PH-START1调控拟南芥种子发育的功能分析
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摘要
种子发育直接关系到作物的产量和品质,虽然文献已报道了多个可调控种子发育的基因,但种子发育的分子机制还未建成完整的调控网络。为鉴定调控种子发育的主效基因、完善种子发育调控网络,本研究在拟南芥(Arabidopsis thaliana)中筛选到可调控种子发育的基因PH-START1 (pleckstrin homolgy-the lipid/sterol-binding StAR-related lipid transfer protein domains 1),在拟南芥中过表达PHSTART1可使结实率降低61.7%、籽粒重下降80%,说明PH-START1是调控种子发育的主效基因。本研究创制了PH-START1的功能获得和缺失的转基因拟南芥植株,通过对转基因植株角果发育及结实率、千粒重分析,确定了其在调控种子发育中的功能;通过解剖镜下对种子胚和胚乳发育的观察,明确目的基因调控种子发育的具体过程;通过对目的基因的表达量和组织定位分析,明确其表达模式。结果发现,野生型种子排列整齐紧密,果荚饱满;PH-START1的T-DNA插入突变体ph-start1的种子排列整齐,占据着角果的整个内部空间,单个角果可结55粒种子,结实率为对照的117%;但过表达PH-START1转基因拟南芥OE-1的种子排列稀疏散乱,其单个角果只有18粒种子,结实率仅为对照的38.30%。进一步分析发现,PHSTART1还影响种子的结构,过表达PH-START1后的种子胚乳细胞明显减少,糊粉层细胞排列散乱,胚发育受阻。对PH-START1的表达模式分析表明,PH-START1在种子中表达量最多,在花的雌蕊中,随着发育时间的延长主要分布在花柱的基部和顶部;在雄蕊中,随着发育时间的延长花丝和花药中的表达量增加,最后主要集中在花药的花粉粒中表达。本研究鉴定到调控种子发育的主效基因PH-START1,明确了其影响种子发育的具体过程及其表达模式,为完善种子发育调控分子网络和为现代分子育种提供了理论依据。
Seed development is directly related to crop yield and quality, although several genes that can regulate seed development have been reported in the literature, the molecular mechanism of seed development has not yet established a complete regulatory network. In order to identify the main genes regulating seeddevelopment and improve the control network of seed development, the gene PH-START1(pleckstrin homolgy-the lipid/sterol-binding St AR-related lipid transfer protein domains 1) was identified that can regulate seed development in Arabidopsis thaliana, over-expression of PH-START1 in Arabidopsis reduced the seed yield by61.7% and the grain weight by 80%, which indicated that PH-START1 was the main gene regulated seed development. The function of the gene in regulating the development of seed was determined by analyzing the growth, seed setting rate and grain weight of the transgenic plants with functional loss and gaining. By observing the development of seed embryo and endosperm, the specific process of gene regulation of seed development was clarified. By analyzing the tissue location of the target gene, the expression pattern was clarified. The wild-type seeds were arranged neatly and tightly and the pods were full under the microscope.The PH-START1 T-DNA insertion mutant, the ph-start1, was arranged neatly, occupying the entire inner space of the siliques, with a single silique producing 55 seeds, with a 117% seed yield. However, the seeds of PHSTART1 transgene OE-1 in A. thaliana were sparse and scattered, with only 18 seeds in a single silique, and only 38.30% of the seed setting rate. Further analysis showed that PH-START1 also affected the structure of the seeds. The endosperm cells of the seeds after over-expression of PH-START1 decreased obviously, and the aleurone layer cells were arranged in disorder and embryo development was blocked. In order to deepen the mechanism of PH-START1, the expression pattern of PH-START1 was analyzed. The results showed that PHSTART1 was the most expressed in seed, and was mainly distributed at the base and top of style with the growth time. In stamens, the expression in filament and anther increased with the increase of development time, and was concentrated in pollen grains. This study identified that the PH-START1 was a main gene that regulates seed development, and clarified its specific process and expression patterns that affected seed development. This result provide a theoretical basis for improving seed development regulatory molecular network and modern molecular breeding.
