利用VIGS技术初步验证WSR1基因功能的研究
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摘要
为进一步了解小麦淀粉合成酶转录因子对淀粉合成的影响,以水稻 RSR1基因为探针,首先利用同源克隆技术和RACE技术获得了小麦 WSR1基因的全长cDNA;生物信息学分析表明,该基因含有AP2保守结构域。随后,以大麦条纹花叶病毒(BSMV)为载体,八氢番茄红素脱氢酶(PDS)基因为指示基因,利用VIGS技术和qRT-PCR技术对 WSR1基因的功能进行研究。结果表明,在接种5 d后, WSR1基因的相对表达量下降到50%, 25 d后下降到12%; WSR1基因沉默后的冀麦325籽粒内支链淀粉、总淀粉和抗性淀粉含量均较对照显著增加;直链淀粉含量、千粒重较对照极显著增加。以上结果表明 WSR1基因负向调控小麦的淀粉合成。
To further understand the effect of wheat starch synthase transcription factors on starch synthesis,the rice RSR1 gene was used as the probe to obtain the homologous RSR1 fragment from wheat genome. The full-length cDNA of the wheat WSR1 gene was cloned using RACE technology. The bioinformatics analysis showed that the gene contains an AP2 conserved domain,namely WSR1. The barley stripe mosaic virus(BSMV) was used as the vector,and the phytoene desaturase(PDS) gene was used as the indicator gene. The function of WSR1 gene was studied by virus induced gene silencing(VIGS) and qRT-PCR. The result showed that after five days of inoculation,the relative expression level of WSR1 gene was reduced to 50%,and remained at a relatively low level(about 12%) after 25 days of inoculation. The contents of amylopectin,total starch and resistant starch were much higher in the WSR1-silenced Jimai 325 than that in the control of Jimai 325 without BSMV:WSR1 transformation. The proportion of amylose was increased from 14.47% to 15.60% and the 1 000-grain weight was increased from 43.1 g to 45.8 g. It was demonstrated that the WSR1 gene negatively regulates starch synthesis in wheat.
To further understand the effect of wheat starch synthase transcription factors on starch synthesis,the rice RSR1 gene was used as the probe to obtain the homologous RSR1 fragment from wheat genome. The full-length cDNA of the wheat WSR1 gene was cloned using RACE technology. The bioinformatics analysis showed that the gene contains an AP2 conserved domain,namely WSR1. The barley stripe mosaic virus(BSMV) was used as the vector,and the phytoene desaturase(PDS) gene was used as the indicator gene. The function of WSR1 gene was studied by virus induced gene silencing(VIGS) and qRT-PCR. The result showed that after five days of inoculation,the relative expression level of WSR1 gene was reduced to 50%,and remained at a relatively low level(about 12%) after 25 days of inoculation. The contents of amylopectin,total starch and resistant starch were much higher in the WSR1-silenced Jimai 325 than that in the control of Jimai 325 without BSMV:WSR1 transformation. The proportion of amylose was increased from 14.47% to 15.60% and the 1 000-grain weight was increased from 43.1 g to 45.8 g. It was demonstrated that the WSR1 gene negatively regulates starch synthesis in wheat.
引文
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[7]刘璇.小麦淀粉糊化特性的关联分析研究[D].郑州:河南农业大学,2014:9. LIU X.Association analysis of starch pasting properties in common wheat [D].Zhengzhou:Henan Agricultural University,2014:9.
[8]于海霞,田纪春.小麦淀粉糊化特性与DArT标记的关联分析[J].作物学报,2012,38(11):1997. YU H X,TIAN J C.Association between starch pasting properties and DArT markers in common wheat [J].Acta Agronomica Sinica,2012,38(11):1997.
[9]李永强,翟红梅,田纪春.蛋白质和淀粉含量对小麦面团流变学特性的影响[J].作物学报,2007,33(6):937. LI Y Q,ZHAI H M,TIAN J C.Effect of protein and starch contents on wheat dough rheological properties [J].Acta Agronomica Sinica,2007,33(6):937.
[10]石培春,王光利,王小国,等.小麦籽粒淀粉主要特性的QTL定位[J].核农学报,2012,26(2):210. SHI P C,WAGN G L,WANG X G,et al.QTL mapping of the major properties of starch in common wheat [J].Journal of Nuclear Agricultural Sciences,2012,26(2):210.
[11]LI S S,JIA J Z,WEI X Y,et al.A intervarietal genetic map and QTL analysis for yield traits in wheat [J].Molecular Breeding,2007(2):167.
