抗双翅目Bt杀虫基因GFMcry11B的人工合成、原核表达及植物转基因的研究
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摘要
稻瘿蚊是我国华南稻区近年来危害越来越严重的水稻双翅目害虫，它属钻蛀害虫，繁殖速度快，化学杀虫剂防治难度较大，并且污染环境。目前常规育种由于受到种质资源稀少和稻瘿蚊生物型发展的限制，还未很好的解决其抗性问题。利用基因工程技术把具有杀稻瘿蚊活性的Bt杀虫晶体蛋白基因转入水稻，培育抗稻瘿蚊水稻新品种成为控制其危害的一条全新途径。
大量的研究表明，将野生型Bt基因直接转入到水稻中后，在水稻中表达量很低甚至不表达，而不具有抗虫育种的实际效果。本研究有目的的利用水稻的偏好密码子对原始Bt基因密码子进行了优化，调整并去除会影响mRNA稳定的序列元件，以期获得高抗双翅目并适合于在水稻中高效表达的新基因，为抗双翅目昆虫的植物基因工程的开展打下基础。其主要工作结果如下：
1．设计并人工合成Bt GFMcryllB杀虫基因。在保证野生型基因氨基酸序列不变的前提下，用水稻的偏好密码子对核苷酸序列进行优化，去掉了原有基因的44处PPSS信号序列、1处内含子切割序列和4处ATTTA序列，AT富集区(≥4个A／T)也由野生型的66处减少到改造后的16处。使G+C含量由原来的32.96％提高到49.43％，达到了植物基因的GC含量水平。改造后的基因与天然的cryllBal基因保持了74％的核苷酸序列同源性，总共修改了725个密码子中的477个，改变幅度达到65.8％。用连续延伸PCR法扩增出了全长的基因，克隆到通用载体pBluescriptⅡSK上，测序结果和预期设计的序列完全一致。
2．构建了该杀虫基因的原核表达载体pGC，在E.coli中获得了高效表达的融合蛋白，并利用亲和层析的方法成功地对其进行了纯化，纯化的蛋白大小与预期一致。
3．完成了双子叶植物表达载体pCSCH的构建，用于烟草的转化。
4．利用纯化的目的蛋白进行兔免疫，得到相应的多克隆抗体，经间接ELISA法检测其效价为1：8000，可以用于转基因植物的ELISA法检测。
5．获得了转GFMcryllB基因的烟草，并对其进行了分子水平和蛋白水平的检测。结果表明，合成的基因已经整合到烟草的基因组中，转基因植株所表达Bt蛋白含量占烟草叶片中可溶性总蛋白含量的0.001％～0.21％。
Rice gall midge (Orseclia oryzae) is an important pest of rice(Oryza saliva L.) in Asia. The larva bores into a rice tiller and eats away its host from the inside.Infestation in a field of susceptible variety can result in complete yield loss. Controlling this pest is very difficult by chemical pesticide and easy to cause environmental pollution. Planting resistant varieties has been considered as the most effective measure for protection against this pest. The practice of regular breeding has not significantly solving the problem of insect-resistance due to lack of germplast resource and limitation of biotype developing. The application of dipteran-specific Bt toxin proteins by means of the technique of genetic engineering in transgenic rice plants provides a new way of controlling this pests.
Poor expression in plants is a well-reported characteristic of wild type Bt insect control proteins results in unsuccessfully commercial use. To increase expression of Bt genes and obtain insect resistance against dipteran-insect hi plants, codon usage of wild type Bt genes is optimized and modified according to plant preferred codons and instability sequence elements is regulated or removed purposefully for efficient plant expression. The main results are summarized as follows:
1. GFMcryllB insecticidal protein gene was designed and synthesized. Modifications and optimization in nucleotide sequence of cryllBal gene did not alter the amino acid sequence of the CryllBal protein. 44 of potential polyadneylylation signal sequence, one of intron cleavage sequence and 4 of ATTTA sequence were removed entirely. A+T rich regions (>4 of consecutive A/T) were decreased from 66 to 16 during GFMcryllB designed. In comparison with the wild type cryllBal gene, the ratio of GC content was increased from 32.96% of the wild type to 49.43% of the synthetic gene, a ratio more close to that of most plant genes. There are 65.8% of codons in wild type gene not suitable for expression in plant were modified to plant favorable condons without amino acid change. The synthesized GFMcryllB Bt gene using successive PCR was cloned into pBluescript IISK vector. Results of sequencing for recombinant plasmid were correct completely.
2. Prokaryotic fusion gene expression vector, pGC, was constructed. A high level of expression of fusion protein in E.coli was detected after IPTG induction and purified proteins were obtained by affinity chromatography with Glutathione Sepharose4B.
3. The purified GFMCryllB proteins were mixed with Freund's complete or incomplete adjuvant as antigen to immune rabbits. ELISA assay revealed that the titer of the prepared antiserum against GFMCryllB protein was as high as 1:8000. Purified IgG labeled with Alkaline Phosphatase by glutaraldehyde one-step method was used as enzyme-linked immunosorbent assay to detect content and level of GFMcryllB gene expression in transgenic plants.
4. Synthesized GFMcryllB gene was cloned into plant expression vector. Recombinant plant expression vector was transferred into tobacco (Nicotiana tabacum L.) plant via Agrobacterium-mediated transformation of tobacco leaf disks.
