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白桦BpCHS3转基因植株耐盐性分析

姜晶 李晓媛 王楚 王芳 姜静

姜晶, 李晓媛, 王楚, 王芳, 姜静. 白桦BpCHS3转基因植株耐盐性分析[J]. 北京林业大学学报, 2019, 41(4): 1-7. doi: 10.13332/j.1000-1522.20180224
引用本文: 姜晶, 李晓媛, 王楚, 王芳, 姜静. 白桦BpCHS3转基因植株耐盐性分析[J]. 北京林业大学学报, 2019, 41(4): 1-7. doi: 10.13332/j.1000-1522.20180224
Jiang Jing, Li Xiaoyuan, Wang Chu, Wang Fang, Jiang Jing. Evaluation of salt tolerant performance of BpCHS3 transgenic plants in Betula platyphylla[J]. Journal of Beijing Forestry University, 2019, 41(4): 1-7. doi: 10.13332/j.1000-1522.20180224
Citation: Jiang Jing, Li Xiaoyuan, Wang Chu, Wang Fang, Jiang Jing. Evaluation of salt tolerant performance of BpCHS3 transgenic plants in Betula platyphylla[J]. Journal of Beijing Forestry University, 2019, 41(4): 1-7. doi: 10.13332/j.1000-1522.20180224

白桦BpCHS3转基因植株耐盐性分析

doi: 10.13332/j.1000-1522.20180224
基金项目: 国家重点研发计划课题(2017YFD0600603)
详细信息
    作者简介:

    姜晶。主要研究方向:林木遗传育种。Email:1187557704@qq.com 地址:150040黑龙江省哈尔滨市香坊区和兴路51号东北林业大学林学院

    责任作者:

    姜静,教授。主要研究方向:林木遗传育种。Email:jiangjing1960@126.com 地址:同上

  • 中图分类号: S792.153

Evaluation of salt tolerant performance of BpCHS3 transgenic plants in Betula platyphylla

  • 摘要: 目的查耳酮合成酶(CHS)是黄酮类化合物生物合成途径中的关键酶,其高表达可以促进黄酮类化合物积累,增强植物抵抗盐碱、干旱等非生物胁迫的能力,开展转BpCHS3白桦耐盐性分析,为阐明该基因的功能提供参考。方法以前期获得的转BpCHS3白桦为试材,进行转基因白桦qRT-PCR及Northern Blot检测,同时测定了叶片中花青素质量分数。NaCl胁迫处理下,分别测定转BpCHS3白桦的盐害指数、叶绿素荧光参数及光合参数。采用qRT-PCR技术,测定转基因株系的类黄酮代谢途径中CHS下游5个关键酶基因以及BpCHS家族成员相对表达量。结果qRT-PCR及Northern Blot检测显示,导入的目标基因BpCHS3在mRNA水平能够表达。转基因白桦组培生根苗在0.3%NaCl胁迫第25天,对照(WT)株系盐害指数高达83%,而转基因白桦平均盐害指数仅为39%;白桦转基因盆栽苗在0.4%NaCl胁迫后,叶绿素荧光参数及光合参数测定显示,NaCl胁迫第9天多数转基因株系最大光化学效率(Fv/Fm)仍为正常值,而WT株系降至0.66,胁迫第9天时实际光化学效率(ΦPSII)和光化学猝灭系数(qP)呈下降趋势,但WT株系的降幅高于转基因株系;NaCl胁迫6 d时,5个转基因株系的净光合速率(Pn)平均值仍高于WT的43.47%。认为盐胁迫下BpCHS3基因在白桦中的过量表达能够维持其较高的光电子传递活性,提高PSⅡ反应中心原初光能转换效率,同时也能维持较高的净光合效率。分别以转BpCHS3白桦cDNA为模板qRT-PCR分析显示,相对WT株系CHS下游的5个关键酶基因在转基因株系中均呈不同程度的上调表达,BpCHS家族成员中只有BpCHS3表达量显著上调,而 BpCHS1和BpCHS2均呈下调表达或与WT株系差异不显著。BpCHS3过表达株系花青素质量分数分析发现,转基因白桦叶片中花青素质量分数均显著低于WT株系,推测BpCHS3的过量表达,对另外2个CHS家族成员产生共抑制,且影响花青素的合成。结论BpCHS3白桦的耐盐性提高,与花青素质量分数多少无关,BpCHS3的过表达可能促进其他黄酮类化合物的积累,从而增强白桦的耐盐能力。