[12]TETLOW I J,DAVIES E J,VARDY K A,et al.Subcellular localization of ADP glucose pyrophosphorylase in developing wheat endosperm and analysis of the plastidial isoform [J].Journal of Experimental Botany,2003,54:1.
[13]HANNAH L C,GREENE T.The complexities of starch biosynthesis in cereal endosperms [M].Springer,Berlin,Heidelberg,2009:287-301.
[14]ZHANG Y,HE Z H,YE G Y,et al.Effect of environment and genotype on bread-making quality of spring-sown spring wheat cultivars in China [J].Euphytica,2004,139(1):75.
[15]SOUZA E J,MARTIN J M,GUTTIERI M J,et al.Influence of genotype,environment,and nitrogen management on spring wheat quality [J].Crop Science,2004,44(2):425.
[16]ZHU Y,HONG M M,WANG Z Y.Identification of the regions involved in protein-protein interaction of OsBP-5 and OsEBP-89 [J].Acta Photophysiologica Sinica,2003,29(5):425.
[17]ROOK F,CORKE F,CARD R,et al.Impaired sucrose-induction mutants reveal the modulation of sugar-induced starch biosynthetic gene expression by abscisic acid signaling [J].The Plant Journal,2001,26(4):421.
[18]FU F F,XUE H W.Coexpression analysis identifies rice starch regulator 1,a rice AP2/EREBP family transcription factor,as a novel rice starch biosynthesis regulator [J].Plant Physiol,2010,154:927.
[19]SCOFIELD S R,HUANG L,BRANDT A S,et al.Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resis-tance pathway [J].Plant Physiol,2005,138:2165.
[20]张增艳,姚乌兰,辛志勇.植物基因功能鉴定新工具-病毒诱导基因沉默技术的研究进展[J].植物遗传资源学报,2006,7(1):100. ZHAGN Z Y,YAO W L,XIN Z Y.Advances on virus-induced gene silencing for gene function characterization in plants [J].Journal of Plant Genetic Resources,2006,7(1):100.
[21]ZHOU H B,LI S F,DENG Z Y,et al.Molecular analysis of three new receptor-like kinase genes from hexaploid wheat and evidence for their participation in the wheat hypersensitive response to stripe rust fungus infection [J].The Plant Journal,2007,52:420.
[22]刘晓东,张增艳,姚乌兰,等.在小麦上实施大麦条斑病毒诱导的基因沉默[J].作物学报,2005,31(11):1518. LIU X D,ZHANG Z Y,YAO W L,et al.Implement of barley stripe mosaic virus-based induced gene silencing in wheat [J].Acta Agronomica Sinica,2005,31(11):1518.
[23]赵丹,赵继荣,黄茜,等.利用BSMV-VIGS技术快速分析小麦 TNBL1基因的抗黄矮病功能[J].作物学报,2011,37(11):2106. ZHAO D,ZHAO J R,HUAGN Q,et al.Functional analysis of TNBL1 gene in wheat defense response to barley yellow dwarf virus using BSMV-VIGS technique [J].Acta Agronomica Sinica,2011,37(11):2106.
[24]李建嫄,张立荣,张娜,等.利用VIGS技术分析 TcLr19中 TaSTKC8基因的抗叶锈性功能[J].河北农业大学学报,2015,38(5):11. ZHAGN J Y,ZHAGN L R,ZHAGN N,et al.Functonal analysis of TaSTKC8 gene on wheat leaf rust resistance in TcLr19 by virus induced gene silencing(VIGS) [J].Journal of Agricultural University of Hebei,2015,38(5):11.
[25]何照花.谷物中直、支链及总淀粉双波长测定法[J].生物化学与生物物理进展,1981(1):70. HE F Z.Double-wavelength determination of amylose,amylopectin and total starch in cereals [J].Progress in Biochemistry and Biophysics,1981(1):70.
[26]戴双,程敦公,李豪圣,等.小麦直、支链淀粉和总淀粉含量的比色快速测定研究[J].麦类作物学报,2008,28(3):442. DAI S,CHENG D G,LI H S,et al.Simultaneous and rapid spectrophotometric determination of amylose,amylopectin and total starch in wheat grain [J].Journal of Triticeae Crops,2008,28(3):442.
[27]张计育,王庆菊,郭忠仁.植物AP2/ERF类转录因子研究进展[J].遗传,2012,34(7):835. ZHANG J Y,WANG Q J,GUO Z R.Progresses on plant AP2/ERF transcription factors [J].Hereditas,2012,34(7):835.
