【作者】 关宁；

【导师】 李聪；

【作者基本信息】 中国农业科学院 ， 草业科学， 2009， 博士

【摘要】 含硫氨基酸(SAAs)对动物产毛和产奶的质量影响很大,饲料中补充SAAs,可使羊毛生长和羊毛中含硫量均显著增加,提高奶牛产奶量。含硫氨基酸在饲料植物中含量较少,特别是半胱氨酸和蛋氨酸,常成为第一限制性必需氨基酸。培育高含硫氨基酸的牧草品种是当前牧草品质改良的研究热点之一。百脉根(Lotus corniculatus L.)是重要的豆科牧草之一,但含硫氨基酸含量较低。通过分子克隆及遗传转化等方法,将外源高含硫氨基酸蛋白基因转入百脉根,提高牧草蛋白中含硫氨基酸含量具有十分重要的意义。玉米醇溶蛋白(zein)是玉米种子中的主要贮藏蛋白质,在胚乳中以蛋白体的形式存在,可分为α-、β-、γ-和δ-zein等四种主要类型。其中β-、γ-和δ-zein富含甲硫氨酸和半胱氨酸,其具有在反刍动物瘤胃中稳定而在肠中降解的特性,可用于牧草品质的改良。研究表明在转基因植株中含硫氨基酸均有提高,但必须使外源蛋白占到总提取蛋白的1%～5%才具有实际的营养价值。因此,zein在转基因植株的表达量仍有待提高。本试验将玉米γ-zein基因和zeolin基因(γ-zein和菜豆球蛋白的融合基因)与植物表达载体pCAMBIA1302的绿色荧光蛋白(GFP)报告基因融合,构建植物表达载体pCBzeolin和pCBzein。通过叶盘法转化模式植物烟草,分析目的基因的表达,验证植物表达载体pCBzeolin和pCBzein的正确性。利用农杆菌介导法将γ-zein基因和zeolin基因导入百脉根中,对转化再生植株进行分子检测和含硫氨基酸含量检测。研究结果如下:1.用限制性内切酶NcoⅠ和BglⅡ从中间载体pMD18zeolin和pMD18zein上切下zeolin基因(约1600bp)和γ-zein基因(约720bp),将其定向连接在经相同酶切的质粒载体pCAMBIA1302上,构建成植物表达载体pCBzeolin和pCBzein,测序并进行序列的生物信息学分析。2.采用冻融法将pCBzeolin和pCBzein导入根癌农杆菌(Agrobacterium tumefaciens)菌株LBA4404,利用该菌株转化烟草和百脉根。3.通过筛选培养和PCR检测,得到转γ-zein基因和zeolin基因的烟草再生植株。目的蛋白表达分析表明γ-zein基因和zeolin基因以蛋白体方式,不均匀地分布在原生质体中,并验证表达载体pCBzeolin和pCBzein的正确性和可行性。4.经过共培养、筛选分化、再生,得到百脉根抗性植株。对抗性植株进行PCR、RT-PCR和斑点杂交检测表明,γ-zein和zeolin基因已经整合到百脉根基因组中,在核酸水平得到表达。5.含硫氨基酸数据分析表明,转zeolin基因百脉根植株含硫氨基酸含量极显著高于转γ-zein基因百脉根植株和非转基因百脉根植株的含量(P<0.01),但转γ-zein基因植株与非转基因植株间差异不显著。更多还原

【Abstract】 Sulphur-containing amino acids (SAAs) play an imimportant role in the efficiency of wool growth in sheep and milk production in dairy animals. The forage rich in sulphur-containing amino acids is benefit to the wool growth, the increase of the content of sulphur in wool, and the enhancement of yield of milk. The forage plants are deficient in sulphur-containing amino acids became the first limiting amino acids, especially cysteine and methionine. Breeding of forage variety containing sulphur-rich amino acid is one of research focus on improvement of forage quality at present.Crowtoe (Lotus corniculatus L.) is one of the most main legume forages which contain little sulphur-containing amino acids. The sulphur-rich protein encoding genes are transferred into crowtoe to improve the content of the sulphur-containing amino acids of forage protein through genetic engineering. Zeins, maize prolamin, the major protein reserves of maize kernels, are accumulated in protein bodies (PBs) arising from the rough endoplasmic reticulum. Zeins can be classified intoα-,β-,γ- andδ-zeins.β-,γ- andδ-zein are rich in the essential sulphur amino acids methionine and cysteine. The zeins are characterized by their stabilization in ruminal and digestibility in intestines, so it can be utilized in forage quality improvement. Though this may be considered a good starting point, further improvements must still be made to significantly increase the nutritional quality of forages, considering that foreign proteins must account for 1%～5% of the total protein in the leaf .The sulphur-containing amino acids genes ofγ-zein and zeolin (fused withγ-zein and phaseolin ) were fused with the report gene GFP from the plant expression vector pCAMBIA1302. The plant expression vectors pCBzeolin and pCBzein were obtained. pCBzeolin and pCBzein were transformed into the model plant tobacco by leaf disc method to prove the validity of pCBzeolin and pCBzein. The expressions ofγ-zein and zeolin gene from transgenic tobaccos were analysesd. pCBzeolin and pCBzein were also transformed into Lotus corniculatus mediated by strain LBA4404. The transformed regeneration plants were testified by molecular detections and analysis of the content of sulphur-containing amino acid. The main results of this study are as following.1. Zeolin gene (about 1600bp) andγ-zein gene (about 720bp) were got by cutting from the middle vectors pMD18zeolin and pMD18zein respectively, and were linked to the expression vector pCAMBIA1302 in the same endonucleases digestion sites. The plant expression vector pCBzeolin and pCBzein were obtained,sequenced and analyzed by bioinformatics software.2. Plasmids pCBzeolin and pCBzein were transferred into Agrobacterium tumefaciens LBA4404 by freeze-thaw method, and then were transformed into tobacco and crowtoe mediated by strain LBA4404.3. After selective culture and PCR analysis, transgenic tobacco regeneration plants were obtained. It was showed thatγ-zein and zeolin accumulated in protein bodies were distributed heterogeneously in the protoplast to prove the validity and feasibilityof pCBzeolin and pCBzein.4. After co-culture, selective differentiation and regeneration, resistant crowtoe plants were obtained. Resistant plants were detected by PCR, RT-PCR and Dot blot. The results have proved that theγ-zein and zeolin genes have been transformed into the genome of L. corniculatus, and expressed on nucleic acid level in the transgenic plants.5. Sulphur-containing amino acid of transgenic crowtoe plants analysis showed that the content of sulphur-amino acid of transgenic zeolin crowtoe plants was significantly higher than that of the content of the transgenicγ-zein crowtoe plants and non-transgenic crowtoe plants (P<0.01), but there was no significant difference between the transgenicγ-zein plants and non-transgenic plants.更多还原