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BpGH3.5基因白桦优良株系选择

于嘉俐 王楚 公欣桐 李继唐 刘桂丰 姜静

于嘉俐, 王楚, 公欣桐, 李继唐, 刘桂丰, 姜静. 转BpGH3.5基因白桦优良株系选择[J]. 北京林业大学学报, 2019, 41(9): 81-89. doi: 10.13332/j.1000-1522.20180434
引用本文: 于嘉俐, 王楚, 公欣桐, 李继唐, 刘桂丰, 姜静. 转BpGH3.5基因白桦优良株系选择[J]. 北京林业大学学报, 2019, 41(9): 81-89. doi: 10.13332/j.1000-1522.20180434
Yu Jiali, Wang Chu, Gong Xintong, Li Jitang, Liu Guifeng, Jiang Jing. Selection of elite transgenic lines of BpGH3.5 in Betula platyphylla[J]. Journal of Beijing Forestry University, 2019, 41(9): 81-89. doi: 10.13332/j.1000-1522.20180434
Citation: Yu Jiali, Wang Chu, Gong Xintong, Li Jitang, Liu Guifeng, Jiang Jing. Selection of elite transgenic lines of BpGH3.5 in Betula platyphylla[J]. Journal of Beijing Forestry University, 2019, 41(9): 81-89. doi: 10.13332/j.1000-1522.20180434

BpGH3.5基因白桦优良株系选择

doi: 10.13332/j.1000-1522.20180434
基金项目: 大学生创新训练项目(201810225105)
详细信息
    作者简介:

    于嘉俐。主要研究方向:林木遗传育种。Email:1044100233@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

    责任作者:

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

Selection of elite transgenic lines of BpGH3.5 in Betula platyphylla

  • 摘要: 目的植物生长素酰胺合成酶基因家族(GH3s)为典型的植物生长素初级/早期响应基因,多数家族基因可通过调节植物体内游离IAA的浓度实现对生长发育的调控。故此,采用基因工程育种技术将BpGH3.5正义链、反义链导入白桦基因组中,预期获得速生转基因白桦新品种。方法以7年生白桦转BpGH3.5基因的54个正、反义链株系及对照(WT)株系为研究对象,测定树高、胸径及材积等生长指标,采用PCR及qRT-PCR技术分别检测转正、反义链各5个株系目标基因的遗传稳定性及相对表达量,同时采用ELISA技术测定游离IAA含量。结果PCR扩增显示,转基因株系中的nptⅡ外源基因均为阳性;qRT- PCR分析显示,5个转正义链株系中BpGH3.5基因表达量均显著高于WT株系,相反,5个转反义链株系中内源BpGH3.5基因表达量均显著下调,即BpGH3.5反义链导入白桦基因组后干扰了白桦BpGH3.5基因的表达。内源游离IAA含量测定显示,转BpGH3.5正义链株系的IAA含量低于或显著低于WT株系,5个转反义链株系均显著高于WT株系(P < 0.01),其IAA含量均值高于WT株系的52.26%。7年生转BpGH3.5白桦的树高、胸径及材积生长性状在株系间的差异达到了极显著水平(P < 0.01),在树高、胸径及材积生长指标高于群体均值的转基因株系中,转反义链株系占80%以上,认为BpGH3.5反义链导入白桦基因组中通过抑制BpGH3.5基因的表达,削弱IAA氨基酸化的能力,进而释放更多游离IAA从而促进白桦的生长。采用主成分分析法,选出10个速生的转反义链株系,这些株系的树高、胸径及材积均值较群体均值分别高8.55%、19.28%、50.42%,材积的平均遗传增益为36.3%。上述入选株系为后续转BpGH3.5白桦的环境释放提供参考。结论BpGH3.5反义链导入白桦基因组中,能够抑制BpGH3.5基因的表达,同时释放更多游离IAA而促进白桦的生长,采用主成分分析法,选出10个优良株系。