     

  • 图  1  BpCHS3白桦qRT-PCR检测

    Figure  1.  Relative quantification of each transgenic line BpCHS3 gene for Betula platyphylla

    图  2  BpCHS3基因白桦Northern Blot检测图谱

    1. WT;2 ~ 4. CHS3-1、CHS3-2、CHS3-4转基因株系。1, WT; 2−4, CHS3-1, CHS3-2, CHS3-4 transgenic lines.

    Figure  2.  Northern Blot pattern of 35S :: BpCHS3 from transgenic birch

    图  3  盐胁迫下参试株系荧光参数及Pn比较

    Fv/Fm. 最大光能转化效率Maximum light energy conversion efficiency;ΦPSII. 实际光化学效率Actual photochemical efficiency;qP. 光化学猝灭系数Photochemical quenching coefficient;Pn. 净光合速率Net photosynthetic rate

    Figure  3.  Comparison of fluorescence parameters and Pn of the tested birch under salt stress

    图  4  转基因株系与WT株系叶片花青素质量分数比较

    Figure  4.  Comparison of anthocyanin mass fractions in leaves between transgenic lines and WT lines

    图  5  黄酮类代谢途径关键酶基因qRT-PCR分析

    Figure  5.  qRT-PCR analysis of key enzyme genes in flavonoid metabolic pathway

    表  1  荧光定量PCR引物序列

    Table  1.   Primer sequences for fluorescent quantitative-PCR

    引物名称 Primer name 上游引物序列 Forward primer sequence (5′−3′) 下游引物序列 Reverse primer sequence (5′−3′)
    18SrRNA ATCTTGGGTTGGGCAGATCG CATTACTCCGATCCCGAAGG
    BpCHS1 CCGTGGAGGAAATCCGAAAGGCTC CGCTCTTGGTGATCCGGAAGTAATA
    BpCHS2 ATGGCGTCCGTCGAAGAAATATTTAA TCGTCTTGGTAGATATAGTTGGATGG
    BpCHS3 GGCATCTCGGACTGGAACTC CACAACGGTCTCGACGGTAA
    BpF3′H CTTGAACCACCGCAACCTCAC CCTCGTCCTCGACCCGAATT
    BpCHI GTATTTGGAGGATACCGCCGTGC GCAAGATCATTGTCACCCGTGTG
    BpDFR1 TGCCGCCTAGTCTTATTACAGCAC GTGAATTGTGGCATCGGCGG
    BpDFR2 CATTGGCGTCTTCCACGTGGCTAC CGACAGCTGATCCACTGGTAGTGT
    BpANR1 AAGGATCGACAATTCCACAAGCAGG CGTATAGCTTGTTGGTGCTGGAGAA
    BpANR2 GGGGGAAGATTGTGATACGGAGGAT GCGAAATCGTAGAGCTTGCTGGTGC
    BpANR3 CGACGAGCAGGCAAGTGACTTTCTC TCGTAGAGCCTGCCGGTGCTGGAG
    BpANS TACTACCCGATCTGCCCTCAGC GGAACACATTTCGCCGTCACCC
    下载: 导出CSV

    表  2  NaCl胁迫下转基因白桦生根情况

    Table  2.   Rooting of transgenic birch under salt stress

    株系
    Strain
    平均主根数
    Average number of primary roots
    平均主根长
    Average main root length/cm
    生根率
    Rooting rate/%
    盐害指数
    Salt damage index/%
    WT 1.2 0.27 ± 0.02c 40 83
    CHS3-1 3.2 1.76 ± 0.03a 100 43
    CHS3-2 2.8 1.66 ± 0.02b 100 35
    注:平均主根长表示方式为“平均值 ± 标准差”,同列不同字母表示差异显著(P < 0.05)。 Notes: data of average main root length were mean ± SD,different lowercases in the same column represent significant differences (P < 0.05).
    下载: 导出CSV
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出版历程
  • 收稿日期:  2018-07-12
  • 修回日期:  2018-07-23
  • 网络出版日期:  2019-04-02
  • 刊出日期:  2019-04-01

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