5. Integration and expression of foreign gene were detected in a number of transgenic tobacco plants by southern blot analysis and ELISA analysis. The results indicated that synthesized GFMcryllB gene was integrated into tobacco genome, expression level of the modified Bt gene reached 0.001%-0.21% of overall soluble proteins.
大量的研究表明，将野生型Bt基因直接转入到水稻中后，在水稻中表达量很低甚至不表达，而不具有抗虫育种的实际效果。本研究有目的的利用水稻的偏好密码子对原始Bt基因密码子进行了优化，调整并去除会影响mRNA稳定的序列元件，以期获得高抗双翅目并适合于在水稻中高效表达的新基因，为抗双翅目昆虫的植物基因工程的开展打下基础。其主要工作结果如下：
1．设计并人工合成Bt GFMcryllB杀虫基因。在保证野生型基因氨基酸序列不变的前提下，用水稻的偏好密码子对核苷酸序列进行优化，去掉了原有基因的44处PPSS信号序列、1处内含子切割序列和4处ATTTA序列，AT富集区(≥4个A／T)也由野生型的66处减少到改造后的16处。使G+C含量由原来的32.96％提高到49.43％，达到了植物基因的GC含量水平。改造后的基因与天然的cryllBal基因保持了74％的核苷酸序列同源性，总共修改了725个密码子中的477个，改变幅度达到65.8％。用连续延伸PCR法扩增出了全长的基因，克隆到通用载体pBluescriptⅡSK上，测序结果和预期设计的序列完全一致。
2．构建了该杀虫基因的原核表达载体pGC，在E.coli中获得了高效表达的融合蛋白，并利用亲和层析的方法成功地对其进行了纯化，纯化的蛋白大小与预期一致。
3．完成了双子叶植物表达载体pCSCH的构建，用于烟草的转化。
4．利用纯化的目的蛋白进行兔免疫，得到相应的多克隆抗体，经间接ELISA法检测其效价为1：8000，可以用于转基因植物的ELISA法检测。
5．获得了转GFMcryllB基因的烟草，并对其进行了分子水平和蛋白水平的检测。结果表明，合成的基因已经整合到烟草的基因组中，转基因植株所表达Bt蛋白含量占烟草叶片中可溶性总蛋白含量的0.001％～0.21％。
Rice gall midge (Orseclia oryzae) is an important pest of rice(Oryza saliva L.) in Asia. The larva bores into a rice tiller and eats away its host from the inside.Infestation in a field of susceptible variety can result in complete yield loss. Controlling this pest is very difficult by chemical pesticide and easy to cause environmental pollution. Planting resistant varieties has been considered as the most effective measure for protection against this pest. The practice of regular breeding has not significantly solving the problem of insect-resistance due to lack of germplast resource and limitation of biotype developing. The application of dipteran-specific Bt toxin proteins by means of the technique of genetic engineering in transgenic rice plants provides a new way of controlling this pests.
Poor expression in plants is a well-reported characteristic of wild type Bt insect control proteins results in unsuccessfully commercial use. To increase expression of Bt genes and obtain insect resistance against dipteran-insect hi plants, codon usage of wild type Bt genes is optimized and modified according to plant preferred codons and instability sequence elements is regulated or removed purposefully for efficient plant expression. The main results are summarized as follows:
1. GFMcryllB insecticidal protein gene was designed and synthesized. Modifications and optimization in nucleotide sequence of cryllBal gene did not alter the amino acid sequence of the CryllBal protein. 44 of potential polyadneylylation signal sequence, one of intron cleavage sequence and 4 of ATTTA sequence were removed entirely. A+T rich regions (>4 of consecutive A/T) were decreased from 66 to 16 during GFMcryllB designed. In comparison with the wild type cryllBal gene, the ratio of GC content was increased from 32.96% of the wild type to 49.43% of the synthetic gene, a ratio more close to that of most plant genes. There are 65.8% of codons in wild type gene not suitable for expression in plant were modified to plant favorable condons without amino acid change. The synthesized GFMcryllB Bt gene using successive PCR was cloned into pBluescript IISK vector. Results of sequencing for recombinant plasmid were correct completely.
2. Prokaryotic fusion gene expression vector, pGC, was constructed. A high level of expression of fusion protein in E.coli was detected after IPTG induction and purified proteins were obtained by affinity chromatography with Glutathione Sepharose4B.
3. The purified GFMCryllB proteins were mixed with Freund's complete or incomplete adjuvant as antigen to immune rabbits. ELISA assay revealed that the titer of the prepared antiserum against GFMCryllB protein was as high as 1:8000. Purified IgG labeled with Alkaline Phosphatase by glutaraldehyde one-step method was used as enzyme-linked immunosorbent assay to detect content and level of GFMcryllB gene expression in transgenic plants.
4. Synthesized GFMcryllB gene was cloned into plant expression vector. Recombinant plant expression vector was transferred into tobacco (Nicotiana tabacum L.) plant via Agrobacterium-mediated transformation of tobacco leaf disks.
5. Integration and expression of foreign gene were detected in a number of transgenic tobacco plants by southern blot analysis and ELISA analysis. The results indicated that synthesized GFMcryllB gene was integrated into tobacco genome, expression level of the modified Bt gene reached 0.001%-0.21% of overall soluble proteins.
引文
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