蓝藻TIM上的一个特异抑制靶位点及其应用 技术领域：
本发明涉及蓝藻TIM(磷酸丙糖异构酶Triosephosphate isomerase)上的一个特异抑制靶位点及其应用，属生物技术领域。
背景技术：
蓝藻(Cyanobacterium)是一类全球分布的光合自养真细菌，因其能形成引起生态灾害的蓝藻“水华”(water bloom)而倍受关注。特别是近年来由于工业和生活废水的污染，使许多淡水湖泊，如中国的滇池、太湖、东湖等一直处于严重的富营养化状态，随之而来的生态恶果是几乎每年都会由于蓝藻爆发而形成严重的蓝藻“水华”，给居民生活及社会经济发展都带来极大影响，国内外都存在类似的问题。尽管人们对这一问题的解决进行了多方面的努力，但迄今还没有很好的治理蓝藻水华的方法。所以，寻找能有效治理蓝藻水华的新途径是十分必要的。
发明内容：
本发明通过分子生物学技术，克隆了三种主要的引起蓝藻“水华”的微囊藻(Microcystis aeruginosa、Microcystis viridis和Microcystis wesenbergii)tim基因地极大部分序列并测序。测得的三条tim基因的部分序列都为663bp(全长约720bp)，并推导出它们编码的TIM氨基酸序列(编码221个氨基酸)。结合已报道的三种蓝藻(Synechocystissp.、Anabaena sp.和Thermosynechococcus elongatus.http：//www.kazusa.or.jp/cyanobase/)的TIM序列和在NCBI Entrez Protein Sequence Database上登录的其它非蓝藻生物的TIM进行复合比对发现：蓝藻的TIM上有一个特异的半胱氨酸位点-Cys176(以Synechocystissp.TIM氨基酸编号为准)。而在非蓝藻生物的TIM中，除蓝色链霉菌(Streptomycescoelicolor)在该位点也是半胱氨酸外，其它都不是半胱氨酸。参考已测定的TIM晶体结构和以Bacillus stearothermophilus TIM的三级结构(PDB entry：1BTM)为模板、蓝藻Synechocystis sp.TIM序列为靶序列进行同源模建都证明：在蓝藻TIM三级结构上，Cys176位于Loop-6的铰链区。Loop-6对维持TIM的功能至关重要：当底物结合时，Loop-6发生相应的较大位移(7.3)并与Loop-7形成新的氢键，确保反应的顺利进行，而Loop-6的运动又是通过Loop-6铰链区的氨基酸二面角的改变实现的。所以，蓝藻TIM的Cys176是一个理想的蓝藻特异抑制剂设计靶位点，可作为设计高效低毒水华抑制剂的应用，更有利于环境保护。
附图说明：
附图为本发明Cys176靶位点在同源模建的蓝藻Synechocystis sp.TIM三级结构上的位置。
图中序号1是TIM的Loop-6、序号2是半胱氨酸-Cys176靶位点在铰链区的位置。
具体实施方式：
1.发明人用PCR方法从三种“水华”微囊藻-Microcystis aeruginosa、Microcystisviridis和Microcystis wesenbergii-的基因组DNA中都扩增得到663bp的tim基因序列并克隆测序，登记于GenBank中(接收号：AY238889、AY238890、AY238891)，进而推导出它们编码的221个氨基酸序列。
2.从日本Kazusa DNA Research Institute建立的蓝藻基因组数据库(网址：http：//www.kazusa.or.jp/cyanobase/)中搜索到三种蓝藻-Synechocystis sp.、Anabaena sp.和Thermosynechococcus elongates-的TIM序列。结果显示，在这三种蓝藻的基因组中各只有单拷贝的tim基因，提示蓝藻糖酵解途径和光合作用途径中3’-磷酸甘油醛和磷酸二羟丙酮之间的转化反应都是由同一TIM催化的。从NCBI Entrez ProteinSequence Database中搜索出已报道的非蓝藻生物TIM的序列共138条(包括了古细菌、真细菌、真核生物的主要类群)。
3.生物信息学分析
将所有的TIM序列进行复合比对，发现在蓝藻TIM中有一个特异的半胱氨酸位点-Cys176。在所有已报道的TIM中，仅蓝藻和蓝色链霉菌的TIM在该位点是半胱氨酸，其它物种的TIM都不是半胱氨酸。
参考PDB数据库(网址：http：//www.rcsb.org/pdb/)登录的已测定的TIM晶体结构发现：蓝藻TIM Cys176位点在三级结构上位于TIM Loop-6的铰链区。以Bacillusstearothermophilus TIM的三级结构(PDB entry：1BTM)为模板、蓝藻Synechocystis sp.TIM序列为靶序列进行同源模建进一步证实了这点。
4.Cys176位点对维持蓝藻TIM功能重要性分析
TIM作为研究酶结构与功能之间关系的模式酶，它的酶动力学、三级结构、酶促反应机制已被详细地研究过。从前人的工作可知：Loop-6对维持TIM的功能至关重要：当底物结合时，Loop-6发生相应的较大位移(7.3)并与Loop-7形成新的氢键，确保反应的顺利进行，而Loop-6的运动又是通过Loop-6铰链区的氨基酸二面角的改变实现的。半胱氨酸的巯基(-SH)反应性能很高，能被多种基团修饰，如巯基试剂等。所以，如用专一修饰蓝藻TIM Cys176的化学试剂修饰该位点，阻碍TIM Loop-6的运动，即可专一地抑制蓝藻TIM的活性，阻断蓝藻糖酵解途径和光合作用途径两条关键代谢途径，从而能十分有效地抑制蓝藻的生长。另外，只有蓝色链霉菌也具有此位点，但该细菌是一种生存于泥土中的放线菌，和引起水华的蓝藻有着不同的生境，因而不影响蓝藻抑制剂的使用。因此，蓝藻TIM的Cys176位点可作为设计高效低毒水华抑制剂的应用。
SEQUENCE LISTING
<110>中国科学院昆明动物研究所
<120>Microcystis aeruginosa的磷酸丙糖异构酶(TIM)基因的部分
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His Leu Asp Glu Thr Pro Asp Asp Arg Glu Val Val Leu Cys Ala
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Phe Thr Ala Leu Ala Val Leu Ser Lys Thr Leu His Gly Gly Arg
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35 40 45
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Glu Lys Val Ile Ile Lys Gln Ile Gln Gly Gly Leu Val Asn Val
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Gln Lys Asn Leu Val Ile Ala Tyr Glu Pro Ile Trp Ala Ile Gly
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160
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Arg Leu Gly Ala Gln Asn Val His Trp Glu Lys Ser Gly Ala Tyr
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Gly Glu Ile Ser Ala Asp Met Leu Thr Glu Ile Gly Val His Tyr
