: 陆地棉, 漆酶基因, 启动子-GUS表达模式分析, 花药高温响应, 创伤诱导

Abstract:

Laccase, a member of the blue copper oxidase protein family, plays an important role in plant lignin synthesis and improving plant resistance to stress. In this study, 104 members of the Laccase gene (GhLAC) family were identified from the upland cotton genome. Phylogenetic tree and tissue expression map were constructed. Twenty genes were randomly selected for qRT-PCR analysis to verify the results of expression heat map. To further explore the role of laccase in cotton, promoter-GUS fusion vectors were transformed into Arabidopsis thaliana. The detailed expression patterns of six members of the Laccase gene family (GhLAC12A, GhLAC14A, GhLAC20A, GhLAC25D, GhLAC59D, and GhLAC63D) were studied by GUS staining in different tissues during different developmental period of transgenic Arabidopsis thaliana. To explore the role of laccase in stress, the expression of the six laccase genes was analyzed by cutting and piercing, and the corresponding genes were analyzed by qRT-PCR using the anther of two cotton strains ‘84021’ (high temperature tolerant) and ‘H05’ (high temperature sensitive) at different stages under normal and high temperature conditions. The results showed that 20 randomly selected genes were differentially expressed in six tissues of root, stem, leaf, petal, anther, and stigma, and the relative expression levels of most genes were consistent with the transcriptome data. The promoter of six laccase genes could drive GUS gene expression in different levels at germination, two-leaf, and four-leaf stages. The trauma treatment indicated that the promoter of GhLAC12A and GhLAC14A significantly improved the ability to drive GUS protein expression in leaves after trauma induction, suggesting that the two genes might be involved in traumatic stress response. In addition, the relative expression levels of the six GhLACs genes were significantly down-regulated after high temperature stress at the tetrad stage and anther dehiscence stage of cotton strain ‘84021’, suggesting GhLACs gene might negatively regulate the high temperature tolerance of cotton anthers. The results of this study provide the reference for further exploring the function of laccase family genes.

Key words: Gossypium hirsutum L., laccase gene, the promoter-GUS expression pattern, anther response to high temperature, wound induced expression

表1

本试验所用引物及用途"

引物名称 Primer name 引物序列 Prime sequence (5°-3°) 用途 Purpose
ubiquitin7-F GAAGGCATTCCACCTGACCAAC 实时荧光定量PCR
ubiquitin7-R CTTGACCTTCTTCTTCTTGTGCTTG qRT-PCR
GhLAC1A-F AATGGCAACAACTTCACATC 实时荧光定量PCR
GhLAC1A-R ACACTCAGAATGGTCCTCGT qRT-PCR
GhLAC12A-F GCTAATCAAACCATTGCTCCTG 实时荧光定量PCR
GhLAC12A-R TCCGACAATCGTCAATCTGTG qRT-PCR
GhLAC14A-F CCTATTGAGGAAGGGACGAG 实时荧光定量PCR
GhLAC14A-R ACCATACTCCGGGATTGGTA qRT-PCR
GhLAC20A-F CCAACTTCTTCGTAGTCGGTAG 实时荧光定量PCR
GhLAC20A-R ATCCCTGGGTTATCTGCTCT qRT-PCR
GhLAC31A-F CCAAGGCGAAACGAATCTTA 实时荧光定量PCR
GhLAC31A-R ATTAGGGCCACCTCCTGTCT qRT-PCR
GhLAC36A-F TGCCAAGAGCATCGTAACCG 实时荧光定量PCR
GhLAC36A-R TCCGTCCGCCCATCCAGTTC qRT-PCR
GhLAC38A-F AGATTCGGGTCCGCAGAGGG 实时荧光定量PCR
GhLAC38A-R CAGCCCAAGCACTGGTAGCG qRT-PCR
GhLAC40A-F GGGTTCTATTTCTAAGCACTTT 实时荧光定量PCR
GhLAC40A-R ACTATCGTTTACGACCAGCA qRT-PCR
GhLAC43A-F AAGAAGGAACATTGTGGTGGCA 实时荧光定量PCR
GhLAC43A-R CCCGGAGTCGATGGGAACTA qRT-PCR
GhLAC6D-F CCAGTTAAACCAGGGAAGAC 实时荧光定量PCR
GhLAC6D-R AGAGCCGGAGTAATGTAAGG qRT-PCR
GhLAC11D-F CCTTAATCCACCAGTATTCTACG 实时荧光定量PCR
GhLAC11D-R AGTTTAGCACAACAGCCCTC qRT-PCR
GhLAC23D-F ACCATTCACTGGCACGGTGTT 实时荧光定量PCR
GhLAC23D-R ACGGATACGATTCATTACGC qRT-PCR
GhLAC25D-F CAATTCCCTGCCTTAGTCCC 实时荧光定量PCR
GhLAC25D-R TTGTCCCAGTCATCGTTTGC qRT-PCR
GhLAC28D-F GGCAGTTACGGTTATCTTGT 实时荧光定量PCR
GhLAC28D-R TTGGGAGGATGATGAGTGGG qRT-PCR
GhLAC54D-F GGGTGTTATCATCTATCAGGG 实时荧光定量PCR
GhLAC54D-R ACATTAAGTGGGACAGGGAC qRT-PCR
GhLAC57D-F TCTCCTGGACAGACCATAGA 实时荧光定量PCR
GhLAC57D-R AGTAGAATAAGCCCTAGCAAC qRT-PCR
GhLAC58D-F TGAGACAGACCCAAGCACAT 实时荧光定量PCR
GhLAC58D-R GACAACGAGGCATGGTAGAT qRT-PCR
GhLAC59D-F ATTGGCAAGGGTTTGAGGAT 实时荧光定量PCR
GhLAC59D-R ACGTAGGTGTAGGGAAAGGATA qRT-PCR
GhLAC61D-F TTGAATGCGATCTGGCTGTT 实时荧光定量PCR
GhLAC61D-R ATGGTGGTCATGGTAGTTGGAATA qRT-PCR
GhLAC63D-F ACTTCCGTGCTCATGTTAGG 实时荧光定量PCR
GhLAC63D-R CAAGAGGCAGACTTGTATTCG qRT-PCR

