里氏木霉纤维素酶基因的克隆及在水稻中的过表达研究
摘要
纤维素是自然界中含量最多的一种有机化合物。开展纤维素酶生物学研究是深入了解纤维素的代谢机制和利用纤维素的基础,特别是在利用木质纤维素生产生物乙醇方面,具有重要的理论意义和应用价值。本实验从里氏木霉(Trichoderma reeseri)中克隆纤维素酶基因,并在水稻中进行过量表达研究,获得如下主要结果。
1.依据发表在GenBank中的基因序列,利用RT-PCR方法,从里氏木霉(Trichoderma reeseri)的cDNA中克隆到了完整的内切β-1,4-葡聚糖酶V(Endo-β-1,4-glucallaseV),内切β-1,4-葡聚糖酶IV(Endo-β-1,4-glucallaseIV)以及外切β-1,4-葡聚糖酶I(β-1,4-glucancellobiohydrolaseI)蛋白质编码区(coding sequence region,CDS)的cDNA。
2.克隆到的里氏木霉EGV基因蛋白质编码区全长726bp,编码242个氨基酸。核酸序列与已发表的里氏木霉AS3.3711菌株的EGV蛋白质编码区cDNA序列相比有一个核苷酸的突变,推导的氨基酸序列也有一个氨基酸的突变,但是没有明显影响到蛋白质的活性。
3.克隆到的里氏木霉EGIV基因蛋白质编码区cDNA全长1032bp,编码344个氨基酸,CBHI基因蛋白质编码区cDNA全长1542bp,编码514个氨基酸。所获得的EGIV基因和CBHI基因核酸序列和已发表的里氏木霉EGIV基因和CBHI基因的蛋白质编码序列相比完全一致。
4.将EGV基因插入到双T-DNA载体pDTB中,构建载体pDTB-HA-EGV。将合成的定位于质体的信号肽Apo-SP插入到载体pDTB-HA-EGV中,构建载体pDTB-Apo-HA-EGV,并且通过红色荧光蛋白在洋葱表皮细胞的瞬时表达验证该信号肽的功能。
5.用农杆菌介导的方法对水稻品种中花11进行转化,分别获得转化pDTB-HA-EGV载体的68个植株和转化pDTB-Apo-HA-EGV载体的97个植株。PCR检测和Southern杂交分析转基因植株,表明目的基因和抗除草剂基因均整合到水稻基因组中。通过对转基因水稻T_1代30个株系共计291棵植株的PCR检测,仅含目的基因的转基因植株27棵,占9.28%;仅含筛选标记的转基因植株20棵,占6.87%。结果表明使用双T-DNA载体共转化法去除转基因植物分子标记的策略有明显的效果。
6.通过Western杂交检测到两个转基因株系的T_0代干枯秸秆和T_1代鲜活叶片中里氏木霉EGV基因发生了表达,有一定的蛋白产物。并且利用刚果红羧甲基纤维素平板检测到它们的蛋白抽提物中存在纤维素酶酶活,证明EGV基因的表达产物发生了正确的折叠,具有一定的活性,能够在一定的条件下降解外切纤维素酶的底物羧甲基纤维素。并且,在室温储藏了3个月的T_0代秸秆中稳定存在。该结果为在作物中定向表达纤维素酶,以期利用于木质纤维素生产生物乙醇提供了一定的证据。
Cellulose is one of the most abundant organic compounds in nature. Carring out of Biology research on cellulase is the base of understanding of the metabolic mechanism of cellulose and utilization of cellulose in depth, especially in the utilization of lignocellulose to produce bioethanol, which has important theoretical significance and application value. In this research, we cloned cellulase gene from Trichoderma reeseri through RT-PCR, and overexpressed it in rice. The main results are as follows:
1. Based on the sequences of cellulase genes in GenBank, Designed specific oligonucletide primers then used the way of the reverse transcriptase-PCR (RT-PCR) to enriching the gene of Endo-β-1,4-glucallaseV, Endo-β-1,4-glucallaseIV andβ-1,4-glucancellobiohydrolaseI.
