小麦金属反应元件结合蛋白cDNA的筛选、克隆与分析
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摘要
已有实验证明金属反应元件（metal response element，MRE）在缺铁胁迫诱
导的基因表达中起到非常重要的作用。本文从Ids1和Ids2基因5’上游的MRE
出发，人工合成长度为74bp的MRE异源三联体。利用~(32)P末端标记的MRE异源三
联体直接筛选小麦缺铁诱导根λgt11-cDNA表达文库，克隆得到了编码其结合蛋
白的长度为279bp的cDNA片段，命名为TaMRE-BP cDNA。该cDNA片段和烟草AVR9
激发子应答蛋白（AVR9 elicitor response protein）cDNA的相应序列有78％
的同源性，TaMRE-BP和烟草AVR9激发子应答蛋白在氨基酸水平上有75％的同源
性。经Northern印迹分析，TaMRE-BP cDNA片段的杂交信号在大约1.5kb处，
其相应基因的转录活性在缺铁处理第9天的根和叶中明显增强，在缺铁处理第
13天的根和叶中稍有减弱，和正常处理时的转录活性相当。Southern印迹分析
结果表明TaMRE-BP cDNA片段的相应基因存在于小麦基因组中。
据推测TaMRE-BP可能作为一种MRE结合蛋白参与应答植物体内Fe/Cu水平
的变化。缺铁胁迫改变植物体内的Fe/Cu水平（植物体内Fe含量下降，Cu含量
上升），进一步诱导该TaMRE-BP cDNA片段相应基因的转录活性，以调节植物体
内的金属营养元素的平衡。该基因的转录活性在缺铁处理第9天的根和叶中明显
增强，而这时麦根酸的分泌量最大，因此该基因的表达和麦根酸的合成可能有某
种联系。TaMRE-BP cDNA片段和烟草AVR9激发子应答蛋白（AVR9 elicitor
response protein）cDNA有78％的同源性，其原因可能是类似AVR9作用的诱
发子改变小麦体内Fe/Cu水平，从而诱导该基因的转录。
Previous experiments have established that a cis-acting element named MIRE
(Metal Response Element, MRE ) plays an important role in Fe-deficiency-induced
gene expression. In this study, three copy of MIRE of 74bp was synthesized and 5?end
32
was labeled by P. A eDNA fragment of 279bp named TaMRE-BP eDNA was cloned
by direct screening Fe-deficient wheat root Xgtl 1-eDNA expression library with the
labeled MIRE. The eDNA exhibits 78% similarity to the eDNA of AVR9 elicitor
response protein and the deduced amino acid sequence exhibits 75% similarity to that
of AVR9 elicitor response protein. Northern blotting reveals that the whole length of
its eDNA is about 1.5kb. On the 9th day of Fe-deficiency treatment, compared with
the normal treatment control, its transcription activity in roots and leaf is apparently
elevated. While 13th day the transcription activety is decreased. Southern blotting
reveals that this gene is wheat genomic gene.
It is assumed that TaMRE-BP response to the change of Fe/Cu level as a
MIRE-binding protein in wheat. Fe deficiency treatment changes Fe/Cu level in plant
( Cu level elevated and Fe level decreased ) . This change tbrther induces the
transcription activity of this gene to regulate the balance of metal level. As its
transcription activity is apparently elevated on the 9th day of Fe-deficiency treatment,
the synthesis of mugineic acid is significantly increased this time. Therefore it抯
assumed that TaMRE-BP has something with the synthesis of mugineie acid. As to its
high similiarity to the cDNA of AVR9 elicitor response like protein. It抯 predicted that
AVR9 like elicitor may indirectly changes Fe/Cu level in wheat, and induces the
transcription activity of this gene.
