金针茹AEG抗性突变株筛选
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摘要
提高农作物中人体与哺乳动物必需氨基酸含量是育种工作的主要研究领域，8种必需氨基酸中的赖氨酸、苏氨酸、甲硫氨酸和异亮氨酸皆以天冬氨酸为共同前体，通过不同的支路来代谢合成，其中赖氨酸还通常是影响氨基酸品质的第一限制性氨基酸，赖氨酸结构类似物S-(2-氨基乙基)-2′半胱氨酸(AEC)在天冬氨酸族代谢途径和含量提高的研究中起着重要作用。本研究首先观察AEC对6种食用菌菌丝生长的影响，探讨其与6种食用菌氨基酸含量的相关性及食用菌间氨基酸代谢的异同，在此基础上，以金针菇单核菌丝产生的粉孢子为材料，通过紫外线诱变，以AEC为选择培养基筛选具有AEC抗性的金针菇突变菌株，并测定突变株与出发菌株氨基酸含量，分析金针菇等食用菌天冬氨酸族氨基酸含量进一步提高的可能性，为食用菌氨基酸品质改良奠定理论与实验基础。
[方法] 1、配制含有不同AEC浓度的基本和完全培养基上，接种金针菇单核菌丝(IM02)，培养观察AEC对菌丝生长的抑制情况，并与AEC对白色茶树菇，榆黄蘑，虎奶菇，杏鲍菇和姬松茸抑制作用进行比较，计算AEC对菌丝生长抑制的IC_(50)；2、观察紫外线和Co~(60)辐射对金针菇粉孢子致死效应；3、利用AEC选择培养基，筛选出经紫外线诱变，具有AEC抗性的金针菇单核菌株，纯化后做抗性的稳定性试验，并通过液体发酵比较抗性菌株和出发菌株菌丝体游离氨基酸和总氨基酸的含量。4、将出发菌株IM02、抗性菌株与金针菇单核菌丝IS02杂交，通过观察有无锁状联合来鉴定菌株。
[结果] 1、AEC浓度在3000 mg．L~(-1)以上，金针菇单核菌丝的生长被完全抑制；在基本培养基上，AEC对金针菇单核菌丝(IM02)，白色茶树菇，榆黄蘑，虎奶菇，杏鲍菇和姬松茸的半数抑制浓度分别为24.8，5754.1，159.4，21.7，1158.8和361.8 mg．L~(-1)；在完全培养基上，AEC对金针菇单核菌丝(IM02)，白色茶树菇，榆黄蘑，虎奶菇和杏鲍菇的半数抑制浓度分别为2792.3，379751.1，402.2，244.9和1576.7 mg．L~(-1)，具有显著的不同；2、紫外线和Co~(60)对金针菇单核菌丝产生粉孢子致死效应分别较符合二次方程Y=3.7833-7.7874X-0.1481X~2和Y=0.4315+0.5768X-0.0008X~2；3、从AEC选择培养基中筛选到一株AEC抗性稳定的突变株A1。氨基酸测定表明，突变株A1在基础培养基发酵的菌丝体游离的异亮氨酸、苏氨酸和天冬氨酸族氨基酸总量分别比出发菌株IM02提高了49.4％、11.1％和17.6％；在完全培养基发酵的菌丝体的游离氨基酸总含量提高了38.3％，其中，异亮氨酸、甲硫氨酸、苏氨酸、赖氨酸和天冬氨酸族氨基酸总量分别提高了78.8％、63.6％、31.3％、14.3％和33.3％。4、杂交试验表明，出发菌株IM02、突变株A1与金针菇单核菌丝IS02均形成了锁状联合。
[结论] 1、所试验的6种食用菌对AEC抗性的显著不同，反映出其代谢途径中相关酶受终产物反馈抑制的敏感程度显著不同，值得对其相关酶(如AK等)作进一步研究。2、AEC抗性菌株的相关游离氨基酸和总氨基酸含量的相应提高，表明利用突变和筛选程序可以选育出高天冬氨酸族含量的食用菌新品种。3、本研究为食用菌氨基酸代谢途径和氮基酸品种改良奠定了理论与实验基础。
Improving amino acids quality is major work of agricultural plants breeding.Threonine, lysine, methionine, and Isoleusine which are biosynthesized through the aspartate family pathway are 4 of 8 essential amino acids for humans and monogastric animals and usually lysine is the first limiting amounts amino acids in many agricultural products. S-(2-aminoethyl)-L-cysteine which is lysine analogue plays a vital role in the study of metobolism of asparate-derived amino acids and elevations of these amino acid content. Edible fungi are abundant in nutritions such as amino acids. Firstly, in this thesis, .inhibitions of lysine analogue S-2-Aminoethyl-L-Cysteine(AEC) on mycelial growth of Agrocybe chaxingufwhitej , Pleurotus citrinopileatus, Pleurotus eryngii, Plewrotus tuber-regium, Agaricus blazi and Flammulina velutipes(IM02)were tested and the relativity of inhibitions effect and amino acids content of above edible fungi was also established. Secondly, a selection scheme based on AEC inhibition was applied to is
olating AEC resistant strains from UV-irradiated oidida of Flammulina velutipes and comparision of free and total amino acids content was conducted. The work was expected to be the theoretical and practical basis of the study of breeding of aspartate family amino acids-rich strain of edible fungi and metobolism of amino acids. 1 .Mycelial growth rate of Agrocybe chaxingufwhite) , Pleurotus citrinopileatus, Pleurotus tuber-regium, Pleurotus eryngii, Agaricus blazei and Flammulina velutipes (IMQ2) was measured when they grow in the minimal medium and complete medium which comprised different concentrations of AEC and ICso of inhibition of AEC on mycelial growth rate were calculated .