Val
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80
Val Ile Gly His Ser Glu Arg Arg Gln Tyr Phe Gly Glu Thr Asp
Glu
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Thr Val Asn Leu Arg Val Ile Ser Ala Gln Lys Gln Gly Leu Ile
Pro
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Ile Ile Cys Val Gly Glu Ser Lys Ala Gln Arg Asp Ala Gly Glu
Thr
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Glu Lys Val Ile Ile Lys Gln Ile Gln Gly Gly Leu Val Asn Val
Asp
130 135 140
Gln Lys Asn Leu Val Ile Ala Tyr Glu Pro Ile Trp Ala Ile Gly
Thr
145 150 155
160
Gly Glu Thr Cys Glu Ser Glu Glu Ala Asn Arg Val Ile Gly Leu
Ile
165 170 175
Arg Gln Gln Leu Asp Asn Pro Glu Val Thr Ile Gln Tyr Gly Gly
Ser
180 185 190
Val Lys Pro Asp Asn Ile Asp Glu Ile Met Ala Gln Ser Gln Ile
Asn
195 200 205
Gly Ala Leu Val Gly Gly Ala Ser Leu Asp Pro Ala Val
210 215 220
注：由于本序列为Microcystis aeruginosa tim基因的绝大部分序列(即未测全)。故在此序列中编号为Cys164的半胱氨酸即为蓝藻Synechocystis sp.TIM的Cys176。
SEQUENCE LISTING
<110>中国科学院昆明动物研究所
<120>Microcystis viridis的磷酸丙糖异构酶(TIM)基因
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<140>1
<141>2003-02-28
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<211>663
<212>DNA
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tcc 48
His Lys Thr Gln Arg Glu Ala Leu Glu Phe Leu Gln Asp Phe Lys
Ser
1 5 10 15
cat cta gat gaa acc ccc gac gac cga gaa gtg gtt ctt tgc gcg
ccc 96
His Leu Asp Glu Thr Pro Asp Asp Arg Glu Val Val Leu Cys Ala
Pro
20 25 30
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Phe Thr Ala Leu Ala Val Leu Ser Lys Thr Leu His Gly Gly Arg
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35 40 45
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Arg Leu Gly Ala Gln Asn Val His Trp Glu Lys Ser Gly Ala Tyr
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Gly Glu Ile Ser Ala Asp Met Leu Thr Glu Ile Gly Val His Tyr
Val
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Thr Val Asn Leu Arg Val Ile Ser Ala Gln Lys Gln Gly Leu Ile
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Ile Ile Cys Val Gly Glu Ser Lys Ala Gln Arg Asp Ala Gly Glu
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gac 432
Glu Lys Val Ile Ile Lys Gln Ile Gln Gly Gly Leu Val Asn Val
Asp
130 135 140
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Gln Lys Asn Leu Val Ile Ala Tyr Glu Pro Ile Trp Ala Ile Gly
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145 150 155
160
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Val Lys Pro Asp Asn Ile Asp Glu Ile Met Ala Gln Ser Gln Ile
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His Lys Thr Gln Arg Glu Ala Leu Glu Phe Leu Gln Asp Phe Lys
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His Leu Asp Glu Thr Pro Asp Asp Arg Glu Val Val Leu Cys Ala
Pro
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Phe Thr Ala Leu Ala Val Leu Ser Lys Thr Leu His Gly Gly Arg
Ile
35 40 45
Arg Leu Gly Ala Gln Asn Val His Trp Glu Lys Ser Gly Ala Tyr
Thr
50 55 60
Gly Glu Ile Ser Ala Asp Met Leu Thr Glu Ile Gly Val His Tyr
Val
65 70 75
80
Val Ile Gly His Ser Glu Arg Arg Gln Tyr Phe Gly Glu Thr Asp
Glu
85 90 95
Thr Val Asn Leu Arg Val Ile Ser Ala Gln Lys Gln Gly Leu Ile
Pro
100 105 110
Ile Ile Cys Val Gly Glu Ser Lys Ala Gln Arg Asp Ala Gly Glu
Thr
115 120 125
Glu Lys Val Ile Ile Lys Gln Ile Gln Gly Gly Leu Val Asn Val
Asp
