【作者】 石凤敏；

【导师】 云锦凤；

【作者基本信息】 内蒙古农业大学 ， 草业科学， 2011， 博士

【摘要】 蒙古冰草（Agropyron mongolicum Keng）是禾本科冰草属小麦族多年生草本植物,不仅具有极高的饲用价值,而且具有很强的抗逆性,富含大量的抗旱、抗寒、耐盐基因,可以为牧草及近缘种作物（水稻、小麦、玉米等）的抗性改良及新品种选育提供宝贵的资源。本研究分离出蒙古冰草MwLEA3基因并进行了功能验证,并首次克隆得到了蒙古冰草类反转录转座子。主要研究结果如下:1.分离了蒙古冰草MwLEA3基因,其全长为705bp,开放阅读框为492bp,编码163个氨基酸。2.构建了蒙古冰草MwLEA3基因的亚细胞定位融合蛋白载体pA7-MwLEA3,并将此载体及pA7-GFP载体质粒转化至洋葱细胞中进行亚细胞定位。试验结果表明,pA7-MwLEA3融合蛋白集中分布在细胞核中,而对照pA7-GFP在整个细胞中均有分布,证明了MwLEA3基因表达的蛋白是核定位蛋白。3.成功构建蒙古冰草MwLEA3基因植物表达载体pCAM-MwLEA3,并经农杆菌介导进行烟草的遗传转化。对经过抗性检测的转基因烟草T₀代植株进行PCR检测、PCR-Southern、RT-PCR鉴定以及干旱胁迫试验。分子检测结果表明,MwLEA3基因已整合到烟草基因组中,并在转录水平正常表达;干旱胁迫试验结果显示转基因烟草植株的抗旱性高于野生型烟草植株。4.对10个株系转基因烟草T₁代的卡那霉素抗性进行研究表明,10个株系中,有7个株系的T₁代的分离比例为3:l,符合孟德尔式遗传规律,这表明MwLEA3基因是单拷贝插入;另外3个株系的T₁代不符合孟德尔式3:1的遗传规律,说明这些植株中的MwLEA3基因可能是多个拷贝插入的。5.构建了35S启动子调控的具有MwLEA3基因反向重复结构的ihpRNA表达载体pART27-MwLEA3（HB）-MwLEA3（EX）,并通过冻融法转化将MwLEA3基因的ihpRNA表达框架成功导入根癌农杆菌LBA4404中。利用带有目的表达载体的农杆菌菌液侵染蒙古冰草愈伤组织,现正在筛选分化培养基上培养,后续试验正在进行中。6.首次克隆得到了蒙古冰草类反转录转座子（MwRRT）基因,该基因片段长度为1005bp,在1～225bp为非编码区,长度为225bp,其氨基酸序列在226～1005bp,共780bp,编码260个氨基酸。核苷酸及氨基酸同源性对比分析发现,所克隆基因与小麦（Triticum aestivum）、水稻（Oryza sativa）、大麦（Hordeum vulgare）、高粱（Sorghum bicolor）的LTR类反转录转座子的核苷酸同源性约70%,氨基酸同源性约50%,GenBank登陆号NO.GQ855806.1。更多还原

【Abstract】 Mongolia Wheatgrass （Agropyron mongolicum Keng）, a perennial grass, belongs to Agropyron genus in Gramineae, not only has high feeding value, but also rich in resistant genes,such as drought-tolerant gene, cold- tolerant gene, salt-tolerant gene and so on. It could provide valuable resource for the improvement of resistance and breeding of forage and its closely related species including rice, wheat, maize and so on. In this paper, the function of the MwLEA3 gene of Mongolia Wheatgrass was verified on the basis of molecular biological identification and physiological and biochemical identification,and MwRRT retrotransposons was cloned from Mongolia Wheatgrass for the first time. The main results are as follows:1. The full length MwLEA3 gene of Mongolia Wheatgrass was cloned by RT-PCR amplification technique. DNAman analysis indicated its open read frame was 492bp, coded 163 amino acids.2. The fusion protein vector pA7-MwLEA3 for subcellular localization was constructed. The fusion protein vector and pA7-GFP vector plasmids were transformed into onion cells. The results showed that the fusion protein pA7-MwLEA3 was mainly distributed in the cell nucleus, while pA7-GFP were distributed in the whole cell. This result was demonstrating that the expressed protein encoded by MwLEA3 was a nuclear localized protein.3. The plant expression vector of pCAM-MwLEA3 was constructed and transformed into tobacco via Agrobacterium infestation. T₀ transformed tobaccos were tested by PCR、PCR-Southern、RT-PCR and drought stress. Molecular detecting results showed that MwLEA3 gene had been transformed into tobacco and expressed. The results of drought stress showed that the transgenic tabaccos had stronger resistance than wide-type ones.4. The results of kanamycin resistant study of 10 lines T₁ generation of transgenic tobacco plants showed that the separation law of T₁ generation of seven lines of transgenic tobacco was 3:1, showing coincidence with Mendelian genetic law. Other three lines did not show seperation ratio of 3:1.5. The ihpRNA expression vector was constructed with inverted repeats of MwLEA3 gene, which was promoted by 35S promoter. The voctor was transferred into Agrobacterium tumefaciens strain LBA4404 with freeze-thaw method. The callus of Mongolia Wheatgrass were infected with Agrobacterium tumfaciens containing the ihpRNA expression vector, and are now being cultured on differentiation medium.6. MwRRT retrotransposons was cloned from Mongolia Wheatgrass for the first time. It had a length of 1005bp, and consisted of a non-coding region from 1bp to 225bp and a coding region from 226bp to 1005bp that encoded 260 amino acids. Sequence comparison analysis with LTR class retrotransposons from wheat （Triticum aestivum）, rice （Oryza sativa）, barley （Hordeum vulgare） and sorghum （Sorghum bicolor） showed that it had about 70% idendity in nucleotide sequence and about 50% homology in amino acid sequence. We registered this gene in GenBank and the accession No. was GQ855806.1.更多还原