Seed development is directly related to crop yield and quality, although several genes that can regulate seed development have been reported in the literature, the molecular mechanism of seed development has not yet established a complete regulatory network. In order to identify the main genes regulating seeddevelopment and improve the control network of seed development, the gene PH-START1(pleckstrin homolgy-the lipid/sterol-binding St AR-related lipid transfer protein domains 1) was identified that can regulate seed development in Arabidopsis thaliana, over-expression of PH-START1 in Arabidopsis reduced the seed yield by61.7% and the grain weight by 80%, which indicated that PH-START1 was the main gene regulated seed development. The function of the gene in regulating the development of seed was determined by analyzing the growth, seed setting rate and grain weight of the transgenic plants with functional loss and gaining. By observing the development of seed embryo and endosperm, the specific process of gene regulation of seed development was clarified. By analyzing the tissue location of the target gene, the expression pattern was clarified. The wild-type seeds were arranged neatly and tightly and the pods were full under the microscope.The PH-START1 T-DNA insertion mutant, the ph-start1, was arranged neatly, occupying the entire inner space of the siliques, with a single silique producing 55 seeds, with a 117% seed yield. However, the seeds of PHSTART1 transgene OE-1 in A. thaliana were sparse and scattered, with only 18 seeds in a single silique, and only 38.30% of the seed setting rate. Further analysis showed that PH-START1 also affected the structure of the seeds. The endosperm cells of the seeds after over-expression of PH-START1 decreased obviously, and the aleurone layer cells were arranged in disorder and embryo development was blocked. In order to deepen the mechanism of PH-START1, the expression pattern of PH-START1 was analyzed. The results showed that PHSTART1 was the most expressed in seed, and was mainly distributed at the base and top of style with the growth time. In stamens, the expression in filament and anther increased with the increase of development time, and was concentrated in pollen grains. This study identified that the PH-START1 was a main gene that regulates seed development, and clarified its specific process and expression patterns that affected seed development. This result provide a theoretical basis for improving seed development regulatory molecular network and modern molecular breeding.
引文
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游晓慧,李威,陶启长,等.2011.WD40重复蛋白家族基因At1g65030调控拟南芥种子的重量与体积[J].植物生理学报,47(7):715-725.(You X H,Li W,Tao Q C e al.2011.At1g65030,a WD40-repeat protein gene,regulates seed mass and size in Arabidopsis[J].Plant Physiology Journal,47(7):715-725.)
周苹.2013.GH3.9基因过表达对拟南芥生长发育的影响研究[D].硕士学位论文.湖南大学.导师:刘选明.pp.11-14.(Zhou P.2013.Effect of GH3.9 gene overexpression on growth and development of Arabidopsis thaliana[D]Thesis for M.S.Hunan University.Supervisor:Liu XM.pp.11-14.)
Bednarek J,Boulaflous A,Girousse C,et al.2012.Down-regulation of the TaGW2 gene by RNA interference results in decreased grain size and weight in wheat[J].Journa of Experimental Botany,63(16):5945-5955.
Disch S,Anastasiou E,Sharma V K,et al.2006.The E3 ubiquitin ligase BIG BROTHER controls arabidopsis organ size in a dosage-dependent manner[J].Current Biology16(3):272-279.
Du L,Li N,Chen L,et al.2014.The ubiquitin receptor DA1regulates seed and organ size by modulating the stability of the ubiquitin-specific protease UBP15/SOD2 in Arabidopsis[J].Plant Cell,26(2):665-677.
Garcia D,Fitz G J,Berger F.2005.Maternal control of integument cell elongation and zygotic control of endosperm growth are coordinated to determine seed size in Arabidopsis[J].Plant Cell,17(1):52-60.
Johnson C S,Kolevski B,Smyth D R.2002.TRANSPARENTTESTA GLABRA2,a trichome and seed coat development gene of Arabidopsis,encodes a WRKY transcription factor[J].Plant Cell,14(6):1359-1375.
Kang X,Li W,Zhou Y,et al.2013.A WRKY transcription factor recruits the SYG1-like protein SHB1 to activate gene expression and seed cavity enlargement[J].PLoSGenetics,9(3):e1003347.
Kang X,Ni M.2006.Arabidopsis SHORT HYPOCOTYL UN-DER BLUE1 contains SPX and EXS domains and acts in cryptochrome signaling[J].Plant Cell,18(4):921-934.
Lemmon M A,Ferguson K M,Aberams C S.2002.Pleckstrin homology domains and the cytoskeleton[J].FEBS Letters,513(1):71-76.
Li N,Li Y.2016.Signaling pathways of seed size control in plants[J].Current Opinion in Plant Biology,33(1):23-32.