2.The death rates of UV-irradiated and Co60 -irradiated oidida of Flammulina velutipes were tested.
3.A selection scheme based on AEC inhibition was applied to isolating AEC resistant strains from UV-irradiated oidida of Flammulina velutipes and free and total amino acids content were determined.
4.The hybridation of AEC-resistant strains and the wild strains with IS02 were conducted to examine the authenticity of these strains.
1 .The results showed that ICso of inhibition of AEC on mycelial growth rate of Agrocybe chaxingufwhite) , Pleurotus citrinopileatus, Pleurotus tuber-regium, Pleurotus eryngii, Agaricus blazei and Flammulina velutipes (IMQ2) in the minimal medium was 5754.1 ? 159.4, 21.7, 1158.8, 361.8 and 24.8mg.L~1 respectively and IC50 of inhibition of AEC on mycelial growth rate of Agrocybe chaxingufwhite , Pleurotus citrinopileatus, Pleurotus tuber-regium, Pleurotus eryngii and Flammulina velutipes(lM02) in the complete medium was 379751.1, 402.2, 244.9,1576.7 and 2792.3mg.L~1, respectively .which were different significantly.
2.The death rates of UV-irradiated and Co60 -irradiated oidida of Flammulina velutipes were in conformity with the quadratic equation Y=3.7833+7.7874X-0.1481X2 and Y=0.4315+ 0.5768X-0.0008X2 through curve estimation.
3. One AEC resistant strain was isolated by screening UV-irradiated oidida of Flammulina velutipes and were confirmed after five replicates. The resistant strain contained much higher concentrations of free threonine, methionine and alanine than the wild strain of IM02.
4.The authenticity of the mutant strain was confirmed by the hybridation test. [Conclusions]
It was estimated that sensitivity of enzymes on end products in the feedback pathway of aspartate family amino acids biosynthesis were different significantly in the edible fungi. The results would cast a promising perspectives on strain improvement work of edible fungi to high aspartate family amino acids.
[方法] 1、配制含有不同AEC浓度的基本和完全培养基上，接种金针菇单核菌丝(IM02)，培养观察AEC对菌丝生长的抑制情况，并与AEC对白色茶树菇，榆黄蘑，虎奶菇，杏鲍菇和姬松茸抑制作用进行比较，计算AEC对菌丝生长抑制的IC_(50)；2、观察紫外线和Co~(60)辐射对金针菇粉孢子致死效应；3、利用AEC选择培养基，筛选出经紫外线诱变，具有AEC抗性的金针菇单核菌株，纯化后做抗性的稳定性试验，并通过液体发酵比较抗性菌株和出发菌株菌丝体游离氨基酸和总氨基酸的含量。4、将出发菌株IM02、抗性菌株与金针菇单核菌丝IS02杂交，通过观察有无锁状联合来鉴定菌株。
[结果] 1、AEC浓度在3000 mg．L~(-1)以上，金针菇单核菌丝的生长被完全抑制；在基本培养基上，AEC对金针菇单核菌丝(IM02)，白色茶树菇，榆黄蘑，虎奶菇，杏鲍菇和姬松茸的半数抑制浓度分别为24.8，5754.1，159.4，21.7，1158.8和361.8 mg．L~(-1)；在完全培养基上，AEC对金针菇单核菌丝(IM02)，白色茶树菇，榆黄蘑，虎奶菇和杏鲍菇的半数抑制浓度分别为2792.3，379751.1，402.2，244.9和1576.7 mg．L~(-1)，具有显著的不同；2、紫外线和Co~(60)对金针菇单核菌丝产生粉孢子致死效应分别较符合二次方程Y=3.7833-7.7874X-0.1481X~2和Y=0.4315+0.5768X-0.0008X~2；3、从AEC选择培养基中筛选到一株AEC抗性稳定的突变株A1。氨基酸测定表明，突变株A1在基础培养基发酵的菌丝体游离的异亮氨酸、苏氨酸和天冬氨酸族氨基酸总量分别比出发菌株IM02提高了49.4％、11.1％和17.6％；在完全培养基发酵的菌丝体的游离氨基酸总含量提高了38.3％，其中，异亮氨酸、甲硫氨酸、苏氨酸、赖氨酸和天冬氨酸族氨基酸总量分别提高了78.8％、63.6％、31.3％、14.3％和33.3％。4、杂交试验表明，出发菌株IM02、突变株A1与金针菇单核菌丝IS02均形成了锁状联合。
[结论] 1、所试验的6种食用菌对AEC抗性的显著不同，反映出其代谢途径中相关酶受终产物反馈抑制的敏感程度显著不同，值得对其相关酶(如AK等)作进一步研究。2、AEC抗性菌株的相关游离氨基酸和总氨基酸含量的相应提高，表明利用突变和筛选程序可以选育出高天冬氨酸族含量的食用菌新品种。3、本研究为食用菌氨基酸代谢途径和氮基酸品种改良奠定了理论与实验基础。
