小麦TaLEC1基因的克隆及其表达特性分析
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摘要
为了探讨LEC1基因在小麦(Triticum aestivum L)非生物胁迫应答中的功能,该研究通过RT-PCR结合RACE技术克隆小麦TaLEC1基因,并采用qRT-PCR方法分析了该基因在小麦不同组织以及不同处理下的表达模式,为深入研究小麦LEC1基因在干旱、高温和高盐胁迫下的响应机制奠定基础。结果表明:(1)成功克隆到小麦TaLEC1基因,该基因cDNA序列全长为1 074 bp,其中5′端非编码区23 bp,开放阅读框为741 bp,3′端非编码区310 bp,编码246个氨基酸,具有典型的CBFD_NFYB结构域。(2)实时荧光定量分析显示,TaLEC1在不同组织间表达差异显著,10 d龄幼苗的叶中表达量最高。(3)TaLEC1基因可被植物激素ABA诱导而上调表达,属于ABA依赖型的表达调控通路。(4)PEG模拟干旱胁迫处理后的0.5～1 h,TaLEC1基因呈上调表达;42℃胁迫处理过程中,TaLEC1基因呈稳定上调表达趋势,并在胁迫处理后12 h和48 h时表达急剧上调,分别为对照的52.8倍和34.5倍;NaCl胁迫处理0.5 h时TaLEC1基因迅速上调表达。研究表明,小麦TaLEC1基因参与ABA依赖的胁迫响应,推测可能在小麦耐受高温胁迫和渗透胁迫过程中发挥着重要的脱水保护功能。
In order to explore the function of LEC1 gene to abiotic stress response in wheat, we cloned a TaLEC1 gene from wheat using RT-PCR combined with RACE technology. The expression patterns of TaLEC1 from different tissues of wheat and under different stress treatments were analyzed by real-time fluorescence quantitative PCR(qRT-PCR). These would be provided a foundation for the drought, high temperature and high salt response mechanism study of LEC1 gene in wheat. The results showed that:(1) the full-length cDNA sequence of TaLEC1 was successfully cloned. The length of cDNA sequence of TaLEC1 is 1 074 bp, contains a 741 bp open reading frame(ORF), with 23 bp in the 5′ UTR and 310 bp in the 3′ UTR. TaLEC1 was predicted to encode a 246 amino acid protein with typical CBFD_NFYB domain.(2) Real time quantitative PCR(qRT-PCR) analysis showed that there were significant differences in the expression levels of TaLEC1 among different tissues and the highest expression was found in 10 d leaves.(3) TaLEC1 could be up-regulated by plant hormone ABA and belongs to ABA-dependent expression regulation pathway.(4) During PEG simulated drought stress, TaLEC1 was induced to up-regulate expression within 0.5～1 h of stress treatment. The expression of TaLEC1 showed a stable up-regulation trend during the whole 42 ℃ stress process and the expression level was sharply up-regulated under 12 h and 48 h of stress treatments, to 52.8 and 34.5 times of that of the control, respectively. TaLEC1 was rapidly up-regulated within 0.5 h of high salt stress treatments. These research have shown that TaLEC1 is involved in ABA-dependent stress response in wheat, suggesting that it may play an important dehydration protection function in wheat tolerance to high temperature and osmotic stress.
