【作者】 孙晓燕；

【导师】 王崇英；

【作者基本信息】 兰州大学 ， 细胞生物学， 2008， 硕士

【摘要】 山黧豆具有许多优良的生物以及农学性状,对环境具有广泛的适应性,一般认为山黧豆中所含的神经毒素ODAP是引起人或动物神经中毒的主要原因,但山黧豆的高营养价值和对环境的广泛适应性是其禁而不绝的主要原因。本研究将不同山黧豆品系的田间生物学性状、毒素含量、抗逆生理生化、蛋白表达谱联合起来进行分析研究,这将为山黧豆种质资源的研究和开发利用提供新的思路,并可为在不影响其抗逆性的同时培育低毒和无毒的山黧豆品系提供重要的参考。主要研究结果概括如下:1通过在生物园中引种30个山黧豆品系,对其毒素含量和花、种子等生殖器官的形态特征的比较分析发现,1)毒素β-ODAP的含量极其不稳定,同一品种在不同年份毒素含量有明显差异,甚至在播种前后也不尽相同,其中8#永寿山黧豆毒素含量最高,6#扁荚山黧豆和9#肉色扁荚山黧豆最低;2)6#、7#阿麻山黧豆、9#的花及种子的形态特征与其他品系差别很大,尤其6#和9#两个品系的抗涝能力明显较弱。2通过对高（8#）、中（7#）、低（9#）毒三个有代表性的品系进行生理生化的研究,结果发现,1)可溶性糖和脯氨酸等小分子渗透物质、SOD和POD等抗氧化酶类在盆栽10d的幼苗中的基础含量均是8#最高,7#次之,9#最低;2)高毒品系8#的茎杆及主根明显粗壮,且侧根多而长,叶片较大,千粒重也明显较高。这表明毒素含量较高的7#和8#品系其抗逆性、产草量、产籽量均优于低毒品系9#。3分别利用SDS-PAGE和2-DE电泳对7#、8#、9#品系的蛋白表达情况进行了比较研究,并重点对2-DE表达谱进行了分析比较,差异分析结果显示,7#和8#、8#和9#、7#和9#两两间分别有21、41、36个差异表达的蛋白点。采用MALDI-TOF/TOF或LTQ（LC-MS-MS）技术对8#和9#品系间的8个有显著差异的蛋白点进行了质谱鉴定,并通过MASCOT、NCBI、SWISS-PROT等数据库对各差异蛋白的功能进行了研究。结果发现:1)低毒品系9#中消失的20kDa伴侣蛋白（20 kDa chaperonin,chloroplast precursor）主要在体内与cpn60家族协同作用,促进核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）大小亚基装配成活性的全酶,利于CO₂的固定;下调的果糖1,6-二磷酸醛缩酶（Fructose-bisphosphate aldolase 2 FDA,chloroplast）主要与叶片淀粉的生物合成能力和蔗糖产量的提高有关,而下调的OSJNEb05K22 5,（Oryza sativa Japonica Group cDNA clone,mRNA sequence）编码蛋白与叶绿体前体、光系统Ⅰ反应中心的亚单位Ⅳ（PSI-E）具73%同源性。显然,这三个蛋白均与碳水化合物的形成有关,其表达量的下调或者消失与9#品系产草量和产籽量相对较低有密切关系。2)新生多肽相关复合体（Nascent polypeptide-associated complex NAC,UBA-like）和核糖核蛋白（ribonucleoprotein RNP）在8#和9#表现为位置的相对迁移,这可能与蛋白的翻译后修饰有关。NAC是一种辅助转录激活因子,具有多种功能,RNP主要在转录水平起作用,与许多细胞进程有关联。二者可能主要在基因的选择性表达过程中起作用,从而使得7#、8#、9#在毒素含量、抗逆性、产草量和产籽量上出现了很大差异。4利用PEG模拟自然干旱对高毒品系8#的耐旱生理机制及与毒素含量的关系进行了初步研究,从而获悉了山黧豆对干旱的部分适应性调节机理。同时了解到毒素含量受干旱的影响程度要远小于微量元素如Fe2+的影响,即毒素并非是山黧豆对抗逆境的一种手段。综合上述实验结果发现,较高毒素品系7#和8#与低毒品系9#的差异主要体现在毒素含量、抗逆性、产草量和产籽量上,所有这些差异都是差异性基因或基因选择性表达的结果。本研究仅初步探讨了毒素含量、抗逆性、作物产量和差异蛋白的关系,更详细的内在相关性还有待进一步研究。更多还原

【Abstract】 Grass pea（Lathyrus sativus L.）has many advantageous biological and agronomic characters as well as extensive tolerance to adverse environment,in spite of neurotoxin ODAP.The biological characters of grass pea in field,ODAP content,physiological and biochemical responses combined with protein expression profile were studied in this thesis,which would provide new clues to the research and development of grass pea.The key results are as follows:1 Thirty species of grass pea were planted in the field.The differences in ODAP content and the morphologic characteristic of both flowers and seeds were compared among different species. The results showed that:1)ODAP content was changeable,and even in the same species.It was highest in 8#,and lowest in 6# and 9#;2)Flowers and seeds of 6#,7# and 9# were quite different from other varieties,and the resistance to waterlogging of 6# and 9# was less strong especially.2 The physiological and biochemical responses among three representative varieties 7#,8#, 9# were studied.The results showed that,1)basal contents of soluble sugar,proline content,SOD and POD were the highest in 8#,lower in 7# and lowest in 9#;2)8# was thicker in stems and roots,bigger in leaf size,higher in 1000-grain weight than 7# and 9#.All above suggested that 7# and 8# were superior to 9# in the aspects of stress resistance,forage yield and grain yield.3 Anaysis of 2-DE showed that there were 21,41 and 36 differentially expressed protein spots in 7#,8# and 9#,respectively.Eight differentially expressed protein spots between the gel of 8# and 9# were identified by MALDI-TOF/TOF and LTQ（LC-MS-MS）and their function were analyzed by searching MASCOT,NCBI database and SWISS-PROT.The results showed that:1) 20 kDa chaperonin（chloroplast precursor）,disappearing in 9#,was normally coordinated with cpn60 family,and promoted the assembly of Rubisco subunit so as to CO₂ fixation; Fructose-bisphosphate aldolase（FDA,chloroplast）,down-regulation in 9#,was related to increasing the production of starch and sucrose,and Oryza sativa Japonica Group cDNA clone OSJNEb05K22 5’ mRNA sequence shared 73%identity to PSI-E（photosystemⅠreaction center subunitⅥ,chloroplast precursor）.Obviously,these three proteins were related to formation of carbohydrates,and thus changes of expression in 9# may be related to the lower forage yield and grain yield.2)Nascent polypeptide-associated complex NAC and ribonucleoprotein（RNP） different in migration between 8# and 9# were both likely to due to post-translational modification. NAC was one kind of auxiliary transcription factor and had many functions.RNP acted at the transcription level,and related to lots of cellular processes.Probably both of them played roles in alternative expression of gene in species 7#,8# and 9#,and led to great differences in toxin content,stress resistance,forage yield and grain yield.4 The drought-resistant responses as well as the relationship between stress and ODAP content were studied.The effect of drought on toxin content was lower than microelement such as Fe²⁺,and it was likely that ODAP played few roles in stress resistance.In summary,there were obvious differences in ODAP content,stress resistance,forage yield and grain yield between high-toxic species 8# and low-toxic 9#,which might be the results of alternative expression of related genes,and further study was necessary for understanding the inherent relevant better.更多还原