甜味蛋白Monellin基因的合成、功能分析及对桑树的遗传转化研究
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摘要
桑树(Morus alba L.)是多年生双子叶森本植物,也是一种重要的经济作物。桑树全身都是宝：桑叶被用作家蚕和畜禽的饲料；桑椹中富含花青素等物质,现已受到越来越多人们的喜爱；桑根、桑皮和桑枝等作为一种传统药材,被用到中药中。随着科技的发展,桑树的价值将得到最大程度的开发利用,因此,选育新桑品种是研究者的重要任务之一。桑树的传统育种,如选择育种、杂交育种等技术已不能满足蚕桑产业向多元化方向发展的需要,现在越来越多的研究者开始了桑树的分子育种相关研究。很多研究表明,外源基因对桑树进行遗传转化是可行的,但如何获得适合的外源基因,提高外源基因的表达量,寻找适合的基因转化方法等都是需要面对和解决的难点。因此,对桑树遗传转化的研究任重而道远。
从西非植物Dioscoreophyllum cumminsii的果实中获得的天然的甜味蛋白Monellin,具有甜度高、热量低等特点。但它的热稳定性较差,其开发生产也受到了产地和资源等因素的限制,难以规模化。因此,研究者运用生物技术的方法,合成了甜味蛋白基因,并在原核与真核表达系统中成功表达,获得的甜味蛋白在酸性环境下最高能耐受60℃高温；在1992年报道了该蛋白在植物中的应用,基因被成功导入莴苣和土豆中,但未能检测出甜味。
如何能够提高该蛋白的表达量和热稳定性,如何能够将monellin基因导入桑树中并得到表达?本研究围绕这些问题开展了较为深入系统的研究,并取得了较好的研究结果,现将研究要点总结如下：
1.一个人工设计合成的monellin基因
本研究根据已报道的Monellin甜蛋白氨基酸序列,通过统计“各物种密码子使用频率数据库codon usage database”信息,兼顾桑树与毕赤酵母密码子的偏爱性,人工设计优化了一条长为294bp的单链monellin基因,并采用重叠PCR的方法合成该基因。将合成的基因核苷酸序列在NCBI中进行比对,结果显示这是一个具有新核苷酸序列的monellin基因。我们将这一基因向NCBI进行了提交,确定基因登录号为GenBank:JQ282905。该基因如能在真核生物和植物的表达系统中表达,则为它扩展应用到其它作物中去提供了新的思路和途径。
2.新monellin基因的真核表达研究
本研究中,选取巴斯德毕赤酵母GS115为宿主菌,以胞外分泌型的载体pPIC9K为基础,构建了两个表达载体pPIC9K-M-E和pPIC9K-M。表达载体pPIC9K-M-E中包含有甜味蛋白monellin基因和绿色荧光蛋白EGFP基因,pPIC9K-M中只包含monellin基因,但它们中均在基因下游带上了组氨酸标签以方便蛋白的分离纯化。构建好的表达载体经线性化后,采用电击法转化入毕赤酵母GS115,经菌落PCR和平板筛选分别获得了阳性酵母表达菌株GS115/pPIC9K-M和GS115/pPIC9K-M-E,利用含G418的平板筛选出基因多拷贝酵母菌株后,在BMGY和BMMY培养基中利用甲醇诱导获得了表达上清。然后采用Ni-NTA亲和柱纯化表达的蛋白,经分析验证两种蛋白均有甜味,约为标准蔗糖的500倍,甜味刺激来的慢去的慢；诱导重组菌GS115/pPIC9K-M-E产生的蛋白在荧光显微镜下能够发出强烈的荧光,诱导重组子GS115/pPIC9K-M获得的蛋白产物能耐80℃高温,pH值在3-12之间时,蛋白降解较少。这些结果证明我们获得的蛋白不但具有甜味,还具有有相对较高的对热稳定性,也有较宽的耐酸碱性。这为它的开发利用提供了条件。由此,我们将获得的monellin基因向国家知识产权局申请了国家发明专利,并于2012年5月获正式授权,专利号为ZL201010042099.3。该基因的获得为桑树及作物遗传转化创新了一个有意义的目的基因。
3.利用根癌农杆菌介导法研究monellin基因对桑树的遗传转化
在农杆菌介导法中需要大量的植物受体材料,为了高效获取组培所需的子叶、下胚轴等外植体,本研究特别探索了一种快速促进桑种子萌发的方法-振荡培养法。实验并分析了静置培养和振荡培养2种培养方式处理下,桑种子萌发状态及萌发过程中内源激素含量和超氧化物歧化酶(SOD)活性的变化。结果显示：振荡培养的桑种子萌发快而整齐,萌发率高达99%,比静置培养分别高9个百分点。经测定,桑种子萌发过程中振荡培养组的SOD活性高于静置培养组。内源激素测定结果显示,IAA和GA均参与了桑种子的萌发过程,且相对较低浓度的IAA和较高浓度的GA能促进桑种子的萌发；当GA含量远高于ABA含量时,能大大促进桑种子的萌发与幼苗的生长。上述实验结果表明,振荡培养比静置培养方式不仅可以提前2天以上获得萌发桑苗,缩短桑树组培时间,还能减少外植体被真菌等感染的机率。
本研究采用根癌农杆菌介导法和花粉介导法对monellin基因在桑树中的遗传转化进行了研究。实验中,构建了植物表达载体pCAMBIA2301-M,以“桂优62号”、“特优2号”、“河南桑”和“嘉陵30号”桑树品种的子叶、下胚轴和丛生芽为遗传转化的受体,经基因转化、诱导培养、生根炼苗等获得了6株基因转化苗,其中“嘉陵30号”和“桂优62号”各3株。经过实验发现,“嘉陵30号”丛生芽和“桂优62号”下胚轴是较好的遗传转化受体,其中共转化215个受体材料,经鉴定获得4株阳性转化苗,转化率为1.9%；而以“河南桑”的组培外植体作为遗传转化受体没有获得阳性转化苗。实验表明,不同品种资源的桑材料用作转化受体时,其遗传转化效果存在较大差异。实验也表明根癌农杆菌介导基因转化桑树是可行的。