In order to explore the function of LEC1 gene to abiotic stress response in wheat, we cloned a TaLEC1 gene from wheat using RT-PCR combined with RACE technology. The expression patterns of TaLEC1 from different tissues of wheat and under different stress treatments were analyzed by real-time fluorescence quantitative PCR(qRT-PCR). These would be provided a foundation for the drought, high temperature and high salt response mechanism study of LEC1 gene in wheat. The results showed that:(1) the full-length cDNA sequence of TaLEC1 was successfully cloned. The length of cDNA sequence of TaLEC1 is 1 074 bp, contains a 741 bp open reading frame(ORF), with 23 bp in the 5′ UTR and 310 bp in the 3′ UTR. TaLEC1 was predicted to encode a 246 amino acid protein with typical CBFD_NFYB domain.(2) Real time quantitative PCR(qRT-PCR) analysis showed that there were significant differences in the expression levels of TaLEC1 among different tissues and the highest expression was found in 10 d leaves.(3) TaLEC1 could be up-regulated by plant hormone ABA and belongs to ABA-dependent expression regulation pathway.(4) During PEG simulated drought stress, TaLEC1 was induced to up-regulate expression within 0.5～1 h of stress treatment. The expression of TaLEC1 showed a stable up-regulation trend during the whole 42 ℃ stress process and the expression level was sharply up-regulated under 12 h and 48 h of stress treatments, to 52.8 and 34.5 times of that of the control, respectively. TaLEC1 was rapidly up-regulated within 0.5 h of high salt stress treatments. These research have shown that TaLEC1 is involved in ABA-dependent stress response in wheat, suggesting that it may play an important dehydration protection function in wheat tolerance to high temperature and osmotic stress.
引文
[1] YADAV D,AHMED I,SHUKLA P,et al.Overexpression of Arabidopsis AnnAt8 alleviates abiotic stress in transgenic Arabidopsis and tobacco[J].Plants,2016,5(2):18-45.
[2] AHUJA I,DE VOS R C,BONES A M,et al.Plant molecular stress responses face climate change[J].Trends in Plant Science,2010,15(12):664-674.
[3] 肖瑞霞,王新国,夏国军,等.小麦逆境胁迫相关基因TaC2DP1的克隆及表达分析[J].中国农业科学,2015,48(8):1 463-1 472.XIAO R X,WANG X G,XIA G J,et al.Cloning and expression analysis of A stress-related TaC2DP1 gene from wheat[J].Scientia Agricultura Sinica,2015,48(8):1 463-1 472.
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[9] KWONG R W,BUI A Q,LEE H,et al.LEAFY COTYLEDON1-LIKE defines a class of regulators essential for embryo development[J].The Plant Cell,2003,15(1):5-18.
[10] XIE Z Y,LI X,GLOVER B J,et al.Duplication and functional diversification of HAP3 genes leading to the origin of the seed-developmental regulatory gene,LEAFY COTYLEDON1 (LEC1),in nonseed plant genomes[J].Molecular Biology and Evolution,2008,25(8):1 581-1 592.
[11] YAMAMOTO A,KAGAYA Y,TOYOSHIMA R,et al.Arabidopsis NF-YB subunits LEC1 and LEC1-LIKE activate transcription by interacting with seed-specific ABRE-binding factors[J].The Plant Journal:for Cell and Molecular Biology,2009,58(5):843-856.
[12] RIDENOUR J B,SMITH J E,BLUHM B H.The HAP complex governs fumonisin biosynthesis and maize kernel pathogenesis in fusarium verticillioides[J].Journal of Food Protection,2016,79(9):1 498-1 507.
[13] NELSON D E,REPETTI P P,ADAMS T R,et al.Plant nuclear factor Y (NF-Y) B subunits confer drought tolerance and lead to improved corn yields on water-limited acres[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104(42):16 450-16 455.
[14] SINHA S,MAITY S N,LU J,et al.Recombinant rat CBF-C,the third subunit of CBF/NFY,allows formation of a protein-DNA complex with CBF-A and CBF-B and with yeast HAP2 and HAP3[J].Proceedings of the National Academy of Sciences of the United States of America,1995,92(5):1 624-1 628.
[15] LOTAN T,OHTO M,YEE K M,et al.Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells[J].Cell,1998,93(7):1 195-1 205.
[16] ZHANG S B,WONG L,MENG L,et al.Similarity of expression patterns of knotted1 and ZmLEC1 during somatic and zygotic embryogenesis in maize (Zea mays L.)[J].Planta,2002,215(2):191-194.
[17] YAZAWA K,TAKAHATA K,KAMADA H.Isolation of the gene encoding Carrot leafy cotyledon1 and expression analysis during somatic and zygotic embryogenesis[J].Plant Physiology and Biochemistry,2004,42(3):215-223.
