高赖氨酸蛋白基因转化银耳研究
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摘要
蛋白质是人的营养要素，它由20多种氨基酸组成，其中有10种氨基酸人不能合成，只能从食物中摄取，称之为必需氨基酸。赖氨酸是十种必需氨基酸之一，赖氨酸具有多种生物学功能，这些功能包括：增进食欲、提高血液中血红蛋白含量；赖氨酸铝盐可治胃溃疡等。人体缺少赖氨酸会出现体重轻、骨骼发育不全、也会降低人体对其它氨基酸的吸收，营养学家把它称为“第一缺乏氨基酸”。
赖氨酸蛋白基因转入具有较高食药用价值的银耳，合成具有生物活性的赖氨酸蛋白，相当于在营养丰富的银耳中添加新的营养保健因子，既提高银耳的营养保健价值，又增加人体对膳食中蛋白质的利用率。
本研究在探索银耳遗传转化体系的基础上，构建了高赖氨酸蛋白基因表达载体，利用限制性内切酶介导整合法(R E M I)，首次把高赖氨酸蛋白基因导入银耳芽孢，获得了转基因菌株。对转基因菌株进行分子检测，证实了外源基因已整合到银耳基因组中。氨基酸含量测定表明，转基因银耳中赖氨酸含量得到不同程度的提高。具体结果如下：
1．银耳原生质体制备与再生条件优化
以芽孢、菌丝体和子实体为材料，通过正交实验，考察了菌株、酶浓度、酶解时间以及酶解温度对原生质体产率和再生率的影响，结果显示：实验材料对原生质体产量影响最大，以芽孢为材料原生质体产量可达到2.75×10~7个／ml，而菌丝体和子实体的原生质体产量仅为2.5×10~6个／ml和1.0×10~6个／ml；在35℃下酶解，原生质体产量最高；溶壁酶浓度在1％～3％之间对原生质体产量影响不大：不同菌株原生质体产量差异不显著。本实验还研究了稳渗剂浓度对原生质体再生率的影响，结果表明，0.5mol／L～0.7 mol／L的KCI对原生质体再生没有显著差异，再生率最高为32.3％。
福建农林大学硕士学位论文
中文摘要
对比紫外线与Y射线对银耳芽抱的诱变效应，Y射线诱变获得了48
个缺陷型菌株，经传代后都被修复，没有获得稳定的突变体，紫外线诱变
后有62个缺陷型菌株，经过传代后获得2株稳定的缺陷型突变体，获得两
个营养缺陷型菌株，其中一株Tausn经鉴定是肌醇缺陷型。突变体Tausn
的生长迟缓期比野生型多3天，生长速度较慢。
3.高赖氨酸蛋白基因转化银耳及分子检测
以pBR一1 ys为中间载体，把pBR一lys的Pubi启动子置换成P35S，构建成
表达载体pCB一1 ys。应用REMI介导转化银耳芽抱，获得众多的转化子，核
rDNA检测结果和营养缺陷型检验结果表明，这些转化子均是银耳芽抱。PCR
检测结果:转化子携带P35S启动子、Lys基因、GUS基因、hPt基因、Tnos
终止子。转基因银耳芽抱对潮霉素的抗性水平可达到巧oug/m1以上，大部
分转化子检测到GUS活性。
4.转基因菌株赖氨酸含量测定
利用氨基酸自动分析仪，对潮霉素抗性较强的6个转基因菌株以及对
照Tausn进行氨基酸分析，结果显示:转基因菌株的赖氨酸含量都有不同
程度的提高，其中有4株提高了11%以上，最高一株为16.57%，而且蛋白
质中氨基酸总量也有较大幅度的提高。
Protein is nutrition to human beings, which consists of twenty different types of amino acids, ten out of which can not be synthesized by human bodies thus have to be taken from food. These types of amino acids are called essential amino acids. Lysine is one of nine essential amino acids. Lysine has many biological functions including: increasing the appetite; enhancing the hemoglobin content in blood and curing peptic ulcer, etc. The lack of lysine in a human being can cause abnormal loss of weight and incomplete growth of bones, and the deficient assimilation of other amino acids, therefore, lysine is called by nutritionist as "the first needed amino acid".
Transformation of lysine-rich protein genes from winged bean to Tremella fuciformis which has high nutritional value may enhance the genetically modified Tremella fuciformis nutritional and functional values. Since lysine-rich protein is a multifunctional protein if the tansgenes express correctly, the utilization of protein will also be enhanced.