4.利用花粉介导法研究monellin基因对桑树的遗传转化
本研究借鉴研究室李军等的花粉介导法将载体pCAMBIA2300-M-E导入“红果1号”和“育2号”杂交桑种子中,共收获1096粒种子,其中200粒种子在萌发期用卡那霉素筛选,获得19株桑苗移栽大田；另外896粒桑种,经振荡培养后播种至大田精心管理。最终,花粉介导法共获得125株遗传转化实生苗,经鉴定有11株阳性转化苗,转化率为1%。这说明本研究采用的花粉介导法也是可行的遗传转化方法。
Mulberry Morus alba L. is a perennial woody dicotyledonous plants, and it is also a kind of important economic tree species in China. Each part of mulberry plant is a treasure:mulberry leaves are used as the natural forage for silkworm Bombyx mori L. and livestock; Mulberry fruits are rich in anthocyanin and other functional substances, so more and more people adore them; Mulberry roots, barks and ramulus mori as the kind of vegetation medicinal materials. were used in traditional Chinese medicine. With the development of science and technology, the value of mulberry will receive a large extent of development and utilization; therefore, breeding new mulberry varieties is one of the most important tasks for researchers. Mulberry traditional breeding, such as selective breeding and cross breeding, can not meet the needs of mulberry breeding. With the development of biotechnology, molecular breeding has become an important direction in mulberry breeding. Many studies showed that the transfer of exogenous genes into mulberry is feasible. However, the synthesis of suitable exogenous gene, the improvement of their expression quantity and the look for suitable transformation method are still problematic. Researchers have heavy responsibilities about mulberry genetic transformation.
Monellin is one of the intensely sweet proteins present in the fruit of West African plant Dioscoreophyllum cumminsii. This natural protein would lose sweetness at high temperatures under acidic pH. Its development and production were affected by the origin or resource, and very difficult to scale. Therefore, the researchers synthesized a monellin gene, which has been expressed in different expression systems, such as E.coli system,B. Subtilis system,S. cerevisiae system,and P. pastoris system. In1997, the single-chain monellin was found to be expressed at a high level in the food yeast Candida utilis, and it only maintains its stability after incubation at60℃for10minutes. Only in1992, monellin was transformed into tomato and lettuce, but it did not show sweet taste.