导的基因表达中起到非常重要的作用。本文从Ids1和Ids2基因5’上游的MRE
出发，人工合成长度为74bp的MRE异源三联体。利用~(32)P末端标记的MRE异源三
联体直接筛选小麦缺铁诱导根λgt11-cDNA表达文库，克隆得到了编码其结合蛋
白的长度为279bp的cDNA片段，命名为TaMRE-BP cDNA。该cDNA片段和烟草AVR9
激发子应答蛋白（AVR9 elicitor response protein）cDNA的相应序列有78％
的同源性，TaMRE-BP和烟草AVR9激发子应答蛋白在氨基酸水平上有75％的同源
性。经Northern印迹分析，TaMRE-BP cDNA片段的杂交信号在大约1.5kb处，
其相应基因的转录活性在缺铁处理第9天的根和叶中明显增强，在缺铁处理第
13天的根和叶中稍有减弱，和正常处理时的转录活性相当。Southern印迹分析
结果表明TaMRE-BP cDNA片段的相应基因存在于小麦基因组中。
据推测TaMRE-BP可能作为一种MRE结合蛋白参与应答植物体内Fe/Cu水平
的变化。缺铁胁迫改变植物体内的Fe/Cu水平（植物体内Fe含量下降，Cu含量
上升），进一步诱导该TaMRE-BP cDNA片段相应基因的转录活性，以调节植物体
内的金属营养元素的平衡。该基因的转录活性在缺铁处理第9天的根和叶中明显
增强，而这时麦根酸的分泌量最大，因此该基因的表达和麦根酸的合成可能有某
种联系。TaMRE-BP cDNA片段和烟草AVR9激发子应答蛋白（AVR9 elicitor
response protein）cDNA有78％的同源性，其原因可能是类似AVR9作用的诱
发子改变小麦体内Fe/Cu水平，从而诱导该基因的转录。
Previous experiments have established that a cis-acting element named MIRE
(Metal Response Element, MRE ) plays an important role in Fe-deficiency-induced
gene expression. In this study, three copy of MIRE of 74bp was synthesized and 5?end
32
was labeled by P. A eDNA fragment of 279bp named TaMRE-BP eDNA was cloned
by direct screening Fe-deficient wheat root Xgtl 1-eDNA expression library with the
labeled MIRE. The eDNA exhibits 78% similarity to the eDNA of AVR9 elicitor
response protein and the deduced amino acid sequence exhibits 75% similarity to that
of AVR9 elicitor response protein. Northern blotting reveals that the whole length of
its eDNA is about 1.5kb. On the 9th day of Fe-deficiency treatment, compared with
the normal treatment control, its transcription activity in roots and leaf is apparently
elevated. While 13th day the transcription activety is decreased. Southern blotting
reveals that this gene is wheat genomic gene.
It is assumed that TaMRE-BP response to the change of Fe/Cu level as a
MIRE-binding protein in wheat. Fe deficiency treatment changes Fe/Cu level in plant
( Cu level elevated and Fe level decreased ) . This change tbrther induces the
transcription activity of this gene to regulate the balance of metal level. As its
transcription activity is apparently elevated on the 9th day of Fe-deficiency treatment,
the synthesis of mugineic acid is significantly increased this time. Therefore it抯
assumed that TaMRE-BP has something with the synthesis of mugineie acid. As to its
high similiarity to the cDNA of AVR9 elicitor response like protein. It抯 predicted that
AVR9 like elicitor may indirectly changes Fe/Cu level in wheat, and induces the
transcription activity of this gene.
引文
1．王琳芳、杨克恭主编，《蛋白质与核酸》，北京医科大学中国协和医科大学联合出版社，343-344
2. ATCHMAKER One-Hybrid System Protocol(PT1031-1)
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4. Ave H, Angeles L, Isolation of DNA-binding proteins by direct screening of cDNA libraries with labeled binding sites. Plant Mol Bio Manual, 1993, B17:1-20
5. Oeda K, Salinas J, Chua N-H, A tobacco bZIP transcription activator (TAF-1) binds to a G-box like motif conserved in plant genes. EMBO J. 1991, 10:1793-1802
6. Lam E, Yuriko K-M, Gilmartin P, Niner B, Bhua N-H, A metal-dependent DNA-binding protein interacts with a constitutive element of a light responsive promoter. Plant Cell, 1990, 2:857-866
7. Perisic O, and Lam E, A tobacco DNA binding protein that interacts with a light responsive box Ⅱ element. Plant Cell, 1992,4:831-838
8. Gilmartin PM, Memelink J, Hiratsuka K, Kay SA, Chua N-H, Characterization of a gene encoding a DNA binding protein with specificity for a light-responsive element.Plant Cell, 1992, 4:839-849
9. Luehrsen KR, De Wet JR, Wslbot V, Transient expression analysis in plans using firegly luciferase reporter gene. Methods Enzymol., 1992, 216:397-414