2. The full-length CDS region of Trichoderma reeseri EGV is 726bp, encoding a polypeptide of 242 amino acids. There is one base difference when compared the EGV CDS to the known sequence of Trichoderma reeseri AS 3.3711 strain EGV CDS, producing one amino acids mutation, but the activity of protein wasn’t changed.
3. The full-length CDS region of Trichoderma reeseri EGIV is1032bp, encoding a polypeptide of 344 amino acids. The full-length CDS region of Trichoderma reeseri is CBHI1542bp, encoding a polypeptide of 514 amino acids. There is no base difference when compared the Trichoderma reeseri EGIV and CBHI CDS to the known sequence respectively.
4.We inserted EGV gene into the pre-build dual T-DNA vector pDTB to construct vector pDTB-HA-EG V, in accordance with co-transformation strategy to removal of molecular markers from transgenic plants. Because of subcellular expression of foreign proteins can increase protein levels and avoid the potential hazards on host,we construct vector pDTB-HA-EGV by inserting signal peptide Apo-SP targeting to the apoplast. We also test the function of Apo-SP by transient expression in onion epidermal cells.
5. Mediated by Agrobacterium, the vector pDTB-HA-EGV and pDTB-HA-EGV were introduced into rice varieties Zhonghua11.We got 68 transgenic plants and 97 transgenic plants respectively. The transgenic plants were verified by PCR and Southern blotting, to display that objective gene and the herbicide resistant gene were integrated into the rice genome. Through PCR of 30 lines totaling 291 plants of T_1 progeny transgenic rice, there are 27 plants contain the objective gene only, accounts for 9.28%; and 20 plants contain selection marker gene only, accounts for 6.87%. The results show the significant effect of taking advantage of dual T-DNA vector to removal molecular markers in accordance with co-transformation strategy.
6. The Trichoderma reesei EGV gene has expression in fresh leaves and dry straw of T_0 progeny of T_1 progeny transgenic rice by Western blot detection. There is cellulase activity in extractive by Congo Red-CMC plate detection, which prove that EGV gene expression product took place correct folding, has some activity, can degradate cellulose substrate CMC on a certain condition. Moreover, the cellulase is stable in T_0 progeny straw after 3 months storage at room temperature. The result provided reliable evidence of subcellular expression of cellulase in corp. and taking advantage of lignocellulose to produce bioethanol.
1.依据发表在GenBank中的基因序列,利用RT-PCR方法,从里氏木霉(Trichoderma reeseri)的cDNA中克隆到了完整的内切β-1,4-葡聚糖酶V(Endo-β-1,4-glucallaseV),内切β-1,4-葡聚糖酶IV(Endo-β-1,4-glucallaseIV)以及外切β-1,4-葡聚糖酶I(β-1,4-glucancellobiohydrolaseI)蛋白质编码区(coding sequence region,CDS)的cDNA。
2.克隆到的里氏木霉EGV基因蛋白质编码区全长726bp,编码242个氨基酸。核酸序列与已发表的里氏木霉AS3.3711菌株的EGV蛋白质编码区cDNA序列相比有一个核苷酸的突变,推导的氨基酸序列也有一个氨基酸的突变,但是没有明显影响到蛋白质的活性。
3.克隆到的里氏木霉EGIV基因蛋白质编码区cDNA全长1032bp,编码344个氨基酸,CBHI基因蛋白质编码区cDNA全长1542bp,编码514个氨基酸。所获得的EGIV基因和CBHI基因核酸序列和已发表的里氏木霉EGIV基因和CBHI基因的蛋白质编码序列相比完全一致。
4.将EGV基因插入到双T-DNA载体pDTB中,构建载体pDTB-HA-EGV。将合成的定位于质体的信号肽Apo-SP插入到载体pDTB-HA-EGV中,构建载体pDTB-Apo-HA-EGV,并且通过红色荧光蛋白在洋葱表皮细胞的瞬时表达验证该信号肽的功能。
5.用农杆菌介导的方法对水稻品种中花11进行转化,分别获得转化pDTB-HA-EGV载体的68个植株和转化pDTB-Apo-HA-EGV载体的97个植株。PCR检测和Southern杂交分析转基因植株,表明目的基因和抗除草剂基因均整合到水稻基因组中。通过对转基因水稻T_1代30个株系共计291棵植株的PCR检测,仅含目的基因的转基因植株27棵,占9.28%;仅含筛选标记的转基因植株20棵,占6.87%。结果表明使用双T-DNA载体共转化法去除转基因植物分子标记的策略有明显的效果。