     

  • 图  1  BpGH3.5转基因白桦中nptⅡ基因检测电泳图谱

    1. Marker DL2000(2 kb、1 kb、0.75 kb、0.50 kb、0.20 kb、0.10 kb);2. 阳性质粒;3. 阴性水对照;4. WT株系;5. G2;6. G7;7. G15;8. G16;9. G20;10. FG8;11. FG9;12. FG10;13. FG12;14. FG15。1, Marker DL2000 (2 kb、1 kb、0.75 kb、0.50 kb、0.20 kb、0.10 kb); 2, positive plasmid; 3, negative control (water); 4, WT line; 5, G2; 6, G7; 7, G15; 8, G16; 9, G20; 10, FG8; 11, FG9; 12, FG10; 13, FG12; 14, FG15.

    Figure  1.  Electrophoresis map of nptII gene in BpGH3.5 transgenic Betula platyphylla

    图  2  转基因株系BpGH3.5基因相对表达量

    不同小写字母代表差异显著性,P < 0.05。下同。Different lowercase letters indicate significant difference, P < 0.05. The same below.

    Figure  2.  Relative expression level of BpGH3.5 gene in transgenic lines

    图  3  转反义链株系内源BpGH3.5相对表达量

    Figure  3.  Relative expression level of endogenous BpGH3.5 in antisense chain

    图  4  转基因株系游离IAA含量比较

    Figure  4.  Comparison of free IAA content in transgenic lines

    表  1  qRT-PCR引物序列

    Table  1.   qRT-PCR primer sequences

    基因 Gene 正向引物(5′—3′) Forward primer (5′−3′) 反向引物(5′—3′) Reverse primer (5′−3′)
    18SrRNA ATCTTGGGTTGGGCAGATCG CATTACTCCGATCCCGAAGG
    BpGH3.5 TCAGATTCTTAGGTTGGGAGCA GGCAGTAGGTGGATTGTTTGTAG
    内源BpGH3.5 Endogenous BpGH3.5 AGCAAGCTCTCAACAGGAAACAG GAATCCGGCGAAGTGTTTGGG
    下载: 导出CSV

    表  2  参试株系间各性状方差分析及遗传参数

    Table  2.   Analysis of variance and genetic parameters of various traits among tested lines

    性状 Trait 平方和
    SS
    自由度
    df
    均方
    MS
    F 检验值
    F
    显著性
    P
    遗传参数 Genetic parameter
    均值
    Mean
    变幅
    Amplitude
    变异系数
    Coefficient of variation/%
    重复力
    Repeatability
    树高 Tree height/m 47.619 54 0.882 3.136** 0.000 5.28 3.20 ~ 6.27 12.73 0.770
    胸径 DBH/cm 45.270 54 0.838 2.169** 0.000 3.91 2.25 ~ 4.98 18.35 0.784
    材积 Volume/m3 0.000 227 54 0.000 1.858** 0.002 0.003 6 0.001 0 ~ 0.006 6 46.06 0.820
    注:**表示在0.01水平上差异显著。Note: ** indicates a significant difference at P < 0.01 level.
    下载: 导出CSV

    表  3  BpGH3.5基因白桦参试性状多重比较

    Table  3.   Multiple comparisons of test traits of transgenic BpGH3.5 for Betula platyphylla