10. Dietrich A, Mayer JE, Hahlbroch K, Fungal elicitor triggers rapid, transient, and specific protein phosphorylation in parsley cell suspension cultures, J. Biol. Chem.1990, 265:6360-68
11. Kao C-Y, Cocciolone SM, Vasil IK, Mccarty DR, Localization and interaction of the cis-acting elements for abscisic acid, VIVIPAROUS1, and light activation of the CI gene of maize. Plant Cell, 1995, 8:1171-79
12． Xiang CB, Miao Z-H, Lam E, Coordinated activation of a tobacco gutahione S-transferase gene by auxins, salicylic acid, methyljasmonate and hydrogen peroxide., Plant Mol. Biol. 1996,32:415-26
13． Moyano E, Marfhez-Garcia JF, Martin C, Apparent redundancy in myb gene function provides gearing for the control of flavonoid biosynthesis in Antirrhinum flowers. Plant Cell, 1996, 8:1519-32
14． Ponticelli AS, Pardee TS, Strubl K, The glutamine-rich activation domains of human Spl do not stimulate transcription in Saccharomyces cerevisiae. Mol. Cell. Biol. 1995, 15:983-88
15． Fan H, Sugiura M, A plant basal in vitro system supporting accurate transcription of both RNA polymerase Ⅱ-and Ⅲ-dependent genes: supplement of green leaf component(s) drives accurate transcription of a light-responsive rbcS gervs. EMBOJ., 1995, 14:1024-31
16． Fan H, Yakura K, Miyanishi M, Sugita M, Sugiura M., In vitro transcription of plant RNA polymerase I-dependent rRNA genes is species-specific. Plant J. 1995, 8:295-98
17． Samach A, Kohalmi SE, Motte P, Dadar, Haughn GW. Divergence of function and regulation of class B floral oran identity genes. Plant Cell, 1997, 9:559-70
18． Ueberlacker B, Klinge B, Weir W, Ectopic expression of the maize homeobox genes ZmHoxla or ZmHoxlb causes pleiotropic alterations in the vegetative and floral development of transgenic tobacco. Plant Cell, 1996, 8:349-62
19． Cannon M, Platz J, O'Leary M, Sookdeo C, Connon F. Organ-specific modulation of gene expression in transgenic plants using antisense RNA. Plant Mol. Biol., 1990, 15:39-47
20． Pnueli L, Hareven D, Broday L, Hurwitz C, Lifschitz E, The TM5 MADS box gene mediates organ differentiation in the three inner whorls of tomato flowers. Plant Cell, 1994,6:175-86
21． Koes R, Souer E, Van Houwelingen A, MurL, Spelt C, et al., Targeted gene inactive in petunia by PCR-based selection of transposon insertion mutants. Proc. Natl. Acad. Sci. USA, 1995, 92:8149-53
22． Kranz H, Denekamp M, Greco R, Jin H-L, Leyva A, et al., Analysis of MYB transcription factors in Arabidopsis thaliana. Int. Conf. Arabidopsis Res. 1997, 8th, Madison, Wis. (Abstr)
23． Gatz C, Chemical control of gene expression. Annu. Rev. Plant Physiol. Plant Mol Biol., 1997,48:89-108
24． Aoyama T, Chun N-H, A glucocorticiod-mediated transcriptional induction system in transgenic plants. Plant J., 1997, 11:605-12
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2. ATCHMAKER One-Hybrid System Protocol(PT1031-1)
3. Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, and Shinozaki K, Two transcription factors, DREB1 and DREB2, with an EREB/AP2 DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression, respectively, in Arabidopsis, Plant Cell, 1998, 10:1391-1406
4. Ave H, Angeles L, Isolation of DNA-binding proteins by direct screening of cDNA libraries with labeled binding sites. Plant Mol Bio Manual, 1993, B17:1-20
5. Oeda K, Salinas J, Chua N-H, A tobacco bZIP transcription activator (TAF-1) binds to a G-box like motif conserved in plant genes. EMBO J. 1991, 10:1793-1802
6. Lam E, Yuriko K-M, Gilmartin P, Niner B, Bhua N-H, A metal-dependent DNA-binding protein interacts with a constitutive element of a light responsive promoter. Plant Cell, 1990, 2:857-866
7. Perisic O, and Lam E, A tobacco DNA binding protein that interacts with a light responsive box Ⅱ element. Plant Cell, 1992,4:831-838
