高山离子芥BADH基因和LEA基因的克隆、表达及功能分析
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摘要
高山离子芥(Chorispora bungeana Fisch．& C．A．Mey)是一种稀有的高山冰缘植物，具有很强抗寒能力，但它不具备特殊的形态结构，被推测它在生理和分子水平上具有特殊的抗冻机制。因此，高山离子芥是研究植物适应冷冻环境和克隆植物抗逆基因的理想材料。本文以高山离子芥为实验材料，克隆到甜菜碱醛脱氢酶基因(CbBADH)和胚胎发生晚期丰富蛋白基因(CbLEA)。对CbBADH基因在不同处理条件下的基因表达模式和甜菜碱的积累进行了研究；通过农杆菌介导的叶盘法将CbLEA基因成功地转化了烟草，并对转基因烟草的抗逆性和CbLEA蛋白的功能进行了分析，内容概括如下：
1．从高山离子芥中克隆了甜菜碱醛脱氢酶基因(CbBADH)，此基因全长1729bp，编码502个氨基酸的蛋白，分子量为54.5KD，等电点为5.30。氨基酸序列分析表明，CbBADH蛋白中含有与酶活性位点和结合位点相关的十肽VSMELGGKSP。CbBADH蛋白与其他已经发现的BADH蛋白的同源性在73.3％～95.4％之间，其中与拟南芥的同源性最高，达到了95.4％，说明克隆到的基因属于BADH基因家族。
2．外源表达和纯化的CbBADH蛋白具有氧化甜菜碱醛的能力，并且在不同的pH值下其酶活性不同，pH 8.0时活性最大。证明CbBADH基因确实是甜菜碱醛脱氢酶的功能基因，其编码的蛋白能催化甜菜碱醛合成甜菜碱，其酶活性不因保守十肽中的L被M代替而受到影响。
3．检测CbBADH基因在冷冻胁迫(4℃、0℃和-4℃)下的表达情况，发现它的转录物在4℃逐渐积累，而在0℃和-4℃的积累更直接和迅速。这表明CbBADH基因的表达与温度密切相关、对温度反应敏感。所以CbBADH基因的表达是高山离子芥传递抵御低温胁迫信号的一个重要保护机制。随着胁迫强度的增大，植物启动这个保护机制就越迅速。这恰恰就反映了高山离子芥具有适应温度剧烈变化的能力。
4．高山离子芥是甜菜碱积累型的植物，在低温胁迫下能够迅速积累甜菜碱以调节细胞的渗透势。在温度和时间梯度上，随着温度的降低甜菜碱的积累迅速增加，并且甜菜碱的积累并不是不断增加的，而是有一定的限度的(在低温胁迫下它增加了3倍)，当达到一定的浓度时，甜菜碱含量就维持在相对稳定的水平。这一结果表明在高山离子芥中，甜菜碱的渗透调节具有一定的局限性，当胁迫强度超过一定范围，它的调节能力不再增强。同时表明，渗透调节只是高山离子芥低温保护机制的一种，但并不是最主要的，在高山离子芥中可能存在其他特殊的低温保护机制。
5．从高山离子芥中克隆了胚胎发生晚期丰富蛋白基因(CbLEA)，此基因全长842bp，编码169个氨基酸的小分子蛋白，其分子量为17.9KD，等电点为6.45。亲水性分析表明该蛋白富含亲水氨基酸，是一种高度亲水性的蛋白，其蛋白二级结构中只含有83.43％的α-螺旋占和16.57％的无规卷曲。CbLEA蛋白除了与拟南芥的LEA蛋白同源性为87.57％外，与其他已经发现的LEA蛋白的同源性都不高，一般在35.84％～52.78％之间，说明克隆到的CbLEA蛋白是一种新的LEA蛋白。
6．通过农杆菌介导法将CbLEA基因转化烟草，经PCR、Real time RT-PCR和Western blot确定了阳性转基因植株，并且检测了转基因植株中CbLEA蛋白的积累量。发现各转基因株系地CbLEA蛋白积累量不同，这就为研究CbLEA蛋白在植物抗逆过程中的作用提供了很好的研究对象。
7．通过对转基因烟草的T_1代幼苗进行生理测定和生长分析发现，在盐和低温胁迫下转基因植株的生长状况都较野生型植株好，转基因植株的种子萌发率、相对含水量和叶绿素含量都比野生型植株高，而其离子渗漏率、丙二醛含量又明显的低于野生型植株。转基因烟草细胞的半致死温度为-3℃，而野生型的仅为-2℃，并且在-2℃的低温下，转基因烟草存活时间较野生型要长。这说明CbLEA是一种真正的抗逆基因，转基因烟草的抗盐和抗冻性明显得到提高。
8．进一步研究发现，转基因烟草抗逆性提高的程度似乎与CbLEA蛋白的积累量呈正相关，CbLEA蛋白积累量越大，转基因烟草的抗逆性越强，反之亦然。这一结果更加充分地证明CbLEA蛋白在胁迫条件下对植物细胞起保护作用。
综上所述，高山离子芥中也存在甜菜碱的渗透调节途径，说明植物在抗寒分子机制方面具有保守性，克隆的基因具有潜在的抗寒功能；同时，甜菜碱的渗透调节具有一定的局限性，渗透调节只是高山离子芥低温保护机制的一种，但并不是最主要的，在高山离子芥中可能存在其他特殊的低温保护机制。转基因植物的抗逆性分析表明高山离子芥CbLEA蛋白在胁迫条件下可能通过结合水分子、维持膜结构而达到保护细胞的作用，高山离子芥的抗逆基因较其它植物的抗逆基因而言在抗冻方面似乎具有显著的优势。
