CN103524434B - 植物乙烯合成途径的小分子抑制剂吡嗪酰胺及其应用 - Google Patents
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Abstract
本发明公开了一种植物乙烯合成途径的小分子抑制剂吡嗪酰胺(Pyrazinamide,简称PZA),其特征在于,其为ACC氧化酶(ACO)的特异性抑制剂,能抑制植物乙烯合成途径中乙烯合成前体ACC转化为乙烯;吡嗪酰胺的结构式如摘要附图所示,分子量为123.12。本发明还提供含有所述吡嗪酰胺的抑制植物乙烯合成途径的培养基。本发明的另一目的是提供所述植物乙烯合成途径的小分子抑制剂吡嗪酰胺在植物生长发育调节中和在植物运输保鲜中的应用。本发明提供的抑制剂吡嗪酰胺可以明显抑制植物乙烯合成,且抑制作用强于已知乙烯合成抑制剂,便于推广应用。
Description
技术领域
本发明涉及生物技术领域,特别是涉及植物乙烯合成途径的小分子抑制剂吡嗪酰胺及其应用。
背景技术
植物激素乙烯对植物生长发育及抵抗外界生物和非生物胁迫具有重要作用。如:乙烯调控种子萌发、叶片衰老、花脱落、果实成熟、以及单性花性别决定和抵御外界胁迫。其中乙烯促进果实成熟和器官脱落的特性,在农业生产中发挥重要作用。如乙烯是农业生产中常用的果实催熟剂主要有效成分,此外,由于乙烯的促进器官衰老脱落的特性,每年蔬菜瓜果花卉采摘后由于乙烯导致的衰老腐败等损失约达30%。传统应用的乙烯抑制剂往往作用位点不特异,如常用的乙烯抑制剂AVG同时也是生长素合成抑制剂且价格昂贵;而常用的银离子型抑制剂除作用位点不特异外,对环境也会造成危害;此外近年来应用的通过抑制乙烯与受体结合的抑制剂并不能直接减少植物乙烯的合成量。目前仍缺乏成本较低、特异性较好、且抑制作用较强的乙烯合成抑制剂。
发明内容
本发明的目的是提供植物乙烯合成途径的小分子抑制剂吡嗪酰胺及其应用。
本发明提供的植物乙烯合成途径的小分子抑制剂吡嗪酰胺(Pyrazinamide,简称PZA)其为ACC氧化酶(ACO)的特异性抑制剂,能抑制植物乙烯合成途径中乙烯合成前体ACC转化为乙烯;吡嗪酰胺的结构式如图1所示,分子量为123.12。
本发明提供的一种抑制植物乙烯合成途径的培养基,其含有吡嗪酰胺。
所述抑制植物乙烯合成途径的培养基中吡嗪酰胺浓度优选为30~50μM(微摩尔每升)的培养基。
所述抑制植物乙烯合成途径的培养基优选为吡嗪酰胺浓度为30~50μM(微摩尔每升)的MS培养基。
进一步地,本发明提供所述植物乙烯合成途径的小分子抑制剂吡嗪酰胺在植物生长发育调节中的应用。
本发明还提供所述植物乙烯合成途径小分子抑制剂吡嗪酰胺在植物运输保鲜中的应用。所述植物主要为瓜果、蔬菜、花卉。
本发明的有益效果在于提供一种植物乙烯合成途径的小分子抑制剂,成本较低、特异性较好地抑制了植物乙烯的合成,直接减少植物乙烯的合成量,为植物生长发育调节以及蔬菜、瓜果、花卉运输保鲜提供了一种新的途径。且本发明的小分子抑制剂吡嗪酰胺可以明显抑制植物乙烯合成,且抑制作用强于已知乙烯合成抑制剂,便于推广应用。
附图说明
图1是吡嗪酰胺的结构式;
图2是实施例2吡嗪酰胺对Col-0和eto1-2表型影响的照片;
图3是实施3施加过量乙烯合成前体ACC基础上吡嗪酰胺对Col-0和eto1-2表型影响的照片;
图4是实施例4吡嗪酰胺对拟南芥黄化苗乙烯合成量影响的柱状态图。
图5是实施例5吡嗪酰胺与AIB对乙烯合成的抑制作用对比图。
具体实施方式
下面结合附图和实施例,对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1筛选抑制植物乙烯合成途径的小分子抑制剂
利用化学遗传学即小分子抑制剂筛选方法,筛选美国加州大学河滨分校提供的含有2000种选自microsource spectrum数据库(SP2000,http://www.msdiscovery.com)的化学小分子。文库中所有小分子均用DMSO配成0.5mg/100μL浓度的母液。相关小分子信息可以从(http://www.msdiscovery.com/downloads.html)下载。该筛选文库中的小分子的结构、分子式、分子量、名称以及功能均已知,小分子筛选测试时的浓度为50~100μM。通过筛选该含2000种小分子的文库(加州大学河滨分校杨贞标惠赠)(参考文献:Girke T,Cheng LC,et al.(2005).“ChemMine.A compound mining database for chemicalgenomics”Plant Physiol.138(2):573-7.),获得了将乙烯合成前体ACC转化为乙烯的ACC氧化酶(ACO)的特异性小分子抑制剂吡嗪酰胺(Pyrazinamide,简称PZA)(分子量:123.12)。
实施例2本发明抑制植物乙烯合成途径的培养基