6.通过Western杂交检测到两个转基因株系的T_0代干枯秸秆和T_1代鲜活叶片中里氏木霉EGV基因发生了表达,有一定的蛋白产物。并且利用刚果红羧甲基纤维素平板检测到它们的蛋白抽提物中存在纤维素酶酶活,证明EGV基因的表达产物发生了正确的折叠,具有一定的活性,能够在一定的条件下降解外切纤维素酶的底物羧甲基纤维素。并且,在室温储藏了3个月的T_0代秸秆中稳定存在。该结果为在作物中定向表达纤维素酶,以期利用于木质纤维素生产生物乙醇提供了一定的证据。
Cellulose is one of the most abundant organic compounds in nature. Carring out of Biology research on cellulase is the base of understanding of the metabolic mechanism of cellulose and utilization of cellulose in depth, especially in the utilization of lignocellulose to produce bioethanol, which has important theoretical significance and application value. In this research, we cloned cellulase gene from Trichoderma reeseri through RT-PCR, and overexpressed it in rice. The main results are as follows:
1. Based on the sequences of cellulase genes in GenBank, Designed specific oligonucletide primers then used the way of the reverse transcriptase-PCR (RT-PCR) to enriching the gene of Endo-β-1,4-glucallaseV, Endo-β-1,4-glucallaseIV andβ-1,4-glucancellobiohydrolaseI.
2. The full-length CDS region of Trichoderma reeseri EGV is 726bp, encoding a polypeptide of 242 amino acids. There is one base difference when compared the EGV CDS to the known sequence of Trichoderma reeseri AS 3.3711 strain EGV CDS, producing one amino acids mutation, but the activity of protein wasn’t changed.
3. The full-length CDS region of Trichoderma reeseri EGIV is1032bp, encoding a polypeptide of 344 amino acids. The full-length CDS region of Trichoderma reeseri is CBHI1542bp, encoding a polypeptide of 514 amino acids. There is no base difference when compared the Trichoderma reeseri EGIV and CBHI CDS to the known sequence respectively.
4.We inserted EGV gene into the pre-build dual T-DNA vector pDTB to construct vector pDTB-HA-EG V, in accordance with co-transformation strategy to removal of molecular markers from transgenic plants. Because of subcellular expression of foreign proteins can increase protein levels and avoid the potential hazards on host,we construct vector pDTB-HA-EGV by inserting signal peptide Apo-SP targeting to the apoplast. We also test the function of Apo-SP by transient expression in onion epidermal cells.
5. Mediated by Agrobacterium, the vector pDTB-HA-EGV and pDTB-HA-EGV were introduced into rice varieties Zhonghua11.We got 68 transgenic plants and 97 transgenic plants respectively. The transgenic plants were verified by PCR and Southern blotting, to display that objective gene and the herbicide resistant gene were integrated into the rice genome. Through PCR of 30 lines totaling 291 plants of T_1 progeny transgenic rice, there are 27 plants contain the objective gene only, accounts for 9.28%; and 20 plants contain selection marker gene only, accounts for 6.87%. The results show the significant effect of taking advantage of dual T-DNA vector to removal molecular markers in accordance with co-transformation strategy.
6. The Trichoderma reesei EGV gene has expression in fresh leaves and dry straw of T_0 progeny of T_1 progeny transgenic rice by Western blot detection. There is cellulase activity in extractive by Congo Red-CMC plate detection, which prove that EGV gene expression product took place correct folding, has some activity, can degradate cellulose substrate CMC on a certain condition. Moreover, the cellulase is stable in T_0 progeny straw after 3 months storage at room temperature. The result provided reliable evidence of subcellular expression of cellulase in corp. and taking advantage of lignocellulose to produce bioethanol.
引文
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