130 135 140
Gln Lys Asn Leu Val Ile Ala Tyr Glu Pro Ile Trp Ala Ile Gly
Thr
145 150 155
160
Gly Glu Thr Cys Glu Ser Glu Glu Ala Asn Arg Val Ile Gly Leu
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165 170 175
Arg Gln Gln Leu Asp Asn Pro Glu Val Thr Ile Gln Tyr Gly Gly
Ser
180 185 190
Val Lys Pro Asp Asn Ile Asp Glu Ile Met Ala Gln Ser Gln Ile
Asn
195 200 205
Gly Ala Leu Val Gly Gly Ala Ser Leu Asp Pro Ala Val
210 215 220
注：由于本序列为Microcystis viridis tim基因的绝大部分序列(即未测全)。故在此序列中编号为Cys164的半胱氨酸即为蓝藻Synechocystissp.TIM的Cys176。
SEQUENCE LISTING
<110>中国科学院昆明动物研究所
<120>Microcystis wesenbergii的磷酸丙糖异构酶(TIM)基因
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<160>2
<170>PatentIn version 3.1
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<222>(1)..(663)
<223>
<300>
<308>GenBank/AY238891
<309>2003-02-20
<313>(1)..(663)
<400>1
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tcc 48
His Lys Thr Gln Arg Glu Ala Leu Glu Phe Leu Gln Asp Phe Lys
Ser
1 5 10 15
cat cta gag gaa acc ccc gac gac cga gaa gta gtt ctt tgc gcg
cct 96
His Leu Glu Glu Thr Pro Asp Asp Arg Glu Val Val Leu Cys Ala
Pro
20 25 30
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att 144
Phe Thr Ala Leu Ala Val Leu Ser Lys Thr Leu His Gly Gly Arg
Ile
35 40 45
cgc ttg ggg gcg caa aat gtc cac tgg gaa aaa tcg ggg gct tat
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Arg Leu Gly Ala Gln Asn Val His Trp Glu Lys Ser Gly Ala Tyr
Thr
50 55 60
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Gly Glu Ile Ser Ala Asp Met Leu Thr Glu Ile Gly Val His Tyr
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65 70 75
80
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Thr Val Asn Leu Arg Val Ile Ser Ala Gln Lys Gln Gly Leu Ile
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Ile Ile Cys Val Gly Glu Ser Lys Ala Gln Arg Asp Ala Gly Glu
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115 120 125
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gac 432
Glu Lys Val Ile Ile Lys Gln Ile Gln Gly Gly Leu Val Asn Val
Asp
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Gln Lys Asn Leu Val Ile Ala Tyr Glu Pro Ile Trp Ala Ile Gly
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160
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180 185 190
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His Leu Glu Glu Thr Pro Asp Asp Arg Glu Val Val Leu Cys Ala
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Ile
35 40 45
Arg Leu Gly Ala Gln Asn Val His Trp Glu Lys Ser Gly Ala Tyr
Thr
50 55 60
Gly Glu Ile Ser Ala Asp Met Leu Thr Glu Ile Gly Val His Tyr
Val
65 70 75
80
Val Ile Gly His Ser Glu Arg Arg Gln Tyr Phe Gly Glu Thr Asp
Glu
85 90 95
Thr Val Asn Leu Arg Val Ile Ser Ala Gln Lys Gln Gly Leu Ile
Pro
100 105 110
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Thr
115 120 125
Glu Lys Val Ile Ile Lys Gln Ile Gln Gly Gly Leu Val Asn Val
Asp
130 135 140
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Thr
145 150 155
160
Gly Glu Thr Cys Glu Ser Glu Glu Ala Asn Arg Val Ile Gly Leu
Ile
165 170 175
Arg Gln Gln Leu Asp Asn Pro Glu Val Thr Ile Gln Tyr Gly Gly
Ser
180 185 190
Val Lys Pro Asp Asn Ile Asp Glu Ile Met Ala Gln Ser Gln Ile
Asn
195 200 205
Gly Ala Leu Val Gly Gly Ala Ser Leu Asp Pro Ala Val
210 215 220
注：由于本序列为Microcystis wesenbergii tim基因的绝大部分序列(即未测全)。故在此序列中编号为Cys164的半胱氨酸即为蓝藻Synechocystis sp.TIM的Cys176。