To improve the expression quantity and thermal stability of Monellin, to transfer and express monellin gene into mulberry, this study has carried out around these problems and then the systematic deep research has been done completely based on reliability. We had achieved good results, and the main points were briefly summed up as follows:
1. Optimization and synthesis of special monellin gene
Firstly, the codon usage frequency and preference index of Pichia pastoris and Morus alba L.were statistical analysised from codon usage database. According to the amino acid sequence of the single-chain monellin which was reported, we designed a fantastic monellin gene by using the codon bias of Pichia pastoris and Morus alba L.. This nucleotide sequence of the monellin gene was analyzed using NCBI Blast, the result showed that this is a new gene. Then This gene was then synthesized by Genscript. The NCBI accession number of this new synthetic gene is JQ282905.
2. Expression of a fantastic monellin gene in Pichia pastoris and its characterization
In this study, the exocytosis Pasteur pichia GS115was selected for host bacterium. pPIC9K was the foundational vector. On that basis, two expression vectors named pPIC9K-M-E and pPIC9K-M were constructed. Expression vector pPIC9K-M-E contained a monellin gene and an enhanced green fluorescent protein (EGFP) gene. The pPIC9K-M only contains a monellin gene. But both vectors have taken a histidine label for separation and purifycation of the protein. The expression vectors were linearized and transformed into P. pastoris GS115strain by electroporation. The positive strains of yeast expression GS115/pPIC9K-M and GS115/pPIC9K-M-E were obtained by colony PCR or flat screening; the positive strain was cultured in BMGY culture medium and induced by methanol. Furthermore, the recombinant protein was purified by Ni-NTA affinity chromatography.
Results showed that the object proteins were successfully expressed in P.pastoris. By a taste test, results showed that the expressed proteins had intense sweetness and it is almost500times sweeter than sucrose by weight. From the fluorescence detection, results demonstrated that the recombinant protein produced by GS115/pPIC9K-M-E strain was easily detectable by its intense fluorescence. The protein was considerably stable between37℃to70℃, and at pH3to pH12. These characterizations made its application of great wide. This fantastic gene was included two species codon bias, thus it can be applied in fermentation industry, also can be used in breeding of mulberry. This monellin gene had obtained the patent of invention in China in2012, and its publicity number is ZL201010042099.3. The gene is a meaningful gene for mulberry and crop genetic transformation.
3. Study on monellin gene transformation in mulberry by agrobacterium-mediated method
In the Agrobacterium-mediated method, mulberry tissue culture is the foundation, and how to quickly get explant is one of the most important links. In order to obtain large number of cotyledons and hypocotyls from germinated mulberry seeds as explants for mulberry tissue culture, the seeds were germinated by shaking cultural. After that, we observed seed germination and measured variations of endogenous phytohormone content and SOD(superoxide dismutase) activity during germination of the seeds under static and shaking cultural conditions. The results showed that the seeds germinated quickly and uniformly under shaking condition. In shaking condition, the seed germination rate was as high as99%, being9percentage points higher over the static condition respectively. SOD activity and endogenous phytohormone content of mulberry seeds during germination were very different between shaking condition and static condition. After analysis, we think the shaking condition is a good way to get explants.
In this study, the agrobacterium tumefaciens-mediated method of genetic transformation were studied. The expression vector named pCAMBIA2301-M was constructed. Using the cotyledons, hypocotyl and cluster bud of four mulberry species named "Guiyou62","Teyou2","Henan" and "Jialing30" as the receptors of transformation, the new monellin gene was transferred into the receptors. After transformation, induction, rooting and hardening of the explants,6plantlets were obtained. Half of them are "Guiyou62", and the others are "Jialing30". We found that the hypocotyl of "Guiyou62" and the cluster buds of "Jialing30" are good genetic transformation receptors.215receptors were used, and then submitted for molecular detection. Finally, we got4positive plantlets, and the genetic transformation rate was1.9%. But the genetic transformation rate of "Henan" was0. The experimental results showed that different receptors had different effect in genetic transformation.