Chorispora bungeana Fisch. & C.A. Mey is a rare and typical alpine subnival plant species that is highly capable of resisting freezing environment. However, it does not possess special morphological characteristics that helped it surviving under freezing environment, physiological and molecular mechanisms were assumed to account for its adaptation to the freezing environment. Therefore, it is a valuable species for stress-related genes cloning and research on the mechanism of cold resistance. In our work, BADH gene (CbBADH) and LEA gene (CbLEA) were cloned from C. bungeana. The pattern of CbBADH expression and accumulation of betaine under various treatments were investigated; the function of CbLEA protein in stress protection was investigated by transgenic approach. The summary is as follows:
1. CbBADH gene is 1729 bp in length with an open reading frame (ORF) of 502 amino acids, corresponding to a protein of predicted molecular mass 54.5 KD and an isoelectric point of 5.30. The deduced amino acid sequence possesses a decapeptide (VSMELGGKSP), which is involved in the enzyme active site and NAD~+ binding. The putative amino acid sequence of the CbBADH showed 73.3-95.4% similarity to the BADH of other plants. All of these indicate that CbBADH gene is the homologus gene of BADH.
2. CbBADH was expressed in and purified from Escherichia coli. Through investigation the function of the enzyme coded by CbBADH, we found that recombinant CbBADH protein showed high activity for the oxidation of betaine aldehyde, and the optimal pH of CbBADH was 8.0. These results indicated that CbBADH gene encoded a functional BADH enzyme, which could catalyze betaine aldehyde to betaine, and the enzyme activity was not affected by the substitution of M for L in the decapeptide.
3. The expression patterns of CbBADH gene were investigated under cold stress. The CbBADH gene transcript progressively increased under 4°C and increased much more distinctly and quickly under 0°C and -4℃. This result suggests there is a close relationship between the expression of CbBADH and cold stress, CbBADH expression is sensitive to low temperature. CbBADH plays an important protective mechanism in signal transduction to resist cold stress in C bungeana. Lower temperature could trigger the expression more quickly exactly, this phenomenon reflects fitly that C. bungeana has the ability to adapt to acute temperature change in the growth environment.