[18] FAMBRINI M,DURANTE C,CIONINI G,et al.Characterization of LEAFY COTYLEDON1-LIKE gene in Helianthus annuus and its relationship with zygotic and somatic embryogenesis[J].Development Genes and Evolution,2006,216(5):253-264.
[19] TAN H L,YANG X H,ZHANG F X,et al.Enhanced seed oil production in canola by conditional expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in developing seeds[J].Plant Physiology,2011,156(3):1 577-1 588.
[20] ELAHI N,DUNCAN R W,STASOLLA C.Modification of oil and glucosinolate content in canola seeds with altered expression of Brassica napus LEAFY COTYLEDON1[J].Plant Physiology and Biochemistry,2016,100:52-63.
[21] HUANG M K,HU Y L,LIU X,et al.Arabidopsis LEAFY COTYLEDON1 controls cell fate determination during post-embryonic development[J].Frontiers in Plant Science,2015,6:955-962.
[22] VERWOERD T C,DEKKER B M,HOEKEMA A.A small-scale procedure for the rapid isolation of plant RNAs[J].Nucleic Acids Research,1989,17(6):321-333.
[23] MORRAN S,EINI O,PYVOVARENKO T,et al.Improvement of stress tolerance of wheat and barley by modulation of expression of DREB/CBF factors[J].Plant Biotechnology Journal,2011,9(2):230-249.
[24] SOLTéSZ A,SMEDLEY M,VASHEGYI I,et al.Transgenic barley lines prove the involvement of TaCBF14 and TaCBF15 in the cold acclimation process and in frost tolerance[J].Journal of Experimental Botany,2013,64(7):1 849-1 862.
[25] 王鼎慧,李永光,李真,等.转GmHAP3-17基因大豆的抗旱性分析[J].大豆科学,2015,34(2):249-254.WANG D H,LI Y G,LI Z,et al.Analysis of drought tolerance in transgenic soybean with GmHAP3-17[J].Soybean Science,2015,34(2):249-254.
[2] AHUJA I,DE VOS R C,BONES A M,et al.Plant molecular stress responses face climate change[J].Trends in Plant Science,2010,15(12):664-674.
[3] 肖瑞霞,王新国,夏国军,等.小麦逆境胁迫相关基因TaC2DP1的克隆及表达分析[J].中国农业科学,2015,48(8):1 463-1 472.XIAO R X,WANG X G,XIA G J,et al.Cloning and expression analysis of A stress-related TaC2DP1 gene from wheat[J].Scientia Agricultura Sinica,2015,48(8):1 463-1 472.
[4] HIRAYAMA T,SHINOZAKI K.Research on plant abiotic stress responses in the post-genome era:past,present and future[J].The Plant Journal:for Cell and Molecular Biology,2010,61(6):1 041-1 052.
[5] SHANMUGAM A,THAMILARASAN S K,PARK J I,et al.Characterization and abiotic stress-responsive expression analysis of SGT1 genes in Brassica oleracea[J].Genome,2016,59(4):243-251.
[6] ZHANG S X,HAIDER I,KOHLEN W,et al.Function of the HD-Zip I gene Oshox22 in ABA-mediated drought and salt tolerances in rice[J].Plant Molecular Biology,2012,80(6):571-585.
[7] ANDLEEB S,AMIN I,BASHIR A,et al.Transient expression of βC1 protein differentially regulates host genes related to stress response,chloroplast and mitochondrial functions[J].Virology Journal,2010,7(1):1-12.
[8] NAKASHIMA K,YAMAGUCHI-SHINOZAKI K.Promoters and Transcription Factors in Abiotic Stress-Responsive Gene Expression[M].Abiotic Stress Adaptation in Plants.2009:199-216.
[9] KWONG R W,BUI A Q,LEE H,et al.LEAFY COTYLEDON1-LIKE defines a class of regulators essential for embryo development[J].The Plant Cell,2003,15(1):5-18.