    株系
    Line
    树高
    Tree height/m
    株系
    Line
    胸径
    DBH/cm
    株系
    Line
    材积
    Volume/m3
    FG15 6.27 ± 0.387a FG9 4.98 ± 1.106a FG15 0.006 6 ± 0.002 8a
    FG12 5.95 ± 0.141ab FG8 4.96 ± 1.524a FG8 0.006 4 ± 0.005 2ab
    FG31 5.75 ± 0.351abc FG15 4.90 ± 1.039abc FG9 0.006 0 ± 0.002 6abc
    FG1 5.70 ± 0.719abc FG4 4.60 ± 0.689abc FG12 0.005 3 ± 0.003 7abcd
    G24 5.70 ± 1.009abc FG14 4.55 ± 0.777abc FG4 0.005 1 ± 0.001 5abcde
    FG5 5.68 ± 0.283abc FG5 4.52 ± 0.782abc FG5 0.005 1 ± 0.002 0abcde
    FG13 5.65 ± 0.581abcd FG12 4.40 ± 1.211abc FG14 0.004 9 ± 0.001 2abcde
    FG14 5.65 ± 0.370abcd FG1 4.35 ± 0.646abc FG1 0.004 6 ± 0.001 3abcdef
    FG20 5.63 ± 0.324abcd FG13 4.30 ± 0.600abc FG13 0.004 5 ± 0.001 4abcdef
    G14 5.63 ± 0.265abcd FG10 4.16 ± 0.885abc FG10 0.004 2 ± 0.002 0abcdefg
    FG18 5.60 ± 0.100abcd FG11 4.16 ± 0.793abc FG11 0.004 2 ± 0.001 5abcdefg
    FG21 5.60 ± 0.472abcd FG2 4.16 ± 0.321abc FG2 0.004 1 ± 0.000 5abcdefg
    FG23 5.60 ± 0.126abcd FG7 4.06 ± 0.879abc FG7 0.004 0 ± 0.002 1abcdefgh
    FG4 5.60 ± 0.346abcd FG16 4.05 ± 0.071abcd G8 0.003 9 ± 0.000 4abcdefgh
    FG9 5.58 ± 0.647abcd G10 4.05 ± 0.451abcd FG16 0.003 8 ± 0.000 3abcdefgh
    G8 5.58 ± 0.462abcd G8 4.05 ± 0.208abcd FG23 0.003 8 ± 0.000 7abcdefgh
    FG25 5.57 ± 0.351abcd FG23 3.95 ± 0.354abcd FG33 0.003 7 ± 0.000 2abcdefgh
    FG27 5.57 ± 0.153abcd G9 3.95 ± 0.208abcd G10 0.003 7 ± 0.000 9abcdefgh
    FG19 5.55 ± 0.173abcd FG33 3.93 ± 0.116abcd FG18 0.003 6 ± 0.000 8abcdefgh
    FG33 5.53 ± 0.283abcd FG28 3.90 ± 0.173abcd FG31 0.003 6 ± 0.000 5bcdefgh
    FG2 5.52 ± 0.141abcd FG32 3.90 ± 0.265abcd G14 0.003 6 ± 0.000 3bcdefgh
    FG11 5.50 ± 0.141abcd G11 3.90 ± 0.283abcd FG19 0.003 5 ± 0.000 8bcdefgh
    FG10 5.48 ± 0.071abcd G14 3.88 ± 0.126abcd FG24 0.003 5 ± 0.000 2bcdefgh
    FG24 5.48 ± 0.451abcd FG24 3.85 ± 0.129abcd FG27 0.003 5 ± 0.000 6bcdefgh
    G22 5.47 ± 0.666abcd G3 3.84 ± 0.167abcde FG28 0.003 5 ± 0.000 2bcdefgh
    FG8 5.44 ± 0.231abcd FG18 3.83 ± 0.404abcde FG32 0.003 5 ± 0.000 4bcdefgh
    FG16 5.40 ± 0.058abcd FG27 3.83 ± 0.208abcde G24 0.003 5 ± 0.000 1bcdefgh
    FG26 5.40 ± 0.071abcd FG3 3.82 ± 0.278abcde FG20 0.003 3 ± 0.000 1cdefgh
    FG29 5.40 ± 0.245abcd FG19 3.80 ± 0.424abcde FG21 0.003 3 ± 0.000 2cdefgh