以小分子抑制剂吡嗪酰胺(Pyrazinamide,简称PZA)制备本发明的抑制植物乙烯合成途径的MS培养基,即配制吡嗪酰胺浓度为30~50μM(微摩尔每升)的MS培养基。对照为常用的MS培养基。
实验材料为拟南芥乙烯过量合成突变株eto1-2,以及乙烯正常表达的拟南芥野生型Col-0。
以抑制植物乙烯合成途径的MS培养基(即添加吡嗪酰胺的MS培养基)和对照MS培养基培养eto1-2和Col-0,于22°培养箱,避光暗培养条件下培养3天。
培养3天后观察,结果见图2(图2为培养基加入本发明的小分子抑制剂吡嗪酰胺的作用,具体为拟南芥野生型Col-0和乙烯突变株eto1-2未施加抑制剂和施加抑制剂的对比表型),表明加了吡嗪酰胺的培养基可明显抑制3天大的拟南芥乙烯过量合成突变株eto1-2黄化苗由于乙烯过量合成导致的根长抑制表型。即该小分子在较低浓度下即可以明显促进乙烯过量合成导致的短根恢复伸长。
实施例3吡嗪酰胺作用靶点的确定
为了确定本发明小分子抑制剂吡嗪酰胺的作用靶点,施加过量的乙烯合成前体ACC后观察小分子抑制剂作用。如果抑制剂作用在乙烯合成途径,则过量乙烯合成前体ACC存在的条件下,小分子抑制剂吡嗪酰胺不再会有明显的抑制乙烯合成作用;但如果抑制剂靶点位于乙烯合成途径下游的乙烯信号传导途径,则在过量乙烯合成前体ACC存在的条件下,抑制剂吡嗪酰胺对乙烯的抑制作用不会受到影响,即过量乙烯合成前体ACC不能影响下游乙烯信号通路抑制剂对乙烯信号的抑制作用。
实验方法参照实施例2,仅在两种培养基中均加入过量乙烯合成前体ACC,加入量为10μM。
实验结果见图3,表明施加过量乙烯合成前体ACC后,本发明小分子对乙烯导致的短根表型无明显作用,即本发明小分子作用靶点位于乙烯合成途径而非下游乙烯信号传导途径。
实施例4吡嗪酰胺作用靶点位置的验证
在分别装有MS培养基、MS+50μM吡嗪酰胺培养基、MS+10ACC培养基、MS+50μM吡嗪酰胺培养基+10μM ACC培养基的密闭西林瓶中培养eto1-2和Col-0,培养3天后(即72小时),利用气象色谱,直接检测本发明小分子抑制剂对拟南芥黄化苗乙烯合成量的抑制情况。
结果如图4所示,发现该抑制剂吡嗪酰胺可以明显抑制拟南芥野生型Col以及乙烯过量合成突变株eto1-2的乙烯合成量。进一步确定本发明抑制剂确实作用于乙烯合成途径,并且由于该抑制剂可以抑制ACC诱导的乙烯合成,说明该抑制剂作用于乙烯合成前体ACC到生成乙烯的过程。
实施例5吡嗪酰胺PZA与乙烯抑制剂AIB作用效果对比
之前有报道发现抑制剂2-氨基异丁酸(α-aminoisobutanoic acid,简称AIB)可以抑制ACC生成乙烯过程,但抑制作用较弱,难以进行实际应用。
对比同样作用于ACC生成乙烯过程的本发明抑制剂PZA与已有抑制剂AIB对乙烯导致的拟南芥黄化苗短根表型影响,具体的实验设计为:
具体实验设计:将测试株拟南芥乙烯过量合成突变体eto1-2和野生型Col以及乙烯下游信号通路组成型激活突变株ctr1-1培养在分别含有同样梯度浓度(0μM,1μM,5μM,10μM,50μM以及100μM)PZA以及AIB的MS培养基上,22°培养箱避光暗培养3天,观察并测量比较相同抑制剂浓度下测试株根长度变化。即抑制剂抑制乙烯合成效果越强,则促进根伸长越明显。对照组野生型Col由于本底乙烯合成量很低,根长变化应不明显;对照组乙烯信号通路组成型激活突变株ctr1-1由于作用于乙烯合成途径下游的信号通路,所以乙烯合成途径的抑制剂应对下游信号通路突变株ctr1-1根神长也无明显作用。图5根长均以拟南芥野生型Col在未施加乙烯合成抑制剂的MS培养基上暗培养3天后的平均根长为参照,即100%,其他测试株以及对照株系的实际平均根长均与Col参照根长进行比对,获得相对根长,便于比较。
实验结果见图5,发现PZA在梯度浓度范围对乙烯过量合成突变株eto1-2根伸长促进作用都明显强于AIB作用,表明PZA对乙烯合成的抑制作用明显强于已知抑制剂AIB。
综上所述,本研究利用化学遗传学方法,筛选到乙烯合成途径ACC生成乙烯过程的小分子抑制剂PZA,该抑制剂可以明显抑制植物乙烯合成,且抑制作用强于已知乙烯合成抑制剂,便于推广应用。
本发明的植物乙烯合成途径小分子抑制剂吡嗪酰胺可在植物运输保鲜中的应用,如防止植物在运输过程中由于产生过量乙烯而导致的衰老、腐败、以及花脱落等。所述植物为需要抑制乙烯合成从而实现保鲜目的的植物,如需要防止衰老腐败及脱落的植物,主要为瓜果、蔬菜、花卉。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (2)
1.一种植物乙烯合成途径的小分子抑制剂吡嗪酰胺在植物生长发育调节中的应用。
2.一种植物乙烯合成途径的小分子抑制剂吡嗪酰胺在植物运输保鲜中的应用。
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