4. Study on monellin gene transformation in mulberry by pollen-mediated method
In this test, we studied on monellin gene transformation in mulberry by pollen-mediated method. The expression vector named pCAMBIA2300-M-E was constructed. The introduction of monellin gene into the hybrid mulberry seed of Red No.1and Breeding No.2was performed using pollen-mediated method referred to Li Jun et al.. A total number of1096hybrid seeds were obtained and200seeds had been selected by kanamicin Other896seeds were cultured by shaking, then they were sowed into field. We obtained125plantlets of monellin gene transformation, and all of them were chosen for molecular detection. Finally, we got11positive plantlets, and the genetic transformation rate was1%.
All the results show that it is feasible to gene transformat into mulberry by Agrobacterium-mediated and pollen-mediated transformation.
从西非植物Dioscoreophyllum cumminsii的果实中获得的天然的甜味蛋白Monellin,具有甜度高、热量低等特点。但它的热稳定性较差,其开发生产也受到了产地和资源等因素的限制,难以规模化。因此,研究者运用生物技术的方法,合成了甜味蛋白基因,并在原核与真核表达系统中成功表达,获得的甜味蛋白在酸性环境下最高能耐受60℃高温；在1992年报道了该蛋白在植物中的应用,基因被成功导入莴苣和土豆中,但未能检测出甜味。
如何能够提高该蛋白的表达量和热稳定性,如何能够将monellin基因导入桑树中并得到表达?本研究围绕这些问题开展了较为深入系统的研究,并取得了较好的研究结果,现将研究要点总结如下：
1.一个人工设计合成的monellin基因
本研究根据已报道的Monellin甜蛋白氨基酸序列,通过统计“各物种密码子使用频率数据库codon usage database”信息,兼顾桑树与毕赤酵母密码子的偏爱性,人工设计优化了一条长为294bp的单链monellin基因,并采用重叠PCR的方法合成该基因。将合成的基因核苷酸序列在NCBI中进行比对,结果显示这是一个具有新核苷酸序列的monellin基因。我们将这一基因向NCBI进行了提交,确定基因登录号为GenBank:JQ282905。该基因如能在真核生物和植物的表达系统中表达,则为它扩展应用到其它作物中去提供了新的思路和途径。
2.新monellin基因的真核表达研究
本研究中,选取巴斯德毕赤酵母GS115为宿主菌,以胞外分泌型的载体pPIC9K为基础,构建了两个表达载体pPIC9K-M-E和pPIC9K-M。表达载体pPIC9K-M-E中包含有甜味蛋白monellin基因和绿色荧光蛋白EGFP基因,pPIC9K-M中只包含monellin基因,但它们中均在基因下游带上了组氨酸标签以方便蛋白的分离纯化。构建好的表达载体经线性化后,采用电击法转化入毕赤酵母GS115,经菌落PCR和平板筛选分别获得了阳性酵母表达菌株GS115/pPIC9K-M和GS115/pPIC9K-M-E,利用含G418的平板筛选出基因多拷贝酵母菌株后,在BMGY和BMMY培养基中利用甲醇诱导获得了表达上清。然后采用Ni-NTA亲和柱纯化表达的蛋白,经分析验证两种蛋白均有甜味,约为标准蔗糖的500倍,甜味刺激来的慢去的慢；诱导重组菌GS115/pPIC9K-M-E产生的蛋白在荧光显微镜下能够发出强烈的荧光,诱导重组子GS115/pPIC9K-M获得的蛋白产物能耐80℃高温,pH值在3-12之间时,蛋白降解较少。这些结果证明我们获得的蛋白不但具有甜味,还具有有相对较高的对热稳定性,也有较宽的耐酸碱性。这为它的开发利用提供了条件。由此,我们将获得的monellin基因向国家知识产权局申请了国家发明专利,并于2012年5月获正式授权,专利号为ZL201010042099.3。该基因的获得为桑树及作物遗传转化创新了一个有意义的目的基因。
3.利用根癌农杆菌介导法研究monellin基因对桑树的遗传转化
在农杆菌介导法中需要大量的植物受体材料,为了高效获取组培所需的子叶、下胚轴等外植体,本研究特别探索了一种快速促进桑种子萌发的方法-振荡培养法。实验并分析了静置培养和振荡培养2种培养方式处理下,桑种子萌发状态及萌发过程中内源激素含量和超氧化物歧化酶(SOD)活性的变化。结果显示：振荡培养的桑种子萌发快而整齐,萌发率高达99%,比静置培养分别高9个百分点。经测定,桑种子萌发过程中振荡培养组的SOD活性高于静置培养组。内源激素测定结果显示,IAA和GA均参与了桑种子的萌发过程,且相对较低浓度的IAA和较高浓度的GA能促进桑种子的萌发；当GA含量远高于ABA含量时,能大大促进桑种子的萌发与幼苗的生长。上述实验结果表明,振荡培养比静置培养方式不仅可以提前2天以上获得萌发桑苗,缩短桑树组培时间,还能减少外植体被真菌等感染的机率。
本研究采用根癌农杆菌介导法和花粉介导法对monellin基因在桑树中的遗传转化进行了研究。实验中,构建了植物表达载体pCAMBIA2301-M,以“桂优62号”、“特优2号”、“河南桑”和“嘉陵30号”桑树品种的子叶、下胚轴和丛生芽为遗传转化的受体,经基因转化、诱导培养、生根炼苗等获得了6株基因转化苗,其中“嘉陵30号”和“桂优62号”各3株。经过实验发现,“嘉陵30号”丛生芽和“桂优62号”下胚轴是较好的遗传转化受体,其中共转化215个受体材料,经鉴定获得4株阳性转化苗,转化率为1.9%；而以“河南桑”的组培外植体作为遗传转化受体没有获得阳性转化苗。实验表明,不同品种资源的桑材料用作转化受体时,其遗传转化效果存在较大差异。实验也表明根癌农杆菌介导基因转化桑树是可行的。
4.利用花粉介导法研究monellin基因对桑树的遗传转化
本研究借鉴研究室李军等的花粉介导法将载体pCAMBIA2300-M-E导入“红果1号”和“育2号”杂交桑种子中,共收获1096粒种子,其中200粒种子在萌发期用卡那霉素筛选,获得19株桑苗移栽大田；另外896粒桑种,经振荡培养后播种至大田精心管理。最终,花粉介导法共获得125株遗传转化实生苗,经鉴定有11株阳性转化苗,转化率为1%。这说明本研究采用的花粉介导法也是可行的遗传转化方法。