Improving amino acids quality is major work of agricultural plants breeding.Threonine, lysine, methionine, and Isoleusine which are biosynthesized through the aspartate family pathway are 4 of 8 essential amino acids for humans and monogastric animals and usually lysine is the first limiting amounts amino acids in many agricultural products. S-(2-aminoethyl)-L-cysteine which is lysine analogue plays a vital role in the study of metobolism of asparate-derived amino acids and elevations of these amino acid content. Edible fungi are abundant in nutritions such as amino acids. Firstly, in this thesis, .inhibitions of lysine analogue S-2-Aminoethyl-L-Cysteine(AEC) on mycelial growth of Agrocybe chaxingufwhitej , Pleurotus citrinopileatus, Pleurotus eryngii, Plewrotus tuber-regium, Agaricus blazi and Flammulina velutipes(IM02)were tested and the relativity of inhibitions effect and amino acids content of above edible fungi was also established. Secondly, a selection scheme based on AEC inhibition was applied to is
olating AEC resistant strains from UV-irradiated oidida of Flammulina velutipes and comparision of free and total amino acids content was conducted. The work was expected to be the theoretical and practical basis of the study of breeding of aspartate family amino acids-rich strain of edible fungi and metobolism of amino acids. 1 .Mycelial growth rate of Agrocybe chaxingufwhite) , Pleurotus citrinopileatus, Pleurotus tuber-regium, Pleurotus eryngii, Agaricus blazei and Flammulina velutipes (IMQ2) was measured when they grow in the minimal medium and complete medium which comprised different concentrations of AEC and ICso of inhibition of AEC on mycelial growth rate were calculated .
2.The death rates of UV-irradiated and Co60 -irradiated oidida of Flammulina velutipes were tested.
3.A selection scheme based on AEC inhibition was applied to isolating AEC resistant strains from UV-irradiated oidida of Flammulina velutipes and free and total amino acids content were determined.
4.The hybridation of AEC-resistant strains and the wild strains with IS02 were conducted to examine the authenticity of these strains.
1 .The results showed that ICso of inhibition of AEC on mycelial growth rate of Agrocybe chaxingufwhite) , Pleurotus citrinopileatus, Pleurotus tuber-regium, Pleurotus eryngii, Agaricus blazei and Flammulina velutipes (IMQ2) in the minimal medium was 5754.1 ? 159.4, 21.7, 1158.8, 361.8 and 24.8mg.L~1 respectively and IC50 of inhibition of AEC on mycelial growth rate of Agrocybe chaxingufwhite , Pleurotus citrinopileatus, Pleurotus tuber-regium, Pleurotus eryngii and Flammulina velutipes(lM02) in the complete medium was 379751.1, 402.2, 244.9,1576.7 and 2792.3mg.L~1, respectively .which were different significantly.
2.The death rates of UV-irradiated and Co60 -irradiated oidida of Flammulina velutipes were in conformity with the quadratic equation Y=3.7833+7.7874X-0.1481X2 and Y=0.4315+ 0.5768X-0.0008X2 through curve estimation.
3. One AEC resistant strain was isolated by screening UV-irradiated oidida of Flammulina velutipes and were confirmed after five replicates. The resistant strain contained much higher concentrations of free threonine, methionine and alanine than the wild strain of IM02.
4.The authenticity of the mutant strain was confirmed by the hybridation test. [Conclusions]
It was estimated that sensitivity of enzymes on end products in the feedback pathway of aspartate family amino acids biosynthesis were different significantly in the edible fungi. The results would cast a promising perspectives on strain improvement work of edible fungi to high aspartate family amino acids.
引文
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