8. Gilmartin PM, Memelink J, Hiratsuka K, Kay SA, Chua N-H, Characterization of a gene encoding a DNA binding protein with specificity for a light-responsive element.Plant Cell, 1992, 4:839-849
9. Luehrsen KR, De Wet JR, Wslbot V, Transient expression analysis in plans using firegly luciferase reporter gene. Methods Enzymol., 1992, 216:397-414
10. Dietrich A, Mayer JE, Hahlbroch K, Fungal elicitor triggers rapid, transient, and specific protein phosphorylation in parsley cell suspension cultures, J. Biol. Chem.1990, 265:6360-68
11. Kao C-Y, Cocciolone SM, Vasil IK, Mccarty DR, Localization and interaction of the cis-acting elements for abscisic acid, VIVIPAROUS1, and light activation of the CI gene of maize. Plant Cell, 1995, 8:1171-79
12． Xiang CB, Miao Z-H, Lam E, Coordinated activation of a tobacco gutahione S-transferase gene by auxins, salicylic acid, methyljasmonate and hydrogen peroxide., Plant Mol. Biol. 1996,32:415-26
13． Moyano E, Marfhez-Garcia JF, Martin C, Apparent redundancy in myb gene function provides gearing for the control of flavonoid biosynthesis in Antirrhinum flowers. Plant Cell, 1996, 8:1519-32
14． Ponticelli AS, Pardee TS, Strubl K, The glutamine-rich activation domains of human Spl do not stimulate transcription in Saccharomyces cerevisiae. Mol. Cell. Biol. 1995, 15:983-88
15． Fan H, Sugiura M, A plant basal in vitro system supporting accurate transcription of both RNA polymerase Ⅱ-and Ⅲ-dependent genes: supplement of green leaf component(s) drives accurate transcription of a light-responsive rbcS gervs. EMBOJ., 1995, 14:1024-31
16． Fan H, Yakura K, Miyanishi M, Sugita M, Sugiura M., In vitro transcription of plant RNA polymerase I-dependent rRNA genes is species-specific. Plant J. 1995, 8:295-98
17． Samach A, Kohalmi SE, Motte P, Dadar, Haughn GW. Divergence of function and regulation of class B floral oran identity genes. Plant Cell, 1997, 9:559-70
18． Ueberlacker B, Klinge B, Weir W, Ectopic expression of the maize homeobox genes ZmHoxla or ZmHoxlb causes pleiotropic alterations in the vegetative and floral development of transgenic tobacco. Plant Cell, 1996, 8:349-62
19． Cannon M, Platz J, O'Leary M, Sookdeo C, Connon F. Organ-specific modulation of gene expression in transgenic plants using antisense RNA. Plant Mol. Biol., 1990, 15:39-47
20． Pnueli L, Hareven D, Broday L, Hurwitz C, Lifschitz E, The TM5 MADS box gene mediates organ differentiation in the three inner whorls of tomato flowers. Plant Cell, 1994,6:175-86
21． Koes R, Souer E, Van Houwelingen A, MurL, Spelt C, et al., Targeted gene inactive in petunia by PCR-based selection of transposon insertion mutants. Proc. Natl. Acad. Sci. USA, 1995, 92:8149-53
22． Kranz H, Denekamp M, Greco R, Jin H-L, Leyva A, et al., Analysis of MYB transcription factors in Arabidopsis thaliana. Int. Conf. Arabidopsis Res. 1997, 8th, Madison, Wis. (Abstr)
23． Gatz C, Chemical control of gene expression. Annu. Rev. Plant Physiol. Plant Mol Biol., 1997,48:89-108
24． Aoyama T, Chun N-H, A glucocorticiod-mediated transcriptional induction system in transgenic plants. Plant J., 1997, 11:605-12
25． Schena M, Lloyd AM, Davis RW, a steroid-inducible gene expression system for plant cells. Proc. Natl. Acad Sci. USA, 1991, 88:10421-25
26． Chen X, Meyerowowitz E, Identification of a gene nagtively regulated by the floral homeotic gene AGAMOUS. Int. conf. Arabidopsis Res., 1997, 8th, Madison, Wis.(Abstr)
27． Sablowski R, Meyerowitz E, Beyond floral homeotic genes: finding their target genes., Int. conf. Arabidopsis Res. 1997, 8th, Madison, Wis.(Abstr)
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