图1

陆地棉、拟南芥和水稻LAC基因家族进化树分析"

表2

陆地棉LAC基因家族信息"

基因ID
Gene ID
染色体
Chr.
基因描述(拟南芥)
Gene description (Arabidopsis)
基因组位置 Genomic position 命名
Name
起始 Start 终止 End
Ghir_A01G021510 A01 Laccase-17 116737831 116742782 GhLAC1A
Ghir_D01G023050 D01 Laccase-17 62359392 62363270 GhLAC1D
Ghir_A01G021950 A01 Laccase-4 117127088 117132069 GhLAC2A
Ghir_D01G023480 D01 Laccase-4 62708521 62717375 GhLAC2D
Ghir_A02G006480 A02 Laccase-21 10086940 10089681 GhLAC3A
Ghir_D02G006860 D02 Laccase-21 9508400 9511507 GhLAC3D
Ghir_A03G005270 A03 Laccase-2 9138986 9141467 GhLAC4A
Ghir_D03G013500 D03 Laccase-2 44314668 44316870 GhLAC4D
Ghir_A03G005280 A03 Laccase-2 9197235 9204292 GhLAC5A
Ghir_D03G013490 D03 Laccase-2 44287872 44290796 GhLAC5D
Ghir_A03G005800 A03 Laccase-4 10413905 10417019 GhLAC6A
Ghir_D03G013060 D03 Laccase-4 43363895 43367056 GhLAC6D
Ghir_A04G009430 A04 Laccase-7 71412678 71434702 GhLAC7A
Ghir_D04G013660 D04 Laccase-7 45073981 45077137 GhLAC7D
Ghir_A04G009470 A04 Laccase-7 71627753 71631136 GhLAC8A
Ghir_D04G013680 D04 Laccase-7 45181487 45184916 GhLAC8D
Ghir_A05G009230 A05 Laccase-4 8476727 8479903 GhLAC9A
Ghir_D05G009240 D05 Laccase-4 7540512 7543586 GhLAC9D
Ghir_A05G010150 A05 Laccase-17 9143475 9146068 GhLAC10A
Ghir_D05G009870 D05 Laccase-17 8212961 8215461 GhLAC10D
Ghir_A05G025140 A05 Laccase-15 25644784 25662157 GhLAC11A
Ghir_D05G025180 D05 Laccase-14 23486473 23489452 GhLAC11D
Ghir_A05G025310 A05 Laccase-14 25939177 25941026 GhLAC12A
Ghir_D05G025230 D05 Laccase-14 23586804 23588738 GhLAC12D
Ghir_A05G025330 A05 Laccase-14 25973262 25976026 GhLAC13A
Ghir_D05G025210 D05 Laccase-14 23560592 23563227 GhLAC13D
Ghir_A05G025340 A05 Laccase-14 25981068 25989683 GhLAC14A
Ghir_D05G025200 D05 Laccase-14 23542194 23557617 GhLAC14D
Ghir_A05G025350 A05 Putative laccase-9 26015294 26017921 GhLAC15A
Ghir_D05G025190 D05 Putative laccase-9 23514531 23517069 GhLAC15D
Ghir_A05G031190 A05 Laccase-22 41692291 41698066 GhLAC16A
Ghir_D05G031070 D05 Laccase-22 33904186 33908534 GhLAC16D
Ghir_A06G012170 A06 Putative laccase-9 67269136 67271306 GhLAC17A
Ghir_D06G012330 D06 Putative laccase-9 29988284 29990547 GhLAC17D
Ghir_A08G021230 A08 Laccase-3 116971139 116973684 GhLAC18A
Ghir_D08G022010 D08 Laccase-3 63494311 63496810 GhLAC18D
Ghir_A09G016340 A09 Laccase-17 72475738 72478310 GhLAC19A
Ghir_D09G015810 D09 Laccase-17 44179260 44181960 GhLAC19D
Ghir_A10G023410 A10 Laccase-22 112560879 112563710 GhLAC20A
Ghir_D10G025960 D10 Laccase-22 66026294 66029136 GhLAC20D
Ghir_A10G024200 A10 Laccase-5 114093313 114095981 GhLAC21A
Ghir_D10G026620 D10 Laccase-5 67161382 67164003 GhLAC21D
Ghir_A11G019640 A11 Laccase-6 27309104 27311987 GhLAC22A
Ghir_D11G019420 D11 Laccase-11 21681414 21682836 GhLAC22D
Ghir_A11G035330 A11 Laccase-17 122965170 122967669 GhLAC23A
Ghir_D11G036190 D11 Laccase-17 72693575 72696286 GhLAC23D
Ghir_A11G035350 A11 Laccase-4 122971778 122974013 GhLAC24A
Ghir_D11G036210 D11 Laccase-17 72700213 72702426 GhLAC24D
Ghir_A11G035490 A11 Laccase-4 123045551 123048240 GhLAC25A
Ghir_D11G036340 D11 Laccase-4 72775350 72777973 GhLAC25D
