植物EPSPS基因家族改组及体外定向分子进化研究

Glyphosate-resistant crop currently takes the biggest share of GM cultivation area all around the world. Thus, the cloning, expression and function analysis of EPSPs become main focuses of modern bio-molecular breeding. Recently, we have cloned the EPSPS gene from Malus domestica (MdEPSPS) by rapid amplification of cDNA ends (RACE). However, wild-type MdEPSPS is not suitable for the development of transgenic glyphosate-resistant crop because of its extremely glyphosate-sensitivety. Thus, we performed DNA shuffling on MdEPSPS, and one mutant (MdEPSPSmutant) which showed highly glyphosate-resistance in transgenic plant was isolated after five rounds of DNA shuffling and screening. But the glyphosate-resistance of MdEPSPSmutant is slightly lower as compared to ZmEPSPSmutant (a mutant EPSPS from Zea mays) which has been utilized to produce the first commercial varieties of glyphosate-resistant maize. So，in this project，excellent chimeric-gene with independent intellectual property rights and higher glyphosate-resistance than MdEPSPSmutant will be obtained by DNA family shuffling used six homologous EPSPS gene resourced different species as template, combined with high-throughput screening system. The three structure of protein encoded by chimeric-gene will be predicted and analyzed using bioinformatics methods. The relationship between mutation amino acid sites/region and the increase of glyphosate-resistance will be discussed. The chimeric-gene will be introduced into the model plants Arabidopsis and Rice and the glyphosate-resistance will be analyzed, which will provide the theory basis for developing transgenic glyphosate-resistant crops with independent intellectual property rights.

抗草甘膦转基因作物是目前全球播种面积最大的转基因作物，因而草甘膦靶酶(EPSPS)基因的克隆及功能验证也就成为现代分子生物育种研究的重点。前期我们采用RACE技术克隆了苹果的EPSPS基因，由于这个基因对草甘膦很敏感，所以我们对其进行DNA改组获得了一个在转基因植物中表现出良好草甘膦抗性的突变体。但与植物源唯一商业化基因-玉米突变体基因相比，其草甘膦抗性略低。因此为使我们所获得的突变体基因能够用于转基因培育，本项目拟以6个不同种属的EPSPS基因为母本基因进行家族DNA改组，结合高通量筛选体系，以期获得可以媲美商业化基因的具有自主知识产权的嵌合突变基因；同时采用生物信息学方法对嵌合突变基因编码蛋白质的三级结构进行分析，探讨突变位点/区域与草甘膦抗性增加的关系；并将突变基因转入模式植物拟南芥和水稻，分析其草甘膦耐受性，为培育具有自主知识产权的抗草甘膦转基因作物奠定理论基础。

迄今为止，只有II类的源于农杆菌CP4的EPSPS基因应用在抗草甘膦转基因作物的培育，但是该基因受到国外跨国公司的专利保护。所以为了进一步增加EPSPS基因的多样性，解决目前生产上唯一应用的农杆菌CP4 EPSPS基因所可能引起的转基因植物的草甘膦抗性降低因素，开发拥有独立产权的自主高抗草甘膦的新型EPSPS基因并对其进行功能分析，这对于我国基因工程研究水平的提升以及拥有知识产权的转基因植物的培育具有重要的理论意义和实践意义。为此本项目通过对6个不同种属的EPSPS基因进行家族DNA改组，结合高通量筛选体系，获得了在大肠杆菌中可以抵抗200mM草甘膦的具有自主知识产权的嵌合突变基因（EPSPSm）；同时通过对该嵌合突变基因进行核苷酸序列测定，发现15个核苷酸位点突变导致了5个氨基酸位点（T41S；T/S113A； S/E115F；G/V245S；R316C）发生突变。定点回复突变显示只有在这5个氨基酸位点共同存在协同作用才能表现出高抗草甘膦特性；然后将酶切后的嵌合突变体基因连接入原核表达载体进行蛋白表达并纯化。同时用纯化出的蛋白开展了动力学参数的测定；随后将该嵌合突变基因以及母本基因（玉米的突变体基因）转入模式植物拟南芥和水稻，筛选得到了转嵌合突变基因和母本基因的拟南芥和水稻的阳性植株。对转基因拟南芥种子进行了萌发和根长草甘膦实验，结果显示当草甘膦浓度为250μM时，转母本基因拟南芥阳性植株不管是根的生长还是种子萌发均已经受到了严重的抑制，而转筛选所获得的嵌合突变基因的阳性植株在草甘膦浓度为750μM时种子萌发和根长生长依然良好。同样的研究结果在转基因水稻中也得到了相应的验证。由此表明，本研究筛选所获得的嵌合突变基因具有培育抗草甘膦植物的潜力，同时筛选所获得的嵌合突变基因还可以作为标记基因应用于转基因植物的高效筛选。