[28]BENNYPAUL H S,MUTTI J S,RUSTGI S,et al.Virus-induced gene silencing(VIGS) of genes expressed in root,leaf and meiotic tissues of wheat [J].Functional & inTegrative Genomics,2012,12(1):143.
[29]马猛.大麦条斑花叶病毒(BSMV)诱导小麦穗部基因有效沉默体系的建立[D].杨凌:西北农林科技大学,2010:19. ZHAGN M.Development of an effective system of virus induced gene silencing by barley stripe mosaic virus(BSMV) in wheat spikes [D].Yangling:Northwest A&F University,2010:19.
[30]YANG H,ZHAO X A,ZHOU Y Q.Starch synthesis in the cereal endosperm [J].Current Opinion in Plant Biology,2003,6(3):215.
[2]班进福,刘彦军,郭进考,等.2008年冀中小麦样品粉质参数状况分析[J].河北农业科学,2010,14(2):67. BAN J F,LIU Y J,GUO J K,et al.Analysis on farinograph paramenters of wheat in central Hebei in 2008 [J].Journal of Hebei Agricultural Sciences,2010,14(2):67.
[3]南富波.病毒诱导的基因沉默体系优化及小麦SBEIIb基因功能的研究[D].石河子:石河子大学,2014:2. NAN F B.Optimization of virus induced gene silencing system and function analysis of wheat(Triticum aestivum L.) SBEIIb [D].Shihezi:Shihezi University,2014:2.
[4]张勇,何中虎.我国春播小麦淀粉糊化特性研究[J].中国农业科学,2002,35(5):471. ZHANG Y,HE Z H.Investigation on paste property of spring-sown Chinese wheat [J].Scientia Agricultura Sinica,2002,35(5):471.
[5]刘霞,谭胜兵,田纪春,等.不同淀粉组分含量小麦品种灌浆过程中淀粉去分支酶的活性及类型[J].中国农业科学,2010,43(4):850. LIU X,TAN S B,TIAN J C,et al.Activity and types of starch debranching enzyme in wheat cultivars containing different starch contents during the grain filling period [J].Scientia Agricultura Sinica,2010,43(4):850.
[6]张洁.淀粉积累规律及含量对小麦品质的影响及黑麦染色体对小麦贮藏蛋白表达的影响研究[D].成都:四川农业大学,2008:13. ZHANG J.The studies on starch aceumulation regularity and the effete of starch on wheat quality and the effete of rye ehromosomes on the expression of storage protein in wheat and rye [D].Chengdu:Sichuan Agricultural University,2008:13.
[7]刘璇.小麦淀粉糊化特性的关联分析研究[D].郑州:河南农业大学,2014:9. LIU X.Association analysis of starch pasting properties in common wheat [D].Zhengzhou:Henan Agricultural University,2014:9.
[8]于海霞,田纪春.小麦淀粉糊化特性与DArT标记的关联分析[J].作物学报,2012,38(11):1997. YU H X,TIAN J C.Association between starch pasting properties and DArT markers in common wheat [J].Acta Agronomica Sinica,2012,38(11):1997.
[9]李永强,翟红梅,田纪春.蛋白质和淀粉含量对小麦面团流变学特性的影响[J].作物学报,2007,33(6):937. LI Y Q,ZHAI H M,TIAN J C.Effect of protein and starch contents on wheat dough rheological properties [J].Acta Agronomica Sinica,2007,33(6):937.
[10]石培春,王光利,王小国,等.小麦籽粒淀粉主要特性的QTL定位[J].核农学报,2012,26(2):210. SHI P C,WAGN G L,WANG X G,et al.QTL mapping of the major properties of starch in common wheat [J].Journal of Nuclear Agricultural Sciences,2012,26(2):210.
[11]LI S S,JIA J Z,WEI X Y,et al.A intervarietal genetic map and QTL analysis for yield traits in wheat [J].Molecular Breeding,2007(2):167.
[12]TETLOW I J,DAVIES E J,VARDY K A,et al.Subcellular localization of ADP glucose pyrophosphorylase in developing wheat endosperm and analysis of the plastidial isoform [J].Journal of Experimental Botany,2003,54:1.
[13]HANNAH L C,GREENE T.The complexities of starch biosynthesis in cereal endosperms [M].Springer,Berlin,Heidelberg,2009:287-301.
[14]ZHANG Y,HE Z H,YE G Y,et al.Effect of environment and genotype on bread-making quality of spring-sown spring wheat cultivars in China [J].Euphytica,2004,139(1):75.
[15]SOUZA E J,MARTIN J M,GUTTIERI M J,et al.Influence of genotype,environment,and nitrogen management on spring wheat quality [J].Crop Science,2004,44(2):425.