Ghir_A13G001780 A13 Laccase-17 2014792 2017130 GhLAC26A
Ghir_D13G002060 D13 Laccase-17 1759037 1761649 GhLAC26D
Ghir_A13G002160 A13 Laccase-17 2565151 2567561 GhLAC27A
Ghir_D13G002440 D13 Laccase-17 2206264 2213052 GhLAC27D
Ghir_A13G002170 A13 Laccase-17 2580835 2585183 GhLAC28A
Ghir_D13G024730 D13 Laccase-2 62656628 62661237 GhLAC28D
Ghir_A13G002350 A13 Laccase-11 2736016 2738294 GhLAC29A
Ghir_D13G002640 D13 Laccase-11 2386833 2389243 GhLAC29D
Ghir_A13G023990 A13 Laccase-2 107564582 107569198 GhLAC30A
Ghir_D11G010590 D11 Laccase-6 9024231 9027561 GhLAC30D
Ghir_A03G005300 A03 Laccase-2 9235739 9242786 GhLAC31A
Ghir_A03G007710 A03 Laccase-11 17682089 17684169 GhLAC32A
Ghir_A04G009070 A04 Laccase-17 69641501 69641989 GhLAC33A
Ghir_A04G009440 A04 Laccase-9 71412678 71415616 GhLAC34A
Ghir_A04G009460 A04 Laccase-7 71525265 71528510 GhLAC35A
Ghir_A05G010190 A05 Laccase-17 9194020 9196191 GhLAC36A
Ghir_A05G025150 A05 Laccase-14 25668096 25669929 GhLAC37A
Ghir_A05G025280 A05 Laccase-14 25869242 25871007 GhLAC38A
Ghir_A05G025290 A05 Laccase-14 25890847 25893461 GhLAC39A
Ghir_A05G025320 A05 Laccase-14 25951588 25954197 GhLAC40A
Ghir_A05G031330 A05 Laccase-12 42716069 42718551 GhLAC41A
Ghir_A06G017280 A06 Laccase-5 116079600 116081862 GhLAC42A
Ghir_A06G017290 A06 Laccase-5 116189142 116197901 GhLAC43A
Ghir_A06G017300 A06 Laccase-5 116223123 116225599 GhLAC44A
Ghir_A06G017320 A06 Laccase-5 116280847 116285027 GhLAC45A
Ghir_A10G009410 A10 Laccase-11 18971904 18974558 GhLAC46A
Ghir_A11G010610 A11 Laccase-6 9769950 9772299 GhLAC47A
Ghir_A12G012190 A12 Laccase-6 80351644 80353970 GhLAC48A
Ghir_A13G003100 A13 Laccase-11 3705196 3707259 GhLAC49A
Ghir_A13G003110 A13 Laccase-11 3713263 3720273 GhLAC50A
Ghir_D03G010220 D03 Laccase-11 35974903 35978183 GhLAC51D
Ghir_D03G013470 D03 Laccase-17 44265628 44266723 GhLAC52D
Ghir_D03G015740 D03 Laccase-3 48218625 48221114 GhLAC53D
Ghir_D04G013650 D04 Laccase-9 44922823 44925840 GhLAC54D
Ghir_D04G013670 D04 Laccase-7 45158044 45161359 GhLAC55D
Ghir_D04G013870 D04 Laccase-4 45597220 45599865 GhLAC56D
Ghir_D05G025170 D05 Laccase-15 23475679 23484348 GhLAC57D
Ghir_D05G025220 D05 Putative laccase-9 23575706 23578427 GhLAC58D
Ghir_D05G027760 D05 Laccase-14 26649634 26651370 GhLAC59D
Ghir_D05G031170 D05 Laccase-12 34446602 34448100 GhLAC60D
Ghir_D05G031210 D05 Laccase-22 34596389 34597068 GhLAC61D
Ghir_D06G018210 D06 Laccase-5 59288750 59291253 GhLAC62D
Ghir_D06G018220 D06 Laccase-5 59357081 59359262 GhLAC63D
Ghir_D06G018230 D06 Laccase-5 59458411 59459122 GhLAC64D
Ghir_D06G018250 D06 Laccase-5 59500342 59502844 GhLAC65D
Ghir_D10G009840 D10 Laccase-11 11987141 11989232 GhLAC66D
Ghir_D10G014410 D10 Laccase-2 25717367 25718684 GhLAC67D
Ghir_D11G019730 D11 Laccase-6 22309695 22312399 GhLAC68D
Ghir_D12G012430 D12 Laccase-6 41466012 41468206 GhLAC69D
Ghir_D13G003370 D13 Laccase-11 3233418 3235743 GhLAC70D
Ghir_D13G003390 D13 Laccase-11 3256191 3258374 GhLAC71D
Ghir_D13G009820 D13 Laccase-8 22859921 22864788 GhLAC72D
Ghir_A08G026500 Scaffold2204 Laccase-7 49182 53499 GhLAC73
Ghir_A03G023780 Scaffold2615 Laccase-3 13682 16169 GhLAC74

