【作者】 沈文飚；

【导师】 万建民；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 粮食安全是一个全球性的战略问题,而粮食贮备是其中重要的一环。已经知道,稻谷在一般贮藏条件下第二年就会产生陈化变质现象,随着贮藏过程中温度和湿度的提高,陈化过程也会迅速加快。据统计,每年我国因为陈化问题损失的包括作为食用和种子的稻谷占总产量的3%。近年来的研究表明,稻谷贮藏期间脂质的降解是导致其品质下降并产生令人不快气味的主要原因之一,其中脂氧合酶（lipoxygenase,LOX,EC1.13.11.12）是脂质降解的关键酶。因此从理论上讲,粮食作物LOX的缺失可以明显的阻止脂质过氧化作用,减缓贮藏粮食氧化变质的速度,保持清新气味,提高耐贮性,上述推测已经在水稻LOX的主要同工酶LOX-3的缺失体与耐贮性关系的研究中得到了初步证实,但是有关LOX-3缺失体的简单筛选方法、水稻种子LOX同工酶分析、LOX-3的基因克隆以及与缺失的关系还有待于进一步的研究。本文首先根据水稻种胚脂氧合酶-3共氧化的次生反应特性及其催化产物的氧化特性,分别建立了筛选水稻种胚LOX-3缺失体的胡萝卜素漂白比色法和I₂-KI,与常用的LOX-3单克隆抗体筛选技术相比,这两种方法具有准确、快速、简单且成本低的特点,可以进一步用于耐储藏水稻的分子育种研究。采用生物化学的手段发现,水稻种子发育过程中的LOX同工酶多态性是非常丰富的,其中存在有分子量明显小于68 kDa的LOX同工酶。根据不同的分离方法,水稻种子发育过程中的LOX同工酶至少是4-9条。与具有LOX-3活性的越光相比,采用梯度PAGE（5-20%）分析发现,LOX-3缺少品种Daw Dam的LOX缺少谱带分别属于第一和第三类,而且至少有4条谱带缺失;采用IEF（pH3.5-10）分析发现,DawDam种子成熟过程中明显缺少pI为4.56的LOX同工酶（Ⅱ）;采用Western blotting分析也发现,LOX-3缺少品种Daw Dam的LOX缺少谱带分别属于两大类。由于LOX-3缺少品种Daw Dam的LOX-3缺少并不是一个同工酶,而是缺少等电点较小的一类LOX,因此建议把种子发育过程中的LOX同工酶划分为LOX-Ⅰ类、LOX-Ⅱ类和LOX-Ⅲ类。进一步的免疫电镜观察发现,越光和Daw Dam的种子发育进程中,LOX蛋白并不出现在油体上,而是分布在蛋白贮藏囊泡（protein storage vacuole,PSV）的四周,因此与底物在空间上是分离的。但是在贮藏过程中,稻谷通常会自身发生细胞结构损伤和化学成分的改变,前者包括脂质体的溶合、细胞膜完整性下降、透性增加和细胞溶质功能的丧失。因此,也会导致LOX与底物的靠近,启动LOX参与的脂质过氧化,催化生成的脂氢过氧化物再被脂氢过氧化物裂解酶（hydroperoxide lyases,HPLs）和脂氢过氧化物异构酶（hydroperoxide isomerases,HPIs）降解或自动氧化为具有挥发性的己醛、戊醛和戊醇等羰基类低分子化合物,从而产生与稻米陈化变质有关的陈米霉味。此外,也不排除LOX作为储藏蛋白或合成传递生物胁迫和非生物胁迫信息信号分子的可能性。进一步克隆并报道一个新的水稻种子脂氧合酶的编码基因OsLOX3,该基因位于水稻第三染色体,并与OsLOX1/2呈基因簇排列。与OsLOX1/2相比,OsLOX3表达产物的等电点是最小的,其在染色体上的位置与本实验室耐贮性的QTL定位结果也是一致的。与OsLOX3相比,发现MiniOsLOX3在第四外元处存在有点突变,因此发生提前终止的现象,其原核表达产物的分子量为28 kDa,但是也具有一定的酶活性。最后,对位于水稻第三染色体的脂氧合酶基因簇进行了生物信息学分析,包括三维结构、结构域分布、进化树分析和表达谱等,并推测水稻种子LOX基因可能存在有可变剪接的可能性。更多还原

【Abstract】 Food security is a global strategy issue and food storage is one of the key rings in this chain. Under normal storage condition, rice grain deterioration and development of a stale flavor often appear in the second year. According to statistics from Ministry of Agriculture, the loss during storage of rough rice for food and seeds accounts for about 3% of the total production per year in China. In recent years, many investigators have demonstrated that the degradation of lipids was mainly responsible for reducing rice quality and producing stale flavor during storage, and lipoxygenase (LOX, EC 1.13.11.12) was the key enzyme for lipid degradation in above processes. Therefore, the absence of LOX enzymes in crops might decrease lipid peroxidation, alleviate the accumulation stale flavor, and thus increase storability. This assumption has been preliminarily clarified in the relationship between LOX-3-less rice and storability. However, the simple method of screening rice embryos for LOX-3-null has not established. Analysis of LOX isozymes in developing rice seeds, cloning of OsLOX3 gene and its relationship with LOX-3-null have not carried out either.In this paper, based on co-oxidation secondary reaction of LOX-3 and the oxidative ability of 9-hydroperoxide, the catalytic product of LOX-3, two reliable, rapid, simple and inexpensive spectrophotometic methods, named as carotene bleaching and iodide-starch methods, were developed to screen rice embryos for LOX-3-null in comparison with the monoclonal antibodies