Li Q,Li L,Yang X,et al.2010.Relationship,evolutionary fate and function of two maize co-orthologs of rice GW2 associated with kernel size and weight[J].BMCPlant Biology,10(3):143-149.
Li Y,Zheng L,Corke F,et al.2008.Control of final seed and organ size by the DA1 gene family in Arabidopsis thaliana[J].Genes&development,22(10):1331-1336.
Luo M,Dennis E S,Berger F,et al.2005,MINISEED3(MINI3),a WRKY family gene,and HAIKU2(IKU2),a leucine-rich repeat(LRR)KINASE gene,are regulators of seed size in Arabidopsis[J].Proceedings of the National Academy of Sciences of the USA,102(48):17531-17536.
Maffucci T,Falasca M.2001.Specificity in pleckstrin homology(PH)domain membrane targeting:A role for a phosphoinositide-protein co-operative mechanism[J].FEBSLetters,506(3):173-179.
Ohto M A,Floyd S K,Fischer R L,et al.2009.Effects of APETALA2 on embryo,endosperm,and seed coat development determine seed size in Arabidopsis[J].Plant Reproduction,22(4):277-289.
Ponting C P,Aravind L.1999.START:A lipid-binding domain in StAR,HD-ZIP and signalling proteins[J].Trends in Biochemical Sciences,24(4):130-132.
Rebecchi M J,Scarlata S.1998.Pleckstrin homology domains:A common fold with diverse functions[J].Annual Review of Biophysics and Biomolecular Structure,27(4):503-528.
Song X J,Huang W,Shi M,et al.2007.A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase[J].Nature Genetics,39(5):623-630.
Wang A,Garcia D,Zhang H,et al.2010.The VQ motif protein IKU1 regulates endosperm growth and seed size in Arabidopsis[J].Plant Journal,63(4):670-679.
Wang H Q,Wang K,Du Q G,et al.2018.Maize Urb2 protein is required for kernel development and vegetative growth by affecting pre-ribosomal RNA processing[J].New Phytologist.doi:10.111/nph.15057.
Xia T,Li N,Dumenil J,et al.2013.The ubiquitin receptor DA1 interacts with the E3 ubiquitin ligase DA2 to regulate seed and organ size in Arabidopsis[J].Plant Cell,25(9):3347-3359.
Xiao Y G,Sun Q B,Kang X J,et al.2016.SHORT HYPOCOT-YL UNDER BLUE1 or HAIKU2 mixexpression alters canola and Arabidopsis seed development[J].New Phytologist,209(2):636-649.
Yamagishi K,Nagata N,Yee K M,et al.2005.TANMEI/EMB2757 encodes a WD repeat protein required for embryo development in Arabidopsis[J].Plant Physiology,139(1):163-173.
Zhou Y,Zhang X,Kang X,et al.2009.SHORT HYPOCOT-YL UNDER BLUE1 associates with MINISEED3 and HAIKU2 promoters in vivo to regulate Arabidopsis seed development[J].Plant Cell,21(1):106-117.
游晓慧,李威,陶启长,等.2011.WD40重复蛋白家族基因At1g65030调控拟南芥种子的重量与体积[J].植物生理学报,47(7):715-725.(You X H,Li W,Tao Q C e al.2011.At1g65030,a WD40-repeat protein gene,regulates seed mass and size in Arabidopsis[J].Plant Physiology Journal,47(7):715-725.)
周苹.2013.GH3.9基因过表达对拟南芥生长发育的影响研究[D].硕士学位论文.湖南大学.导师:刘选明.pp.11-14.(Zhou P.2013.Effect of GH3.9 gene overexpression on growth and development of Arabidopsis thaliana[D]Thesis for M.S.Hunan University.Supervisor:Liu XM.pp.11-14.)
Bednarek J,Boulaflous A,Girousse C,et al.2012.Down-regulation of the TaGW2 gene by RNA interference results in decreased grain size and weight in wheat[J].Journa of Experimental Botany,63(16):5945-5955.
Disch S,Anastasiou E,Sharma V K,et al.2006.The E3 ubiquitin ligase BIG BROTHER controls arabidopsis organ size in a dosage-dependent manner[J].Current Biology16(3):272-279.