Mulberry Morus alba L. is a perennial woody dicotyledonous plants, and it is also a kind of important economic tree species in China. Each part of mulberry plant is a treasure:mulberry leaves are used as the natural forage for silkworm Bombyx mori L. and livestock; Mulberry fruits are rich in anthocyanin and other functional substances, so more and more people adore them; Mulberry roots, barks and ramulus mori as the kind of vegetation medicinal materials. were used in traditional Chinese medicine. With the development of science and technology, the value of mulberry will receive a large extent of development and utilization; therefore, breeding new mulberry varieties is one of the most important tasks for researchers. Mulberry traditional breeding, such as selective breeding and cross breeding, can not meet the needs of mulberry breeding. With the development of biotechnology, molecular breeding has become an important direction in mulberry breeding. Many studies showed that the transfer of exogenous genes into mulberry is feasible. However, the synthesis of suitable exogenous gene, the improvement of their expression quantity and the look for suitable transformation method are still problematic. Researchers have heavy responsibilities about mulberry genetic transformation.
Monellin is one of the intensely sweet proteins present in the fruit of West African plant Dioscoreophyllum cumminsii. This natural protein would lose sweetness at high temperatures under acidic pH. Its development and production were affected by the origin or resource, and very difficult to scale. Therefore, the researchers synthesized a monellin gene, which has been expressed in different expression systems, such as E.coli system,B. Subtilis system,S. cerevisiae system,and P. pastoris system. In1997, the single-chain monellin was found to be expressed at a high level in the food yeast Candida utilis, and it only maintains its stability after incubation at60℃for10minutes. Only in1992, monellin was transformed into tomato and lettuce, but it did not show sweet taste.
To improve the expression quantity and thermal stability of Monellin, to transfer and express monellin gene into mulberry, this study has carried out around these problems and then the systematic deep research has been done completely based on reliability. We had achieved good results, and the main points were briefly summed up as follows:
1. Optimization and synthesis of special monellin gene
Firstly, the codon usage frequency and preference index of Pichia pastoris and Morus alba L.were statistical analysised from codon usage database. According to the amino acid sequence of the single-chain monellin which was reported, we designed a fantastic monellin gene by using the codon bias of Pichia pastoris and Morus alba L.. This nucleotide sequence of the monellin gene was analyzed using NCBI Blast, the result showed that this is a new gene. Then This gene was then synthesized by Genscript. The NCBI accession number of this new synthetic gene is JQ282905.