4. Like other higher plants, C. bungeana accumulated betaine in response to low temperature stress. Lower temperature could trigger the accumulation more quickly exactly. Moreover, the increase in the endogenous betaine levels in C. bungeana seemed to have a maximum level, which increased 3-fold, under different low temperature stresses. These results implied that the accumulation of betaine was one but not the main protective mechanisms in C. bungeana to resist cold stress, other special cold-hardiness mechanism still existed in C. bungeana
5. CbLEA gene is 842 bp in length with an open reading frame (ORF) of 169 amino acids, corresponding to a protein of predicted molecular mass 17.9 KD and an isoelectric point of 6.45. Hydrophilicity prediction shows CbLEA is a highly hydrophilic small protein. The secondary structure analysis shows CbLEA protein contains 83.43% alpha helix and 16.57% random coil. CbLEA protein shares lower similarity with LEAs from other plants (35.84%~52.78%) except A.thalian, indicating that CbLEA is a novel LEA gene.
6. CbLEA gene was over-expressed in tobacco by Agrobacterium-mediated leaf disc transformation with a construct containing the CbLEA ORF under control of CaMV 35S promoter. The transgenic tobacco plants were confirmed by polymerase chain reaction, real time RT-PCR and Western bolt analysis. To investigate the function CbLEA protein, the three transgenic lines were chosen to test whether over-expression of the CbLEA protein increased their ability for tolerance against stress, based on accumulated amounts of CbLEA protein.
7. Under stress condition, the transgenic T_1 plants show better performance than non-transgenic plants, and maintaine higher RWC and chlorophyll content, less MDA and EL, when compared with non-transgenic plants. Moreover, the LT50 (the killing temperature for 50% of the cells) values was significantly altered in transgenic plant, which was declined to -3°C, the LT50 value is -2°C in non-transgenic plants, and the transgenic plants survived longer time than non-transgenic plants at the constant freezing treatment of -2°C. These results indicated that CbLEA gene was an excellent stress-tolerance gene, and the transgenic tobacco plants exhibited significantly increased tolerance to salinity and cold.
8. Furthermore, the extent of increased stress tolerance shows a positive correlation to accumulation level of the CbLEA protein, indicating that the CbLEA gene product function in cell protection under stress condition.
In conclusion, the accumulation of betaine was also found in C. bungeana, indicating that there is a conservative mechanism in plant cold-resisting process. However, the increase in the endogenous betaine levels in C. bungeana seemed to have a maximum level under cold stress, and the accumulation of betaine was one but not the main protective mechanisms in C. bungeana to resist cold stress, other special cold-hardiness mechanism still existed in C. bungeana. Tthe analysis of stress tolerance in transgenic plant show that the CbLEA protein may protein cell through binding water, maintaining membrane structure, the stress-tolerance gene from C. bungeana seem to be more efficient than the stress-tolerance gene from other plant in cold tolerance.
