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东北白桦BpPAT1基因的表达模式及耐盐性分析

田增智 贺子航 王智博 张群 王超 及晓宇

田增智, 贺子航, 王智博, 张群, 王超, 及晓宇. 东北白桦BpPAT1基因的表达模式及耐盐性分析[J]. 北京林业大学学报. doi: 10.12171/j.1000-1522.20200302
引用本文: 田增智, 贺子航, 王智博, 张群, 王超, 及晓宇. 东北白桦BpPAT1基因的表达模式及耐盐性分析[J]. 北京林业大学学报. doi: 10.12171/j.1000-1522.20200302
Tian Zengzhi, He Zihang, Wang Zhibo, Zhang Qun, Wang Chao, Ji Xiaoyu. Expression and Salt tolerance Analysis of BpPAT1 Gene in Betula platyphylla[J]. Journal of Beijing Forestry University. doi: 10.12171/j.1000-1522.20200302
Citation: Tian Zengzhi, He Zihang, Wang Zhibo, Zhang Qun, Wang Chao, Ji Xiaoyu. Expression and Salt tolerance Analysis of BpPAT1 Gene in Betula platyphylla[J]. Journal of Beijing Forestry University. doi: 10.12171/j.1000-1522.20200302

东北白桦BpPAT1基因的表达模式及耐盐性分析

doi: 10.12171/j.1000-1522.20200302

Expression and Salt tolerance Analysis of BpPAT1 Gene in Betula platyphylla

  • 摘要:   目的  GRAS家族是植物特有的具有高度保守羧基末端的转录因子家族,已有研究表明GRAS转录因子是植物胁迫反应中关键的转录调节因子之一。本研究拟对白桦中GRAS转录因子基因BpPAT1基因是否具有耐盐能力进行分析,为阐明白桦GRAS转录因子响应盐胁迫的分子调控机制奠定基础,进一步丰富木本植物GRAS转录因子响应逆境胁迫分子机制的研究。  方法  从盐胁迫白桦转录组数据中筛选并获得了1条GRAS转录因子基因,将其命名为BpPAT1。利用蛋白多序列比对及系统进化树来分析BpPAT1与其他GRAS家族蛋白的亲缘关系。利用实时荧光定量PCR(qRT-PCR)技术分析盐胁迫及非胁迫条件下白桦根、茎和叶组织中BpPAT1的表达模式,初步鉴定其是否响应盐胁迫。为了进一步分析BpPAT1的抗逆功能,构建其植物过表达载体(pROKII-BpPAT1)与抑制表达载体(pFGC5941-BpPAT1),利用农杆菌介导的高效瞬时遗传转化体系,获得BpPAT1基因瞬时过表达、抑制表达及对照白桦植株。在盐胁迫下分别对BpPAT1瞬时表达及对照植株的耐盐相关生理指标进行测定,鉴定BpPAT1是否具有耐盐能力。  结果  多序列比对及系统进化树分析结果表明BpPAT1蛋白具有GRAS家族的序列特征,且与拟南芥中AtPAT1蛋白的亲缘关系较近。qRT-PCR结果表明:在盐胁迫6 h后,BpPAT1在白桦植株中的表达量显著上升(P < 0.05),说明该基因能响应盐胁迫。抗逆生理指标的测定结果表明:在白桦中过表达BpPAT1能够使过氧化物酶(POD)及超氧化物歧化酶(SOD)活性显著增强(P < 0.05);同时增加了白桦组织中的脯氨酸含量,降低了电解质渗透率及丙二醛含量。  结论  白桦BpPAT1基因能响应盐胁迫,过表达BpPAT1显著增强了白桦POD、SOD酶活性和脯氨酸含量,降低了电解质渗透率及丙二醛含量,进而提高了ROS清除能力,有效提高了白桦的耐盐能力。

     

  • 图  1  白桦BpPAT1蛋白的多序列比对分析(A);及系统进化树分析(B)

    AtPAT1. 拟南芥(NP_001332482.1);QsGRAS. 欧洲栓皮栎(XP_023916980.1);MrGRAS. 杨梅(KAB1208676.1);JrGRAS. 胡桃(XP_018849898.1);VvGRAS. 葡萄(XP_002272334.1);JcGRAS20. 麻风树(XP_012081428.1);CfGRAS. 土瓶草(GAV74587.1);DlGRAS54. 龙眼(AGE44291.1);TcGRAS. 可可(EOX93442.1)。AtPAT1, Arabidopsis thaliana(NP_001332482.1); QsGRAS, Quercus suber(XP_023916980.1); MrGRAS, Morella rubra(KAB1208676.1); JrGRAS, Juglans regia(XP_018849898.1);VvGRAS, Vitis vinifera(XP_002272334.1); JcGRAS20, Jatropha curcas(XP_012081428.1); CfGRAS, Cephalotus follicularis(GAV74587.1); DlGRAS54, Dimocarpus longan(AGE44291.1); TcGRAS, Theobroma cacao(EOX93442.1).