2. Expression of a fantastic monellin gene in Pichia pastoris and its characterization
In this study, the exocytosis Pasteur pichia GS115was selected for host bacterium. pPIC9K was the foundational vector. On that basis, two expression vectors named pPIC9K-M-E and pPIC9K-M were constructed. Expression vector pPIC9K-M-E contained a monellin gene and an enhanced green fluorescent protein (EGFP) gene. The pPIC9K-M only contains a monellin gene. But both vectors have taken a histidine label for separation and purifycation of the protein. The expression vectors were linearized and transformed into P. pastoris GS115strain by electroporation. The positive strains of yeast expression GS115/pPIC9K-M and GS115/pPIC9K-M-E were obtained by colony PCR or flat screening; the positive strain was cultured in BMGY culture medium and induced by methanol. Furthermore, the recombinant protein was purified by Ni-NTA affinity chromatography.
Results showed that the object proteins were successfully expressed in P.pastoris. By a taste test, results showed that the expressed proteins had intense sweetness and it is almost500times sweeter than sucrose by weight. From the fluorescence detection, results demonstrated that the recombinant protein produced by GS115/pPIC9K-M-E strain was easily detectable by its intense fluorescence. The protein was considerably stable between37℃to70℃, and at pH3to pH12. These characterizations made its application of great wide. This fantastic gene was included two species codon bias, thus it can be applied in fermentation industry, also can be used in breeding of mulberry. This monellin gene had obtained the patent of invention in China in2012, and its publicity number is ZL201010042099.3. The gene is a meaningful gene for mulberry and crop genetic transformation.
3. Study on monellin gene transformation in mulberry by agrobacterium-mediated method
In the Agrobacterium-mediated method, mulberry tissue culture is the foundation, and how to quickly get explant is one of the most important links. In order to obtain large number of cotyledons and hypocotyls from germinated mulberry seeds as explants for mulberry tissue culture, the seeds were germinated by shaking cultural. After that, we observed seed germination and measured variations of endogenous phytohormone content and SOD(superoxide dismutase) activity during germination of the seeds under static and shaking cultural conditions. The results showed that the seeds germinated quickly and uniformly under shaking condition. In shaking condition, the seed germination rate was as high as99%, being9percentage points higher over the static condition respectively. SOD activity and endogenous phytohormone content of mulberry seeds during germination were very different between shaking condition and static condition. After analysis, we think the shaking condition is a good way to get explants.
In this study, the agrobacterium tumefaciens-mediated method of genetic transformation were studied. The expression vector named pCAMBIA2301-M was constructed. Using the cotyledons, hypocotyl and cluster bud of four mulberry species named "Guiyou62","Teyou2","Henan" and "Jialing30" as the receptors of transformation, the new monellin gene was transferred into the receptors. After transformation, induction, rooting and hardening of the explants,6plantlets were obtained. Half of them are "Guiyou62", and the others are "Jialing30". We found that the hypocotyl of "Guiyou62" and the cluster buds of "Jialing30" are good genetic transformation receptors.215receptors were used, and then submitted for molecular detection. Finally, we got4positive plantlets, and the genetic transformation rate was1.9%. But the genetic transformation rate of "Henan" was0. The experimental results showed that different receptors had different effect in genetic transformation.
4. Study on monellin gene transformation in mulberry by pollen-mediated method
In this test, we studied on monellin gene transformation in mulberry by pollen-mediated method. The expression vector named pCAMBIA2300-M-E was constructed. The introduction of monellin gene into the hybrid mulberry seed of Red No.1and Breeding No.2was performed using pollen-mediated method referred to Li Jun et al.. A total number of1096hybrid seeds were obtained and200seeds had been selected by kanamicin Other896seeds were cultured by shaking, then they were sowed into field. We obtained125plantlets of monellin gene transformation, and all of them were chosen for molecular detection. Finally, we got11positive plantlets, and the genetic transformation rate was1%.
All the results show that it is feasible to gene transformat into mulberry by Agrobacterium-mediated and pollen-mediated transformation.
引文
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