    G5 5.38 ± 0.451abcd FG31 3.80 ± 0.283abcde FG3 0.003 3 ± 0.000 3cdefgh
    FG28 5.37 ± 0.205abcde G13 3.80 ± 0.316abcde G3 0.003 3 ± 0.000 1cdefgh
    FG32 5.37 ± 1.174abcde G24 3.80 ± 0.000abcde G5 0.003 3 ± 0.000 4cdefgh
    FG7 5.36 ± 0.208abcde G6 3.78 ± 0.236abcde G11 0.003 2 ± 0.000 5cdefgh
    G17 5.30 ± 0.141abcde G5 3.76 ± 0.321abcde G6 0.003 2 ± 0.000 5cdefgh
    G23 5.30 ± 0.690abcde FG6 3.74 ± 0.478abcde G9 0.003 2 ± 0.000 4cdefgh
    G10 5.25 ± 0.424abcde FG21 3.70 ± 0.100bcde FG25 0.003 1 ± 0.000 4cdefgh
    FG3 5.20 ± 0.409abcde G4 3.70 ± 0.557bcde G13 0.003 1 ± 0.000 6cdefgh
    G4 5.20 ± 0.436abcde FG20 3.67 ± 0.058bcde G4 0.003 1 ± 0.000 7cdefgh
    G6 5.20 ± 1.686abcde FG17 3.60 ± 0.283cde G12 0.003 0 ± 0.001 5cdefgh
    G3 5.18 ± 0.367bcde G12 3.60 ± 0.993cde FG17 0.002 9 ± 0.000 6defgh
    G18 5.10 ± 0.354bcdef G18 3.60 ± 0.566cde FG26 0.002 9 ± 0.000 5defgh
    FG17 5.05 ± 0.171bcdefg FG25 3.57 ± 0.116cde G17 0.002 9 ± 0.000 3defgh
    G19 5.05 ± 0.495bcdefg G23 3.57 ± 0.493cde G18 0.002 9 ± 0.000 8defgh
    G13 5.00 ± 0.200bcdefg G17 3.55 ± 0.071cde G22 0.002 9 ± 0.001 0defgh
    G11 4.90 ± 0.200bcdefg FG26 3.50 ± 0.265cde G23 0.002 9 ± 0.000 7defgh
    WT 4.87 ± 0.702cdefg WT 3.47 ± 0.252cde FG29 0.002 8 ± 0.001 0defgh
    G9 4.83 ± 1.980cdefg G22 3.43 ± 0.493cde FG6 0.002 8 ± 0.000 7defgh
    G12 4.76 ± 0.332cdefg FG29 3.40 ± 0.566cde WT 0.002 6 ± 0.000 6defgh
    FG6 4.60 ± 0.205defgh FG30 3.40 ± 0.100cde G19 0.002 5 ± 0.000 1defgh
    G15 4.60 ± 0.392defgh G15 3.37 ± 0.322cde G15 0.002 3 ± 0.000 5defgh
    FG30 4.33 ± 0.126efgh G7 3.37 ± 0.153cde FG30 0.002 2 ± 0.000 2efgh
    G16 4.13 ± 0.492fgh G19 3.35 ± 0.071cde G7 0.001 9 ± 0.000 4fgh
    G20 4.07 ± 0.141ghi G16 2.80 ± 0.781def G16 0.001 7 ± 0.001 2fgh
    G7 3.73 ± 0.586hi G20 2.60 ± 0.781ef G20 0.001 4 ± 0.000 9gh
    G2 3.20 ± 0.515i G2 2.25 ± 0.919f G2 0.001 0 ± 0.001 1h
    均值 Mean 5.28 ± 0.67 均值 Mean 3.91 ± 0.72 均值 Mean 0.003 6 ± 0.001 7
    注:数字表示方式为平均值 ± 标准差;不同处理变量差异采用Duncans multiple-range差异性检验进行分析;不同的小写字母表示处理间差异达到显著水平P < 0.05。Notes: data were mean ± SE; different lowercases represent significant differences among treatments using Duncans multiple-range test, P < 0.05.
    下载: 导出CSV

