转BpGH3.5基因白桦优良株系选择
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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。
[Objective] The auxinamide synthase gene family(GH3 s) 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. [Method] In 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. [Result] PCR showed that the npt II 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.[Conclusion] Transgenic 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.
[Objective] The auxinamide synthase gene family(GH3 s) 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. [Method] In 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. [Result] PCR showed that the npt II 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.[Conclusion] Transgenic 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.
引文
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[2]Mu H Z, Liu Z J, Lin L, et al. Transcriptomic analysis of phenotypic changes in birch(Betula platyphylla)autotetraploids[J]. International Journal of Molecular Sciences, 2012, 13:13012-13029.
[3]姜静,杨光,祝泽兵,等.白桦强化种子园优良家系选择[J].东北林业大学学报, 2011, 39(1):1-4.Jiang J, Yang G, Zhu Z B, et al. Family selectionfrom intensive seed orchard of Betula platyphylla[J]. Journal of Northeast Forestry University, 2011, 39(1):1-4.
[4]刘超逸,刘桂丰,方功桂,等.四倍体白桦木材纤维性状比较及优良母树选择[J].北京林业大学学报,2017,39(2):9-15.Liu C Y,Liu G F,Fang G G,et al.Comparison of tetraploid Betula platyphylla wood fiber traits and selection of superior seed trees[J].Journal of Beijing Forestry University,2017,39(2):9-15.
[5]刘宇,徐焕文,张广波,等.白桦半同胞子代多点生长性状测定及优良家系选择[J].北京林业大学学报,2017,39(3):7-15.Liu Y,Xu H W,Zhang G B,et al.Multipoint growth trait test of half-sibling offspring and excellent family selection of Betula platyphylla[J].Journal of Beijing Forestry University,2017,39(3):7-15.
[6]徐焕文,刘宇,李志新,等.5年生白桦杂种子代多点稳定性分析及优良家系选择[J].北京林业大学学报,2015,37(12):24-31.Xu H W,Liu Y,Li Z X,et al.Analysis of the stability and superiority of five-year-old birch crossbreed families based on a multi-site test[J].Journal of Beijing Forestry University,2015,37(12):24-31.
[7]Huang H J,Wang S,Jiang J,et al.Overexpression of BpAP1induces early flowering and produces dwarfism in Betula platyphylla×B.pendula[J].Physiol Plant,2014,151:495-506.
[8]詹亚光,王玉成,王志英,等.白桦的遗传转化及转基因植株的抗虫性[J].植物生理与分子生物学学报,2003,29(5):380-386.Zhan Y G,Wang Y C,Wang Z Y,et al.Genetic transformation of Betula platyphylla and Insect resistance of the transgenic plants[J].Journal of Plant Physiology and Molecular Biology,2003,29(5):380-386.
[9]李园园,杨光,韦睿,等.转TabZIP基因白桦的获得及耐盐性分析[J].南京林业大学学报(自然科学版),2013,37(5):6-12.Li Y Y,Yang G,Wei R,et al.TabZIP transferred Betula platyphylla generation and salt tolerance analysis[J].Journal of Nanjing Forestry University(Natural Sciences Edition),2013,37(5):6-12.
[10]Zhang W B,Wei R,Chen S,et al.Functional characterization of CCR in birch(Betula platyphylla×Betula pendula)through overexpression and suppression analysis[J].Physiologia Plantarum,2015,154:283-296.
[11]陈继英,刘超逸,王朔,等.白桦BpTOPP1基因功能[J].东北林业大学学报,2018,46(8):13-19.Chen J Y,Liu C Y,Wang S,et al.A preliminary study on Function of BpTOPP 1 Gene in Betulla platyphylla×B.pendula[J].Journal of Northeast Forestry University,2018,46(8):13-19.
[12]范志勇,姜晶,王芳,等.转BpCHS基因过量表达白桦叶片和韧皮部色素含量及植株表型分析[J].东北林业大学学报,2018,46(6):8-13.Fan Z Y,Jiang J,Wang F,et al.Overexpression of BpCHSconfers changes of pigment content in leaves and phloem and other phenotypic traits in transgenic birch[J].Journal of Northeast Forestry University,2018,46(6):8-13.
[13]Yang G,Chen S,Wang S,et al.BpGH3.5,an early auxinresponse gene,regulates root elongation in Betula platyphylla×Betula pendula[J].Plant Cell Tissue and Organ Culture,2015,120(1):239-250.
[14]Guilfoyle T J,Ulmasov T,Hagen G.The ARF family of transcription factors and their role in plant hormone-responsive transcription[J].Cellular and Molecular Life Sciences,1998,54(7):619-627.
[15]Liscum E,Reed J W.Genetics of Aux/IAA and ARF action in plant growth and development[J].Plant Molecular Biology,2002,49(3-4):387-400.
[16]黎颖,左开井,唐克轩.植物GH3基因家族的功能研究概况[J].植物学报,2008,25(5):507-515.Li Y,Zuo K J,Tang K X.A survey of functional studies of the GH3 gene family in plants[J].Chinese Bulletin of Botany,2008,25(5):507-515.
[17]Mellor N,Band LR,Pěn?ík A,et al.Dynamic regulation of auxin oxidase and conjugating enzymes AtDAO1 and GH3 modulates auxin homeostasis[J].Proceedings of the National Academy of Sciences of the United States of America,2016,113(39):11022-11027.
[18]Takase T,Nakazawa M,Ishikawa A,et al.ydk1-D,an auxinresponsive GH3 mutant that is involved in hypocotyl and root elongation[J].The Plant Journal,2004,37:471-483.
[19]Nakazawa M,Yabe N,Ichikawa T,et al.DFL1,an auxinresponsive GH3 gene homologue,negatively regulates shoot cell elongation and lateral root formation,and positively regulates the light response of hypocotyl length[J].The Plant Journal,2001,25:213-221.
[20]刘晓东,李月,王若仲,等.过表达GH3-5提高拟南芥抗旱的分子机制[J].南京农业大学学报,2016,39(4):557-562.Liu X D,Li Y,Wang R Z,et al.Molecular mechanism of drought tolerance conferred by overexpression of GH3-5[J].Journal of Nanjing Agricultural University,2016,39(4):557-562.
[21]刘晓东,王若仲,焦彬彬,等.拟南芥IAA酰胺合成酶GH3-6负调控干旱和盐胁迫的反应[J].植物学报,2016,51(5):586-593.Liu X D,Wang R Z,Jiao B B,et al.Indole acetic acid-amido synthetase GH3-6 negatively regulates response to drought and salt in Arabidopsis[J].Bulletin of Botany,2016,51(5):586-593.
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