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    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

    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。

       

      Abstract:
      ObjectiveThe auxinamide synthase gene family (GH3s) is a typical auxin primary/early response gene. Most family genes can regulate growth and development by regulating the concentration of free IAA in plants. Therefore, genetic engineering was used to introduce the BpGH3.5 sense and antisense strand into the Betula platyphylla genome in order to obtain fast-growing transgenic Betula platyphylla variety.
      MethodIn total, 54 sense, antisense and control (WT) transgenic lines of BpGH3.5 were used in the study. Tree height, DBH and volume were measured. The genetic stability and relative expression of the target gene of five sense and antisense lines were detected by PCR and qRT-PCR, respectively. The free IAA content was determined by enzyme-linked immunosorbent assay.
      ResultPCR showed that the nptII exogenous genes in the transgenic lines were all positive. qRT-PCR analysis showed that the BpGH3.5 gene was significantly higher in the five sense lines than in the WT line. In contrast, the endogenous BpGH3.5 was significantly down-regulated in the five antisense lines. The genome interfered the expression of BpGH3.5 in Betula platyphylla. The content of endogenous free IAA showed that the IAA content of the transgenic BpGH3.5 sense lines was significantly lower than that of the WT line. IAA content of the five antisense lines was significantly higher than that of the WT line (P< 0.01), and their average IAA content was 52.26% higher than the WT line. The differences in tree height, DBH and volume growth of 7-year-old transgenic BpGH3.5 Betula platyphylla were significant among lines (P< 0.01). Height, DBH and volume of transgenic lines were higher than the population mean, the antisense lines accounted for above 80%. In conclusion, transgenic BpGH3.5 antisense lines of Betula platyphylla can inhibit the expression of BpGH3.5, and reduce the ability of amino acid production resulting release more free IAA to promote the growth of Betula platyphylla. Ten fast growing trees were selected from antisense lines using principal component analysis. The average tree height, DBH and volume of these lines were 8.55%, 19.28%, and 50.42% higher than the population mean, respectively. The average genetic gain of tree volume was 36.3%. Results of selected lines provide useful information for future release transgenic BpGH3.5 lines in Betula platyphylla.
      ConclusionTransgenic BpGH3.5 antisense lines of Betula platyphylla can inhibit the expression of BpGH3.5, and release more free IAA to promote the growth of Betula platyphylla. Ten fast growing trees were selected from antisense lines using principal component analysis.

       

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