[16]ZHU Y,HONG M M,WANG Z Y.Identification of the regions involved in protein-protein interaction of OsBP-5 and OsEBP-89 [J].Acta Photophysiologica Sinica,2003,29(5):425.
[17]ROOK F,CORKE F,CARD R,et al.Impaired sucrose-induction mutants reveal the modulation of sugar-induced starch biosynthetic gene expression by abscisic acid signaling [J].The Plant Journal,2001,26(4):421.
[18]FU F F,XUE H W.Coexpression analysis identifies rice starch regulator 1,a rice AP2/EREBP family transcription factor,as a novel rice starch biosynthesis regulator [J].Plant Physiol,2010,154:927.
[19]SCOFIELD S R,HUANG L,BRANDT A S,et al.Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resis-tance pathway [J].Plant Physiol,2005,138:2165.
[20]张增艳,姚乌兰,辛志勇.植物基因功能鉴定新工具-病毒诱导基因沉默技术的研究进展[J].植物遗传资源学报,2006,7(1):100. ZHAGN Z Y,YAO W L,XIN Z Y.Advances on virus-induced gene silencing for gene function characterization in plants [J].Journal of Plant Genetic Resources,2006,7(1):100.
[21]ZHOU H B,LI S F,DENG Z Y,et al.Molecular analysis of three new receptor-like kinase genes from hexaploid wheat and evidence for their participation in the wheat hypersensitive response to stripe rust fungus infection [J].The Plant Journal,2007,52:420.
[22]刘晓东,张增艳,姚乌兰,等.在小麦上实施大麦条斑病毒诱导的基因沉默[J].作物学报,2005,31(11):1518. LIU X D,ZHANG Z Y,YAO W L,et al.Implement of barley stripe mosaic virus-based induced gene silencing in wheat [J].Acta Agronomica Sinica,2005,31(11):1518.
[23]赵丹,赵继荣,黄茜,等.利用BSMV-VIGS技术快速分析小麦 TNBL1基因的抗黄矮病功能[J].作物学报,2011,37(11):2106. ZHAO D,ZHAO J R,HUAGN Q,et al.Functional analysis of TNBL1 gene in wheat defense response to barley yellow dwarf virus using BSMV-VIGS technique [J].Acta Agronomica Sinica,2011,37(11):2106.
[24]李建嫄,张立荣,张娜,等.利用VIGS技术分析 TcLr19中 TaSTKC8基因的抗叶锈性功能[J].河北农业大学学报,2015,38(5):11. ZHAGN J Y,ZHAGN L R,ZHAGN N,et al.Functonal analysis of TaSTKC8 gene on wheat leaf rust resistance in TcLr19 by virus induced gene silencing(VIGS) [J].Journal of Agricultural University of Hebei,2015,38(5):11.
[25]何照花.谷物中直、支链及总淀粉双波长测定法[J].生物化学与生物物理进展,1981(1):70. HE F Z.Double-wavelength determination of amylose,amylopectin and total starch in cereals [J].Progress in Biochemistry and Biophysics,1981(1):70.
[26]戴双,程敦公,李豪圣,等.小麦直、支链淀粉和总淀粉含量的比色快速测定研究[J].麦类作物学报,2008,28(3):442. DAI S,CHENG D G,LI H S,et al.Simultaneous and rapid spectrophotometric determination of amylose,amylopectin and total starch in wheat grain [J].Journal of Triticeae Crops,2008,28(3):442.
[27]张计育,王庆菊,郭忠仁.植物AP2/ERF类转录因子研究进展[J].遗传,2012,34(7):835. ZHANG J Y,WANG Q J,GUO Z R.Progresses on plant AP2/ERF transcription factors [J].Hereditas,2012,34(7):835.
[28]BENNYPAUL H S,MUTTI J S,RUSTGI S,et al.Virus-induced gene silencing(VIGS) of genes expressed in root,leaf and meiotic tissues of wheat [J].Functional & inTegrative Genomics,2012,12(1):143.
[29]马猛.大麦条斑花叶病毒(BSMV)诱导小麦穗部基因有效沉默体系的建立[D].杨凌:西北农林科技大学,2010:19. ZHAGN M.Development of an effective system of virus induced gene silencing by barley stripe mosaic virus(BSMV) in wheat spikes [D].Yangling:Northwest A&F University,2010:19.
[30]YANG H,ZHAO X A,ZHOU Y Q.Starch synthesis in the cereal endosperm [J].Current Opinion in Plant Biology,2003,6(3):215.