technique. Thus, above two methods could be used in breeding for storable rice cultivars.There are several LOX isozymes in maturing rice seeds, including some isozymes with molecular weight less than 68 kDa. According to different biochemical separating methods, there exist at least 4-9 isozyme bands. For example, in comparison with rice cultivar (Koshihikari, Japanese rice cultivar) with normal LOX-3 activity, the LOX-3-null cultivar Daw Dam reported by Suzuki et al (1996a, 2000) lack of the first and third types of LOX isozymes by native gradient PAGE (5-20%), also at least four bands disappeared. Furthermore, by using IEF methods (pH3.5-10), LOX isozyme (type II, pI4.56) could not be easily detected in Daw Dam. Meanwhile, two types of LOX isozymes disappeared confirmed by using western blotting method. In view of the fact that the lack of LOX isozymes in Daw Dam was the type of LOX isozyme with lowest pI rather than a single isozyme, we suggested that the LOX isozymes during rice maturation process was classified as three type, named as type I, II and III.In maturing rice seeds, the majority of LOX protein was present in protein storage vacuole (PSV) rather than oil body of both wild-type and LOX-3-null seeds, suggesting that LOX is separated from its substrate. However, physical damage during storage caused direct contact of the lipase with triacylglycerols, contributing the release of free fatty acids, especially the increase of the polyunsaturated fatty acid. Furthermore, LOX could catalyze the oxidation of polyunsaturated fatty acids, including linoleic and linolenic acids, into conjugated hydroperoxy fatty acids. Hydroperoxides were further transformed by autooxidation and enzymatic degradation, including hydroperoxide lyases (HPLs) and hydroperoxide isomerases (HPIs), into various volatile carbonyl compounds, such as hexanal, pentanal and pentanol, adding off-flavor which decreased the quality of strored rice and seriously influenced its storability. Additionally, the possibility that rice embryo LOXs may function as seed storage proteins or be involved in the synthesis signal molecule responsible for abiotic and biotic stresses could not be easily ruled out.Furthermore, cloning of a novel LOX gene OsLOX3 from developing rice seeds, which was located in Chromosome 3, also formed LOX gene cluster with OsLOX1/2. In comparison with the expression products of OsLOX1/2, OsLOX3 exhibited the lowest pI. The location of OsLOX3 in chromosome 3 was also consistent with QTL position related to rice storability reported by our research groups. The expression, purification and characterize of Mini0sL0X3 were also carried out. The 1 bp deletion of adenine residue, which causes a frame-shift, was at the 5’ region of the fourth exon, leads to the product of MiniOsLOX3 with molecular weight being 28 kDa. Meanwhile, lower LOX activity was also detected. Finally, the bioinformatical analysis of LOX gene cluster located in chromosome 3 also illusted three-dimensional structures, domain distribution, phylogenetic analysis and the expressions profiles of the deduced OsLOX1/2/3. Additionally, we deduced that there exists the possibility of alternative splicing.更多还原