转录因子转化丹参发根体系及果糖磷酸酶基因的克隆研究
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摘要
目的:为深入研究丹参中迷迭香酸代谢途径的调控,通过发根农杆菌介导叶盘法将AtMYB4转化丹参发根体系,进一步研究代谢途径中关键酶基因的关系以及AtMYB4对各基因表达水平的影响;通过对丹参中特异果糖磷酸酶基因的克隆研究,为深入明确丹参中迷迭香酸的生物合成提供依据。
方法:通过RT-PCR获取转录因子AtMYB4后,构建植物表达载体AtMYB4+pBI121和AtMYB4+pCAMBIA1304+,通过农杆菌菌株C58C1介导叶盘法转化丹参成熟叶片,获得无菌的转基因发根体系。采用实时定量PCR技术对丹参迷迭香酸代谢途径中关键酶编码基因的表达水平进行考察,考察AtMYB4对代谢途径中相关基因表达的影响与调控程度。选用cDNA末端快速扩增法对果糖磷酸酶基因进行克隆,并进行相关生物信息学分析分析。
结果:AtMYB4转录因子的转入,在不同程度上激活了代谢途径中的关键酶基因的表达,促使内源基因SmPAL、Sm4CL1、Sm4CL2、SmTAT、SmRAS与对照相比,分别提高了1.91倍、2.04倍、2.11倍、1.67倍、1.82倍;并对其旁路途径进行了相应的抑制,SmC4H、SmHPPD、SmHPPR的表达仅为对照的48.5%、65.9%和73.5%。采用cDNA末端快速扩增法,首次从丹参中克隆出特异果糖磷酸酶基因(fructose-bisphosphate aldolase,英文缩写为SmFBA,登录号为FJ 540907);其全长cDNA为1390 bp,包含1065 bp的开放式阅读框并编码355个氨基酸,等电点为5.60、分子量为37.78 kDa。其编码的氨基酸序列与其他物种有较高的同源性。基因组DNA序列显示其含有3个外显子和2个内含子;实时定量PCR分析结果显示其为组成型表达,但在根中表达最高。
结论:丹参发根体系为利用基因工程手段来提高目标产物的含量提供了平台,本课题采用转录因子AtMYB4调控迷迭香酸代谢途径中关键编码酶基因的表达,结果显示,确实在一定程度上激活了代谢途径并且对旁路途径抑制进行了相应的抑制;其次,果糖磷酸酶基因是糖酵解途径中的关键酶基因,它的成功克隆,为后续研究丹参中糖酵解途径的相关机制和基因间的相互关系提供了前提保证,为进一步研究丹参中的次生代谢途径及重要代谢产物提供理论依据。
Objective: The transcriptional factor AtMYB4 gene was transformed into the hair roots of Salvia miltiorrhiza Bunge to investigate the regulation of AtMYB4 on the metabolism of water-soluble components in S. miltiorrhiza. A novel fructose-bisphosphate aldolase gene was cloned from S. miltiorrhiza by using of rapid amplification of cDNA ends and bioinformatics analysis was performenced.
Methods: RT-PCR was carried out to abtain AtMYB4, and the plant expression vector AtMYB4 + pBI121 and AtMYB4 + pCAMBIA1304+ were constructed then. Mature leaves of S. miltiorrhiza were used as explants and Agrobacterium-mediated transformated by strain C58C1, followed by co-culture, removing bacteria, liquid ultimately sterile culture of transgenic hairy roots. Applying real-time PCR technique to detect the expression level of the key genes in the rosmarinic acid metabolic pathway and inspect AtMYB4 related metabolic pathways and regulation of gene expression level. By using of rapid amplification of cDNA ends to clone the novel fructose phosphatase gene, and analyzed with the software.
Results: Danshen phenolic acids are mainly rosmarinic acid and its derivatives as lithospermate B. Two paralle branches initialed the biosynthetic pathway. Integrating AtMYB4 into S. miltiorrhiza hairy root could active the expression of key enzymes in the pathway respectively, promoting endogenous genes PAL, 4CL1, 4CL2, TAT, RAS increased 1.91, 2.04, 2.11, 1.67 and 1.81 times respectively compared with control, and repressed the by path, reducing the expression of C4H, HPPD, HPPR by 48.5%, 5.9% and 73.5%.
We cloned novel fructose-bisphosphate aldolase gene (designated as SmFBA, GenBank accession number FJ 540907) cloned from S. miltiorrhiza firstly. Its full-length cDNA was 1kb and 390 bp. Both the open reading frame was 1,065 bp and encoded 355 amino acid residues of a protein. The deduced protein had isoelectric point (pI) of 5.60 and a calculated molecular weight of about 37.78 kDa. SmFBA protein had high homology and identity with other plant FBAs. The SmFBA genomic DNA sequence was also obtained, revealing SmFBA had three exons and two introns. Real-time quantitative PCR analysis showed that SmFBA expressed constitutively in all tested organs with the highest expression level in roots.
Conclusion: The cloning and functional analysis of fructose-bisphosphate aldolase gene will help us to transfer the interrelated genes into S. miltiorrhiza or other model plants and comprehend the metabolic flux in transgenic plant in future. This study helped us to understand EMP pathway, because SmFBA was the key gene in EMP pathway. It could give us a theory and foundation for next study in future.