图2

陆地棉LAC基因表达热图 A: GhLAC基因家族在‘H05’根、茎、叶、花瓣、花药与柱头中的表达热图。B: 不同组织中高量表达的基因。"

图3

GhLAC基因家族部分成员组织表达分析 R: 根; S: 茎; L: 叶; P: 花瓣; St: 柱头; A: 花药。误差值代表3个生物学重复的标准差, GhUBQ7 (Gh_A11G011460)为内参基因。通过ANOVA方法分析表达差异显著性, 不同的字母代表在0.05概率水平差异显著。"

图4

6个GhLACs基因启动子驱动GUS基因在种子萌发时期的表达 Stage 1: 种子萌发初期; Stage 2: 种子萌发后期。RH: 根毛; Ra: 幼根。图中标尺均为200 μm。"

图5

6个GhLACs基因启动子驱动GUS基因在二叶期和四叶期的表达模式分析 Stage 1: 二叶期; Stage 2: 四叶期。Tr: 表皮毛。标尺为1 mm。(A~F)和(G~L): ProGhLAC12A、ProGhLAC14A、ProGhLAC20A、ProGhLAC25D、ProGhLAC59D和ProGhLAC63D在二叶期(A~F)和四叶期(G~L)的表达模式分析; (M~R)和(S~X): ProGhLAC12A、ProGhLAC14A、ProGhLAC20A、ProGhLAC25D、ProGhLAC59D和ProGhLAC63D在四叶期的叶(M~R)和根(S~X)的表达模式分析。"

图6

GhLACs基因启动子驱动GUS基因在成熟期花序和叶片中的表达分析 (A~F)、(G~L)分别代表了GhLAC12A、GhLAC14A、GhLAC20A、GhLAC25D、GhLAC59D与GhLAC63D基因启动子驱动GUS基因在花期和成熟期叶部的表达情况。An: 花药。标尺为1 mm。"

图7

二叶期和四叶期创伤诱导表达模式分析 A: 二叶期创伤诱导表达模式分析; B: 四叶期创伤诱导表达模式分析。CK: 对照组; C: 切割组; P: 刺洞组。红色箭头代表切割处理, 白色箭头代表刺洞处理。标尺为2 mm。"

图8

6个GhLAC基因家族成员高温诱导表达分析 GhLAC基因在2个陆地棉品系‘84021’ (耐高温品系)、‘H05’ (敏高温品系)中TS (四分体时期; 花蕾长度6~7 mm)、TDS (绒毡层降解时期; 花蕾长度9~14 mm)和ADS (花药开裂时期; 花蕾长度大于24 mm)的花药中的荧光定量检测。HN: 常温下的‘H05’; HH: 高温下的‘H05’; 8N: 常温下的‘84021’; 8H: 高温下的‘84021’。误差值代表3个生物学重复的标准差, GhUBQ7 (Gh_A11G011460)为内参基因。使用t检验进行统计学分析, 星号表示有显著差异(*P < 0.05, **P < 0.01)。"

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