In the study, on the basis of studying the system of genetic transformation of Tremella fuciformis, a lysine-rich protein gene expression vector was constructed, and then transferred into Tremella fuciformis spores with Restriction Enzyme-Mediated DNA Integration (REMI). Molecular evidence demonstrated that lysine-rich protein gene had been integrated into the genome of transgenic Tremella fuciformis strains. Amino acids content testing results also confirmed that lysine content of transgenic strains was elevated comparably.
1. Optimization of preparation and regeneration condition of Tremella fuciformis protoplasts
Orthogonal design was used for researching the high yield protoplast of Tremella fuciformis. The results showed that the materials (conidia, mycelia and fruit bodies) were the major factor affecting protoplast yield. Using conidia as material, the yield of protoplast could reach 2.75x107/ml, while using mycelia and fruit body material, the yield of protoplast was only 2.5x106/ml and 1.0x106/ml respectively. The optimal digesting temperature was 35.The lywallzyme concentration ranging from 1% to 3%, did not affect the yield of protoplasts significantly. There was also no
significant difference in the yield of protoplasts from different strains. The effect of osmotic stabilizer (KCI) concentration on regeneration of protoplasts was also studied. The results showed that there was no significant difference in the rate of protoplasts regeneration in different concentrations. KCI concentration from 0.5mol/L to 0.7 mol/L did not show significant difference to regeneration of protoplasts: the highest protoplasts regeneration rate was 32.3% in.
2. Selection of auxotrophic strains
Induced effects on the spores of Tremella fuciformis by ultraviolet and r-ray radiations were compared. 48 auxotrophic strains from r-rayed were obtained, but the mutants were not steady. 62 auxotrophic strains were obtained from ultraviolet radiation and two were steady auxotrophs. One of two strains Tau811 was inositol-defect mutant by identification. Lag phase of growth of Tau811 was three days more than wild stains.
3. Transformation of lysine-rich protein gene to Tremella fuciformis and its molecular evidence
P35S was incorporated into pBR-lys plasmid instead of promoter Pubi to construct expression vector pCB-lys. Transformation of lysine-rich protein gene to Tremella fuciformis produced many transformants. The results of the test of nuclear rDNA and deficiency diagnosis of nutrients showed that these transformants were yeast-like-conidia. These transformants bring with 35S promoter, Lys gene, GUS gene, hpt gene, nos terminator by PCR determination. The resistance to hygromycin in transformants reached 150ug/ml, and the determination of GUS activity of transformants was positive.
4. Determination of the lysine content of transgenic strains
Using Automatic Amino Acid Analyzer, Amino acids content determination of non-transgenic strain Tau811 and six transgenic strains with high resistance to hygromycin, showed that: the ly
赖氨酸蛋白基因转入具有较高食药用价值的银耳，合成具有生物活性的赖氨酸蛋白，相当于在营养丰富的银耳中添加新的营养保健因子，既提高银耳的营养保健价值，又增加人体对膳食中蛋白质的利用率。
本研究在探索银耳遗传转化体系的基础上，构建了高赖氨酸蛋白基因表达载体，利用限制性内切酶介导整合法(R E M I)，首次把高赖氨酸蛋白基因导入银耳芽孢，获得了转基因菌株。对转基因菌株进行分子检测，证实了外源基因已整合到银耳基因组中。氨基酸含量测定表明，转基因银耳中赖氨酸含量得到不同程度的提高。具体结果如下：
1．银耳原生质体制备与再生条件优化
以芽孢、菌丝体和子实体为材料，通过正交实验，考察了菌株、酶浓度、酶解时间以及酶解温度对原生质体产率和再生率的影响，结果显示：实验材料对原生质体产量影响最大，以芽孢为材料原生质体产量可达到2.75×10~7个／ml，而菌丝体和子实体的原生质体产量仅为2.5×10~6个／ml和1.0×10~6个／ml；在35℃下酶解，原生质体产量最高；溶壁酶浓度在1％～3％之间对原生质体产量影响不大：不同菌株原生质体产量差异不显著。本实验还研究了稳渗剂浓度对原生质体再生率的影响，结果表明，0.5mol／L～0.7 mol／L的KCI对原生质体再生没有显著差异，再生率最高为32.3％。
福建农林大学硕士学位论文
中文摘要
对比紫外线与Y射线对银耳芽抱的诱变效应，Y射线诱变获得了48
个缺陷型菌株，经传代后都被修复，没有获得稳定的突变体，紫外线诱变
后有62个缺陷型菌株，经过传代后获得2株稳定的缺陷型突变体，获得两
个营养缺陷型菌株，其中一株Tausn经鉴定是肌醇缺陷型。突变体Tausn
的生长迟缓期比野生型多3天，生长速度较慢。
3.高赖氨酸蛋白基因转化银耳及分子检测
以pBR一1 ys为中间载体，把pBR一lys的Pubi启动子置换成P35S，构建成
表达载体pCB一1 ys。应用REMI介导转化银耳芽抱，获得众多的转化子，核
rDNA检测结果和营养缺陷型检验结果表明，这些转化子均是银耳芽抱。PCR
检测结果:转化子携带P35S启动子、Lys基因、GUS基因、hPt基因、Tnos
终止子。转基因银耳芽抱对潮霉素的抗性水平可达到巧oug/m1以上，大部
分转化子检测到GUS活性。
4.转基因菌株赖氨酸含量测定
利用氨基酸自动分析仪，对潮霉素抗性较强的6个转基因菌株以及对
照Tausn进行氨基酸分析，结果显示:转基因菌株的赖氨酸含量都有不同
程度的提高，其中有4株提高了11%以上，最高一株为16.57%，而且蛋白
