【作者】 牛一丁；

【导师】 扈廷茂； 哈斯阿古拉；

【作者基本信息】 内蒙古大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 本论文主要对苜蓿中的DREB类转录因子基因进行了研究。分别以4℃寒冷胁迫处理的黄花苜蓿、紫花苜蓿总RNA为模板,运用RACE技术和RT-PCR方法,扩增得到黄花苜蓿逆境胁迫诱导相关转录因子MfDREB1基因、MfDREB1s基因和紫花苜蓿逆境胁迫诱导相关转录因子MsDREB1基因全长cDNA。将以上3个基因cDNA进行克隆与序列分析,序列分析表明MfDREB1基因cDNA长952 bp,包含一个651 bp的开放阅读框,编码一条含216个氨基酸的多肽,分子量约为24.6 kDa,等电点为5.95;MfDREB1s基因cDNA长744bp,包含一个555bp的开放阅读框,编码一条含184个氨基酸的多肽,分子量约为20.8kDa,等电点为9.11;MsDREB1基因cDNA长902bp,包含一个长651 bp的开放阅读框,编码一条含216个氨基酸的多肽,分子量约为24.9 kDa,等电点为6.11。Protein Blast数据显示,以上3条多肽均属于EREBP/AP2家族DNA结合蛋白的典型成员。进一步分别克隆了MfDREB1、MfDREB1s和MsDREB1的基因组DNA,序列分析表明以上3个基因均无内含子。基因组Southern blot分析表明,MfDREB1和MfDREB1s基因在黄花苜蓿基因组中共有3个拷贝,MsDREB1基因在紫花苜蓿基因组中以2拷贝存在。Northern blot结果表明MfDREB1和MfOREB1s基因的表达可受低温胁迫的诱导。拟南芥rd29A基因的表达受干旱、高盐及低温的诱导。rd29A基因启动子区中含有与上述环境胁迫应答相关的DRE顺式作用元件,该启动子可以感受环境胁迫,启动下游报告基因的表达。本研究以拟南芥基因组DNA为模板,扩增得到长937bp的rd29A启动子片段。为下一步外源基因在转基因植物中的诱导表达奠定基础。为建立简便易行、费用低廉的紫花苜蓿基因转化技术,以紫花苜蓿（Medicago sativa L.）为受体材料,以含Gus基因的植物双元表达载体为外源基因供体进行了花粉管通道法转基因的研究。授粉后分别于6h、9h、12h、20h、24h、28h、32h、48h时滴加DNA溶液,待荚果成熟后收获转化种子。对T₀代植株进行PCR检测,结果显示授粉后20至48h范围内导入均可得到较高的转化频率,其中授粉后32h滴加DNA溶液所得的转化率最高（16.7%）。本研究对紫花苜蓿组织培养作了系统研究,建立了一套高效稳定的紫花苜蓿再生体系。运用农杆菌介导法将超表达和诱导表达MfDREB1基因的植物双元表达载体pPZP221（35S-MfDREB-nos）、pPZP221（RD29AP-MfDREB-nos）导入紫花苜蓿,PCR检测表明外源基因己整合到受体基因组中。转基因苜蓿中MfDREB1的超表达使得转基因植株的抗寒性比对照植株有所增强。更多还原

【Abstract】 DREB transcription factors play an important role in tolerance to abiotic stress of plant.In this paper mainly we carried out researches of the DREB type transcription factors in Medicago.Three new DRE-binding protein genes MfDREB1 cDNA,MfDREB1s cDNA and MsDREB1 cDNA that encoded EREBP/AP2 type transcription factors were isolated by RACE and RT-PCR from Medicago falcata L.and Medicago sativa L.seedlings.The MfDREB1 has a open reading frame of 651bp,which coded 216 amino acid residues and the putative protein was predicted to have a molecular weight of 24.6 kDa,pl 5.95.The MfDREB1s has a open reading frame of 555bp,the putative protein is 184 amino acid long with a predicted molecular weight of 20.8 kDa,pl 9.11.The MsDREB1 has a open reading frame of 651bp, encoding 216 amino acid residues and the putative protein with a predicted molecular weight of 24.9 kDa,pl 6.11.The Protein Blast data revealed that the three proteins can be classified as typical members of the EREBP/AP2 family of DNA-binding proteins.The comparison of the MfDREB1 cDNA,MfDREB1s cDNA and MsDREB1 cDNA with their corresponding genes in genomic DNA showed that the size and nucleotide sequence of the cDNA were identical with that of the genomic DNA.This suggested that the genomic MfDREB gene,MfDREB1s gene and MsDREB1 gene have no introns.Southern blot analysis indicated that the MfDREB gene and MfDREB1s gene have three copies in Medicago falcate genome,the MsDREB1 is a double-copy gene in alfalfa.Northern blot analysis indicated that the MfDREB gene and MfDREB1s gene were induced by low temperature stress.Arabidopsis rd29A gene was induced with drought,high salt and cold treatments.There are two DRE/CRT(a DNA cis-regulatory sequence) element in the region of the rd29A promoter which was responsive to low temperature,drought and high-salt stress.The rd29A promoter has the ability to perceive abiotic stress and respond to it by activating downstream report gene.We amplified a 937bp promoter region of the rd29A gene promoter from the Arabidopsis genomic DNA by PCR.This cloned promoter region may be useful for the stress-inducible expression of foreign gene in transgenic plant.In order to establish a simple and practical transformation technique with a high transformation rate in alfalfa(Medicago sativa L.),the pollen-tube pathway transformation method has been developed.The alfalfa cultivar ’Algonguin’ was used as recipient and the binary vector pPZP221 containing a GUS(β-glucuronidase) gene was used as the donor DNA of transgene.The donor DNA solution was applied to the stigma at 6,9, 12,20,24,28,32 and 48 hour after hand-pollination,respectively.PCR results showed that the transformation rates were high from 20h to 48h, with the highest rate of 16.7%at 32h after pollination.The paper established a efficient regenerative system of alfalfa tissue culture.The binary vector pPZP221(35S-MfDREB-nos) and pPZP221(RD29AP-MfDREB-nos) were transformed into alfalfa by Agrobacterium infiltration respectively.The PCR analysis suggested that the foreign gene was inserted into the recipient’s genome.The over—expression of MfDREB1 activated expression of downstream genes in transgenic alfalfa, resulting in enhanced tolerance to low-temperature stress.更多还原