Du L,Li N,Chen L,et al.2014.The ubiquitin receptor DA1regulates seed and organ size by modulating the stability of the ubiquitin-specific protease UBP15/SOD2 in Arabidopsis[J].Plant Cell,26(2):665-677.
Garcia D,Fitz G J,Berger F.2005.Maternal control of integument cell elongation and zygotic control of endosperm growth are coordinated to determine seed size in Arabidopsis[J].Plant Cell,17(1):52-60.
Johnson C S,Kolevski B,Smyth D R.2002.TRANSPARENTTESTA GLABRA2,a trichome and seed coat development gene of Arabidopsis,encodes a WRKY transcription factor[J].Plant Cell,14(6):1359-1375.
Kang X,Li W,Zhou Y,et al.2013.A WRKY transcription factor recruits the SYG1-like protein SHB1 to activate gene expression and seed cavity enlargement[J].PLoSGenetics,9(3):e1003347.
Kang X,Ni M.2006.Arabidopsis SHORT HYPOCOTYL UN-DER BLUE1 contains SPX and EXS domains and acts in cryptochrome signaling[J].Plant Cell,18(4):921-934.
Lemmon M A,Ferguson K M,Aberams C S.2002.Pleckstrin homology domains and the cytoskeleton[J].FEBS Letters,513(1):71-76.
Li N,Li Y.2016.Signaling pathways of seed size control in plants[J].Current Opinion in Plant Biology,33(1):23-32.
Li Q,Li L,Yang X,et al.2010.Relationship,evolutionary fate and function of two maize co-orthologs of rice GW2 associated with kernel size and weight[J].BMCPlant Biology,10(3):143-149.
Li Y,Zheng L,Corke F,et al.2008.Control of final seed and organ size by the DA1 gene family in Arabidopsis thaliana[J].Genes&development,22(10):1331-1336.
Luo M,Dennis E S,Berger F,et al.2005,MINISEED3(MINI3),a WRKY family gene,and HAIKU2(IKU2),a leucine-rich repeat(LRR)KINASE gene,are regulators of seed size in Arabidopsis[J].Proceedings of the National Academy of Sciences of the USA,102(48):17531-17536.
Maffucci T,Falasca M.2001.Specificity in pleckstrin homology(PH)domain membrane targeting:A role for a phosphoinositide-protein co-operative mechanism[J].FEBSLetters,506(3):173-179.
Ohto M A,Floyd S K,Fischer R L,et al.2009.Effects of APETALA2 on embryo,endosperm,and seed coat development determine seed size in Arabidopsis[J].Plant Reproduction,22(4):277-289.
Ponting C P,Aravind L.1999.START:A lipid-binding domain in StAR,HD-ZIP and signalling proteins[J].Trends in Biochemical Sciences,24(4):130-132.
Rebecchi M J,Scarlata S.1998.Pleckstrin homology domains:A common fold with diverse functions[J].Annual Review of Biophysics and Biomolecular Structure,27(4):503-528.
Song X J,Huang W,Shi M,et al.2007.A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase[J].Nature Genetics,39(5):623-630.
Wang A,Garcia D,Zhang H,et al.2010.The VQ motif protein IKU1 regulates endosperm growth and seed size in Arabidopsis[J].Plant Journal,63(4):670-679.
Wang H Q,Wang K,Du Q G,et al.2018.Maize Urb2 protein is required for kernel development and vegetative growth by affecting pre-ribosomal RNA processing[J].New Phytologist.doi:10.111/nph.15057.
Xia T,Li N,Dumenil J,et al.2013.The ubiquitin receptor DA1 interacts with the E3 ubiquitin ligase DA2 to regulate seed and organ size in Arabidopsis[J].Plant Cell,25(9):3347-3359.
Xiao Y G,Sun Q B,Kang X J,et al.2016.SHORT HYPOCOT-YL UNDER BLUE1 or HAIKU2 mixexpression alters canola and Arabidopsis seed development[J].New Phytologist,209(2):636-649.
Yamagishi K,Nagata N,Yee K M,et al.2005.TANMEI/EMB2757 encodes a WD repeat protein required for embryo development in Arabidopsis[J].Plant Physiology,139(1):163-173.
Zhou Y,Zhang X,Kang X,et al.2009.SHORT HYPOCOT-YL UNDER BLUE1 associates with MINISEED3 and HAIKU2 promoters in vivo to regulate Arabidopsis seed development[J].Plant Cell,21(1):106-117.