    Figure  1.  Multiple sequence alignment analysis of Betula platyphylla BpPAT1 protein (A) and phylogenetic tree analysis (B)

    图  2  盐胁迫条件下BpPAT1基因的表达模式

    Figure  2.  Expression pattern of BpPAT1 in B. platyphylla under salt stress.

    图  3  BpPAT1基因在瞬时表达白桦植株中的表达情况

    A. 盐胁迫下不同转化时间对照植株及瞬时过表达植株BpPAT1基因的表达水平;B. 正常条件下与盐胁迫后对照植株及瞬时转化植株BpPAT1基因的表达水平;Control. 对照植株;OE. 瞬时过表达植株;IE. 瞬时抑制表达植株;*. 显著性差异(P < 0.05)。下同。A, the expression levels of BpPAT1 gene in control plants and transient overexpressed plants at different transformation time under salt stress; B, the expression level of BpPAT1 gene in control plants and transient transformed plants under normal conditions and salt stress. Control, Control plant; OE, Transient overexpression plant; IE, Transient inhibitory expression plant. Asterisks indicate significant difference (P < 0. 05). The same below.

    Figure  3.  Expression of BpPAT1 Gene in transient expression plants of B. platyphylla under Salt stress

    图  4  正常条件及盐胁迫后瞬时表达OE、IE及control白桦植株中ROS水平(B,D)及POD(A)、SOD(C)活性分析

    Figure  4.  Analysis of ROS level (B,D) and activities of POD (A) and SOD (C) in OE, IE and control B. platyphylla plants under normal condition and salt stress

    图  5  正常条件及盐胁迫下OE、IE及Control白桦植株中电解质渗透率(A)及MDA含量(B)分析

    Figure  5.  Electrolyte leakage (A) and MDA contents (B) analysis in OE, IE and Control B. platyphylla plants under normal condition and salt stress

    图  6  正常条件及盐胁迫下OE、IE及Control白桦植株中脯氨酸含量分析

    Figure  6.  Proline content analysis in OE,IE and Control B. platyphylla plants under normal condition and salt stress

    表  1  引物序列

    Table  1.   Primers used in this study

    用途 Application引物名称 Primer name引物序列(5′-3′) Primer sequence(5′-3′)
    q-BpPAT1-F TACTGCTGCATTCTATCCAC
    q-BpPAT1-R ACTTACCAAGAAGCTCATG
    q-BpPAT1-OE-F CCCCACATCCGCATAACA
    q-BpPAT1-OE-R CCCAGGTCGAATCCCAAG
    q-BpPAT1-IE-F ACGAACGGTGTTGCACTT
    q-BpPAT1-IE-R GAGCCATAGGTATTGTCAGG
    实时荧光定量 PCR Quantitative Real-time PCR Actin-F TGAGAAGAGCTATGAGTTGC
    Actin-R GTAGATCCACCACTAAGCAC
    Tubulin-F TCAACCGCCTTGTCTCTCAGG
    Tubulin-R TGGCTCGAATGCACTGTTGG
    基因克隆 Gene cloning BpPAT1-F- GCTCTAGAATGTCCAACGGATTGTACTATC
    BpPAT1-R- GGGTACCTCACTTCCATGCACAAGCAG
    载体构建 Vector verification pROKⅡ-F AGACGTTCCAACCACGTCTT
    pROKⅡ-R CCAGTGAATTCCCGATCTAG
    pFGC5941-cisF CGCTCGAGTATAAGAGCT
    pFGC5941-cisR ACCTTCCCACAATTCGTCGG
    pFGC5941-antiF GCATGCTATGCATTCAAT
    pFGC5941-antiR CGTGCACAACAGAATTGAAAGC
    pFGC5941-BpPAT1-cisF CCCATGGCAGCTATGCTACAATGATAG
    pFGC5941-BpPAT1-cisR TTGGCGCGCCTCCACATATAGAAGAGCCAT
    pFGC5941-BpPAT1-antiF CTCTAGACAGCTATGCTACAATGATAG
    pFGC5941-BpPAT1-antiR CGGATCCTCCACATATAGAAGAGCCAT
    下载: 导出CSV
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