方法:通过RT-PCR获取转录因子AtMYB4后,构建植物表达载体AtMYB4+pBI121和AtMYB4+pCAMBIA1304+,通过农杆菌菌株C58C1介导叶盘法转化丹参成熟叶片,获得无菌的转基因发根体系。采用实时定量PCR技术对丹参迷迭香酸代谢途径中关键酶编码基因的表达水平进行考察,考察AtMYB4对代谢途径中相关基因表达的影响与调控程度。选用cDNA末端快速扩增法对果糖磷酸酶基因进行克隆,并进行相关生物信息学分析分析。
结果:AtMYB4转录因子的转入,在不同程度上激活了代谢途径中的关键酶基因的表达,促使内源基因SmPAL、Sm4CL1、Sm4CL2、SmTAT、SmRAS与对照相比,分别提高了1.91倍、2.04倍、2.11倍、1.67倍、1.82倍;并对其旁路途径进行了相应的抑制,SmC4H、SmHPPD、SmHPPR的表达仅为对照的48.5%、65.9%和73.5%。采用cDNA末端快速扩增法,首次从丹参中克隆出特异果糖磷酸酶基因(fructose-bisphosphate aldolase,英文缩写为SmFBA,登录号为FJ 540907);其全长cDNA为1390 bp,包含1065 bp的开放式阅读框并编码355个氨基酸,等电点为5.60、分子量为37.78 kDa。其编码的氨基酸序列与其他物种有较高的同源性。基因组DNA序列显示其含有3个外显子和2个内含子;实时定量PCR分析结果显示其为组成型表达,但在根中表达最高。
结论:丹参发根体系为利用基因工程手段来提高目标产物的含量提供了平台,本课题采用转录因子AtMYB4调控迷迭香酸代谢途径中关键编码酶基因的表达,结果显示,确实在一定程度上激活了代谢途径并且对旁路途径抑制进行了相应的抑制;其次,果糖磷酸酶基因是糖酵解途径中的关键酶基因,它的成功克隆,为后续研究丹参中糖酵解途径的相关机制和基因间的相互关系提供了前提保证,为进一步研究丹参中的次生代谢途径及重要代谢产物提供理论依据。
Objective: The transcriptional factor AtMYB4 gene was transformed into the hair roots of Salvia miltiorrhiza Bunge to investigate the regulation of AtMYB4 on the metabolism of water-soluble components in S. miltiorrhiza. A novel fructose-bisphosphate aldolase gene was cloned from S. miltiorrhiza by using of rapid amplification of cDNA ends and bioinformatics analysis was performenced.
Methods: RT-PCR was carried out to abtain AtMYB4, and the plant expression vector AtMYB4 + pBI121 and AtMYB4 + pCAMBIA1304+ were constructed then. Mature leaves of S. miltiorrhiza were used as explants and Agrobacterium-mediated transformated by strain C58C1, followed by co-culture, removing bacteria, liquid ultimately sterile culture of transgenic hairy roots. Applying real-time PCR technique to detect the expression level of the key genes in the rosmarinic acid metabolic pathway and inspect AtMYB4 related metabolic pathways and regulation of gene expression level. By using of rapid amplification of cDNA ends to clone the novel fructose phosphatase gene, and analyzed with the software.
Results: Danshen phenolic acids are mainly rosmarinic acid and its derivatives as lithospermate B. Two paralle branches initialed the biosynthetic pathway. Integrating AtMYB4 into S. miltiorrhiza hairy root could active the expression of key enzymes in the pathway respectively, promoting endogenous genes PAL, 4CL1, 4CL2, TAT, RAS increased 1.91, 2.04, 2.11, 1.67 and 1.81 times respectively compared with control, and repressed the by path, reducing the expression of C4H, HPPD, HPPR by 48.5%, 5.9% and 73.5%.
We cloned novel fructose-bisphosphate aldolase gene (designated as SmFBA, GenBank accession number FJ 540907) cloned from S. miltiorrhiza firstly. Its full-length cDNA was 1kb and 390 bp. Both the open reading frame was 1,065 bp and encoded 355 amino acid residues of a protein. The deduced protein had isoelectric point (pI) of 5.60 and a calculated molecular weight of about 37.78 kDa. SmFBA protein had high homology and identity with other plant FBAs. The SmFBA genomic DNA sequence was also obtained, revealing SmFBA had three exons and two introns. Real-time quantitative PCR analysis showed that SmFBA expressed constitutively in all tested organs with the highest expression level in roots.
Conclusion: The cloning and functional analysis of fructose-bisphosphate aldolase gene will help us to transfer the interrelated genes into S. miltiorrhiza or other model plants and comprehend the metabolic flux in transgenic plant in future. This study helped us to understand EMP pathway, because SmFBA was the key gene in EMP pathway. It could give us a theory and foundation for next study in future.
引文
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[3] Grotewold E, Chamberlin M, Snook M, et al. Engineering secondary metabolism in maize cells ectopic expression of transcription factors [J]. Plant Cell, 1998, 10:721-740.
[4] Gandikota M, de Kochko A, Chen L, et al. Development of transgenic rice plants expressing maize anthocyanin genes and increased blast resistance [J]. Mol Breed, 2001, 7(1):73-83.
[5] Borevitz JO, Xia Y, Blount J, et al. Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis [J]. Plant Cell, 2000, 12:2383-2393.
[6]柳丽,张洪泉.丹参活性成分的现代中药药理研究进展[J].中国野生植物资源,2003,22(6):1-4.
[7] Zhou LM, Zuo Z, Moses S. Danshen: an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use [J].J Clin Pharmacol, 2005, 45:1345-1359.
[8]梁勇,羊裔明,袁淑兰.丹参酮药理作用研究进展[J].中草药,2000,31(4):304.
[9] Hac Young Chung.丹参药理学[M].国外医学中医中药分册,1988,20:10.
[10]刘平,刘乃明,徐列明,等.丹参酸乙对四氯化碳体外损伤原代培养大鼠肝细胞的直接保护作用[J].中国中药杂志,1997;22(5):303.
[11]康永.中药药理学[M].北京:科学出版社,2001.
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