质中氨基酸总量也有较大幅度的提高。
Protein is nutrition to human beings, which consists of twenty different types of amino acids, ten out of which can not be synthesized by human bodies thus have to be taken from food. These types of amino acids are called essential amino acids. Lysine is one of nine essential amino acids. Lysine has many biological functions including: increasing the appetite; enhancing the hemoglobin content in blood and curing peptic ulcer, etc. The lack of lysine in a human being can cause abnormal loss of weight and incomplete growth of bones, and the deficient assimilation of other amino acids, therefore, lysine is called by nutritionist as "the first needed amino acid".
Transformation of lysine-rich protein genes from winged bean to Tremella fuciformis which has high nutritional value may enhance the genetically modified Tremella fuciformis nutritional and functional values. Since lysine-rich protein is a multifunctional protein if the tansgenes express correctly, the utilization of protein will also be enhanced.
In the study, on the basis of studying the system of genetic transformation of Tremella fuciformis, a lysine-rich protein gene expression vector was constructed, and then transferred into Tremella fuciformis spores with Restriction Enzyme-Mediated DNA Integration (REMI). Molecular evidence demonstrated that lysine-rich protein gene had been integrated into the genome of transgenic Tremella fuciformis strains. Amino acids content testing results also confirmed that lysine content of transgenic strains was elevated comparably.
1. Optimization of preparation and regeneration condition of Tremella fuciformis protoplasts
Orthogonal design was used for researching the high yield protoplast of Tremella fuciformis. The results showed that the materials (conidia, mycelia and fruit bodies) were the major factor affecting protoplast yield. Using conidia as material, the yield of protoplast could reach 2.75x107/ml, while using mycelia and fruit body material, the yield of protoplast was only 2.5x106/ml and 1.0x106/ml respectively. The optimal digesting temperature was 35.The lywallzyme concentration ranging from 1% to 3%, did not affect the yield of protoplasts significantly. There was also no
significant difference in the yield of protoplasts from different strains. The effect of osmotic stabilizer (KCI) concentration on regeneration of protoplasts was also studied. The results showed that there was no significant difference in the rate of protoplasts regeneration in different concentrations. KCI concentration from 0.5mol/L to 0.7 mol/L did not show significant difference to regeneration of protoplasts: the highest protoplasts regeneration rate was 32.3% in.
2. Selection of auxotrophic strains
Induced effects on the spores of Tremella fuciformis by ultraviolet and r-ray radiations were compared. 48 auxotrophic strains from r-rayed were obtained, but the mutants were not steady. 62 auxotrophic strains were obtained from ultraviolet radiation and two were steady auxotrophs. One of two strains Tau811 was inositol-defect mutant by identification. Lag phase of growth of Tau811 was three days more than wild stains.
3. Transformation of lysine-rich protein gene to Tremella fuciformis and its molecular evidence
P35S was incorporated into pBR-lys plasmid instead of promoter Pubi to construct expression vector pCB-lys. Transformation of lysine-rich protein gene to Tremella fuciformis produced many transformants. The results of the test of nuclear rDNA and deficiency diagnosis of nutrients showed that these transformants were yeast-like-conidia. These transformants bring with 35S promoter, Lys gene, GUS gene, hpt gene, nos terminator by PCR determination. The resistance to hygromycin in transformants reached 150ug/ml, and the determination of GUS activity of transformants was positive.
4. Determination of the lysine content of transgenic strains
Using Automatic Amino Acid Analyzer, Amino acids content determination of non-transgenic strain Tau811 and six transgenic strains with high resistance to hygromycin, showed that: the ly
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