[10] XIE Z Y,LI X,GLOVER B J,et al.Duplication and functional diversification of HAP3 genes leading to the origin of the seed-developmental regulatory gene,LEAFY COTYLEDON1 (LEC1),in nonseed plant genomes[J].Molecular Biology and Evolution,2008,25(8):1 581-1 592.
[11] YAMAMOTO A,KAGAYA Y,TOYOSHIMA R,et al.Arabidopsis NF-YB subunits LEC1 and LEC1-LIKE activate transcription by interacting with seed-specific ABRE-binding factors[J].The Plant Journal:for Cell and Molecular Biology,2009,58(5):843-856.
[12] RIDENOUR J B,SMITH J E,BLUHM B H.The HAP complex governs fumonisin biosynthesis and maize kernel pathogenesis in fusarium verticillioides[J].Journal of Food Protection,2016,79(9):1 498-1 507.
[13] NELSON D E,REPETTI P P,ADAMS T R,et al.Plant nuclear factor Y (NF-Y) B subunits confer drought tolerance and lead to improved corn yields on water-limited acres[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104(42):16 450-16 455.
[14] SINHA S,MAITY S N,LU J,et al.Recombinant rat CBF-C,the third subunit of CBF/NFY,allows formation of a protein-DNA complex with CBF-A and CBF-B and with yeast HAP2 and HAP3[J].Proceedings of the National Academy of Sciences of the United States of America,1995,92(5):1 624-1 628.
[15] LOTAN T,OHTO M,YEE K M,et al.Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells[J].Cell,1998,93(7):1 195-1 205.
[16] ZHANG S B,WONG L,MENG L,et al.Similarity of expression patterns of knotted1 and ZmLEC1 during somatic and zygotic embryogenesis in maize (Zea mays L.)[J].Planta,2002,215(2):191-194.
[17] YAZAWA K,TAKAHATA K,KAMADA H.Isolation of the gene encoding Carrot leafy cotyledon1 and expression analysis during somatic and zygotic embryogenesis[J].Plant Physiology and Biochemistry,2004,42(3):215-223.
[18] FAMBRINI M,DURANTE C,CIONINI G,et al.Characterization of LEAFY COTYLEDON1-LIKE gene in Helianthus annuus and its relationship with zygotic and somatic embryogenesis[J].Development Genes and Evolution,2006,216(5):253-264.
[19] TAN H L,YANG X H,ZHANG F X,et al.Enhanced seed oil production in canola by conditional expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in developing seeds[J].Plant Physiology,2011,156(3):1 577-1 588.
[20] ELAHI N,DUNCAN R W,STASOLLA C.Modification of oil and glucosinolate content in canola seeds with altered expression of Brassica napus LEAFY COTYLEDON1[J].Plant Physiology and Biochemistry,2016,100:52-63.
[21] HUANG M K,HU Y L,LIU X,et al.Arabidopsis LEAFY COTYLEDON1 controls cell fate determination during post-embryonic development[J].Frontiers in Plant Science,2015,6:955-962.
[22] VERWOERD T C,DEKKER B M,HOEKEMA A.A small-scale procedure for the rapid isolation of plant RNAs[J].Nucleic Acids Research,1989,17(6):321-333.
[23] MORRAN S,EINI O,PYVOVARENKO T,et al.Improvement of stress tolerance of wheat and barley by modulation of expression of DREB/CBF factors[J].Plant Biotechnology Journal,2011,9(2):230-249.
[24] SOLTéSZ A,SMEDLEY M,VASHEGYI I,et al.Transgenic barley lines prove the involvement of TaCBF14 and TaCBF15 in the cold acclimation process and in frost tolerance[J].Journal of Experimental Botany,2013,64(7):1 849-1 862.
[25] 王鼎慧,李永光,李真,等.转GmHAP3-17基因大豆的抗旱性分析[J].大豆科学,2015,34(2):249-254.WANG D H,LI Y G,LI Z,et al.Analysis of drought tolerance in transgenic soybean with GmHAP3-17[J].Soybean Science,2015,34(2):249-254.
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