1．从高山离子芥中克隆了甜菜碱醛脱氢酶基因(CbBADH)，此基因全长1729bp，编码502个氨基酸的蛋白，分子量为54.5KD，等电点为5.30。氨基酸序列分析表明，CbBADH蛋白中含有与酶活性位点和结合位点相关的十肽VSMELGGKSP。CbBADH蛋白与其他已经发现的BADH蛋白的同源性在73.3％～95.4％之间，其中与拟南芥的同源性最高，达到了95.4％，说明克隆到的基因属于BADH基因家族。
2．外源表达和纯化的CbBADH蛋白具有氧化甜菜碱醛的能力，并且在不同的pH值下其酶活性不同，pH 8.0时活性最大。证明CbBADH基因确实是甜菜碱醛脱氢酶的功能基因，其编码的蛋白能催化甜菜碱醛合成甜菜碱，其酶活性不因保守十肽中的L被M代替而受到影响。
3．检测CbBADH基因在冷冻胁迫(4℃、0℃和-4℃)下的表达情况，发现它的转录物在4℃逐渐积累，而在0℃和-4℃的积累更直接和迅速。这表明CbBADH基因的表达与温度密切相关、对温度反应敏感。所以CbBADH基因的表达是高山离子芥传递抵御低温胁迫信号的一个重要保护机制。随着胁迫强度的增大，植物启动这个保护机制就越迅速。这恰恰就反映了高山离子芥具有适应温度剧烈变化的能力。
4．高山离子芥是甜菜碱积累型的植物，在低温胁迫下能够迅速积累甜菜碱以调节细胞的渗透势。在温度和时间梯度上，随着温度的降低甜菜碱的积累迅速增加，并且甜菜碱的积累并不是不断增加的，而是有一定的限度的(在低温胁迫下它增加了3倍)，当达到一定的浓度时，甜菜碱含量就维持在相对稳定的水平。这一结果表明在高山离子芥中，甜菜碱的渗透调节具有一定的局限性，当胁迫强度超过一定范围，它的调节能力不再增强。同时表明，渗透调节只是高山离子芥低温保护机制的一种，但并不是最主要的，在高山离子芥中可能存在其他特殊的低温保护机制。
5．从高山离子芥中克隆了胚胎发生晚期丰富蛋白基因(CbLEA)，此基因全长842bp，编码169个氨基酸的小分子蛋白，其分子量为17.9KD，等电点为6.45。亲水性分析表明该蛋白富含亲水氨基酸，是一种高度亲水性的蛋白，其蛋白二级结构中只含有83.43％的α-螺旋占和16.57％的无规卷曲。CbLEA蛋白除了与拟南芥的LEA蛋白同源性为87.57％外，与其他已经发现的LEA蛋白的同源性都不高，一般在35.84％～52.78％之间，说明克隆到的CbLEA蛋白是一种新的LEA蛋白。
6．通过农杆菌介导法将CbLEA基因转化烟草，经PCR、Real time RT-PCR和Western blot确定了阳性转基因植株，并且检测了转基因植株中CbLEA蛋白的积累量。发现各转基因株系地CbLEA蛋白积累量不同，这就为研究CbLEA蛋白在植物抗逆过程中的作用提供了很好的研究对象。
7．通过对转基因烟草的T_1代幼苗进行生理测定和生长分析发现，在盐和低温胁迫下转基因植株的生长状况都较野生型植株好，转基因植株的种子萌发率、相对含水量和叶绿素含量都比野生型植株高，而其离子渗漏率、丙二醛含量又明显的低于野生型植株。转基因烟草细胞的半致死温度为-3℃，而野生型的仅为-2℃，并且在-2℃的低温下，转基因烟草存活时间较野生型要长。这说明CbLEA是一种真正的抗逆基因，转基因烟草的抗盐和抗冻性明显得到提高。
8．进一步研究发现，转基因烟草抗逆性提高的程度似乎与CbLEA蛋白的积累量呈正相关，CbLEA蛋白积累量越大，转基因烟草的抗逆性越强，反之亦然。这一结果更加充分地证明CbLEA蛋白在胁迫条件下对植物细胞起保护作用。
综上所述，高山离子芥中也存在甜菜碱的渗透调节途径，说明植物在抗寒分子机制方面具有保守性，克隆的基因具有潜在的抗寒功能；同时，甜菜碱的渗透调节具有一定的局限性，渗透调节只是高山离子芥低温保护机制的一种，但并不是最主要的，在高山离子芥中可能存在其他特殊的低温保护机制。转基因植物的抗逆性分析表明高山离子芥CbLEA蛋白在胁迫条件下可能通过结合水分子、维持膜结构而达到保护细胞的作用，高山离子芥的抗逆基因较其它植物的抗逆基因而言在抗冻方面似乎具有显著的优势。
Chorispora bungeana Fisch. & C.A. Mey is a rare and typical alpine subnival plant species that is highly capable of resisting freezing environment. However, it does not possess special morphological characteristics that helped it surviving under freezing environment, physiological and molecular mechanisms were assumed to account for its adaptation to the freezing environment. Therefore, it is a valuable species for stress-related genes cloning and research on the mechanism of cold resistance. In our work, BADH gene (CbBADH) and LEA gene (CbLEA) were cloned from C. bungeana. The pattern of CbBADH expression and accumulation of betaine under various treatments were investigated; the function of CbLEA protein in stress protection was investigated by transgenic approach. The summary is as follows:
1. CbBADH gene is 1729 bp in length with an open reading frame (ORF) of 502 amino acids, corresponding to a protein of predicted molecular mass 54.5 KD and an isoelectric point of 5.30. The deduced amino acid sequence possesses a decapeptide (VSMELGGKSP), which is involved in the enzyme active site and NAD~+ binding. The putative amino acid sequence of the CbBADH showed 73.3-95.4% similarity to the BADH of other plants. All of these indicate that CbBADH gene is the homologus gene of BADH.