    表  4  特征根及标准化特征向量

    Table  4.   Characteristic roots and standardized eigenvectors

    主成分
    Principal
    component
    特征根
    Characteristic
    root
    方差贡献率
    Variance contribution rate/%
    累积贡献率
    Cumulative contribution rate/%
    性状
    Trait
    因子载荷
    Factor
    loading
    特征向量
    Standardized eigenvector
    1 2.651 88.351 88.351 树高 Tree height/m 0.882 0.542
    2 0.315 10.492 98.842 胸径 DBH/cm 0.964 0.592
    3 0.035 1.158 100 材积 Volume/m3 0.971 0.596
    下载: 导出CSV

    表  5  参试株系综合评价

    Table  5.   Comprehensive evaluation of dtested clones

    株系 Line Y
    Y value
    1级通直度比率
    Ratio of level 1
    straightness/%
    综合评价
    Comprehensive
    evaluation
    株系 Line Y
    Y value
    1级通直度比率
    Ratio of level 1
    straightness/%
    综合评价
    Comprehensive
    evaluation
    FG15 3.950 100.00 优秀 Good FG20 0.072 100.00 良好 Better
    FG8 3.091 85.71 优秀 Good G5 −0.069 85.71 一般 Common
    FG9 3.035 100.00 优秀 Good G3 −0.173 100.00 一般 Common
    FG12 2.337 100.00 优秀 Good FG3 −0.177 83.33 一般 Common
    FG4 2.117 100.00 优秀 Good FG25 −0.214 100.00 一般 Common
    FG5 2.102 100.00 优秀 Good G6 −0.278 100.00 一般 Common
    FG14 1.999 100.00 优秀 Good G11 −0.427 100.00 一般 Common
    FG1 1.650 100.00 优秀 Good G4 −0.434 100.00 一般 Common
    FG13 1.487 80.00 优秀 Good G9 −0.444 100.00 一般 Common
    FG10 1.010 83.33 优秀 Good G13 −0.507 100.00 一般 Common
    FG11 0.991 100.00 良好 Better G23 −0.573 100.00 一般 Common
    FG2 0.976 100.00 良好 Better FG26 −0.587 100.00 一般 Common
    G8 0.797 100.00 良好 Better G22 −0.590 100.00 一般 Common
    FG7 0.645 85.71 良好 Better G17 −0.613 100.00 一般 Common
    FG23 0.644 100.00 良好 Better FG29 −0.746 66.67 一般 Common
    FG16 0.564 50.00 良好 Better G18 −0.752 100.00 一般 Common
    FG31 0.507 100.00 良好 Better FG17 −0.802 66.67 一般 Common
    G14 0.497 100.00 良好 Better G12 −1.034 83.33 较差 Less
    FG33 0.483 100.00 良好 Better FG6 −1.137 100.00 较差 Less
    G24 0.403 100.00 良好 Better WT −1.296 100.00 较差 Less
    G10 0.394 80.00 良好 Better G19 −1.314 100.00 较差 Less
    FG18 0.361 100.00 良好 Better G15 −1.844 100.00 较差 Less
    FG27 0.310 100.00 良好 Better FG30 −2.130 100.00 较差 Less
    FG19 0.255 100.00 良好 Better G7 −2.923 80.00 较差 Less
    FG24 0.244 100.00 良好 Better G16 −3.307 80.00 较差 Less
    FG32 0.195 100.00 良好 Better G20 −3.765 100.00 较差 Less
    FG28 0.195 100.00 良好 Better G2 −5.255 33.33 较差 Less
    FG21 0.078 100.00 良好 Better
    下载: 导出CSV
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出版历程
  • 收稿日期:  2018-12-25
  • 修回日期:  2019-03-20
  • 网络出版日期:  2019-09-06
  • 刊出日期:  2019-09-01

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