2. CbBADH was expressed in and purified from Escherichia coli. Through investigation the function of the enzyme coded by CbBADH, we found that recombinant CbBADH protein showed high activity for the oxidation of betaine aldehyde, and the optimal pH of CbBADH was 8.0. These results indicated that CbBADH gene encoded a functional BADH enzyme, which could catalyze betaine aldehyde to betaine, and the enzyme activity was not affected by the substitution of M for L in the decapeptide.
3. The expression patterns of CbBADH gene were investigated under cold stress. The CbBADH gene transcript progressively increased under 4°C and increased much more distinctly and quickly under 0°C and -4℃. This result suggests there is a close relationship between the expression of CbBADH and cold stress, CbBADH expression is sensitive to low temperature. CbBADH plays an important protective mechanism in signal transduction to resist cold stress in C bungeana. Lower temperature could trigger the expression more quickly exactly, this phenomenon reflects fitly that C. bungeana has the ability to adapt to acute temperature change in the growth environment.
4. Like other higher plants, C. bungeana accumulated betaine in response to low temperature stress. Lower temperature could trigger the accumulation more quickly exactly. Moreover, the increase in the endogenous betaine levels in C. bungeana seemed to have a maximum level, which increased 3-fold, under different low temperature stresses. These results implied that the accumulation of betaine was one but not the main protective mechanisms in C. bungeana to resist cold stress, other special cold-hardiness mechanism still existed in C. bungeana
5. CbLEA gene is 842 bp in length with an open reading frame (ORF) of 169 amino acids, corresponding to a protein of predicted molecular mass 17.9 KD and an isoelectric point of 6.45. Hydrophilicity prediction shows CbLEA is a highly hydrophilic small protein. The secondary structure analysis shows CbLEA protein contains 83.43% alpha helix and 16.57% random coil. CbLEA protein shares lower similarity with LEAs from other plants (35.84%~52.78%) except A.thalian, indicating that CbLEA is a novel LEA gene.
6. CbLEA gene was over-expressed in tobacco by Agrobacterium-mediated leaf disc transformation with a construct containing the CbLEA ORF under control of CaMV 35S promoter. The transgenic tobacco plants were confirmed by polymerase chain reaction, real time RT-PCR and Western bolt analysis. To investigate the function CbLEA protein, the three transgenic lines were chosen to test whether over-expression of the CbLEA protein increased their ability for tolerance against stress, based on accumulated amounts of CbLEA protein.
7. Under stress condition, the transgenic T_1 plants show better performance than non-transgenic plants, and maintaine higher RWC and chlorophyll content, less MDA and EL, when compared with non-transgenic plants. Moreover, the LT50 (the killing temperature for 50% of the cells) values was significantly altered in transgenic plant, which was declined to -3°C, the LT50 value is -2°C in non-transgenic plants, and the transgenic plants survived longer time than non-transgenic plants at the constant freezing treatment of -2°C. These results indicated that CbLEA gene was an excellent stress-tolerance gene, and the transgenic tobacco plants exhibited significantly increased tolerance to salinity and cold.
8. Furthermore, the extent of increased stress tolerance shows a positive correlation to accumulation level of the CbLEA protein, indicating that the CbLEA gene product function in cell protection under stress condition.
In conclusion, the accumulation of betaine was also found in C. bungeana, indicating that there is a conservative mechanism in plant cold-resisting process. However, the increase in the endogenous betaine levels in C. bungeana seemed to have a maximum level under cold stress, and the accumulation of betaine was one but not the main protective mechanisms in C. bungeana to resist cold stress, other special cold-hardiness mechanism still existed in C. bungeana. Tthe analysis of stress tolerance in transgenic plant show that the CbLEA protein may protein cell through binding water, maintaining membrane structure, the stress-tolerance gene from C. bungeana seem to be more efficient than the stress-tolerance gene from other plant in cold tolerance.
引文
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