大豆胰蛋白酶抑制剂和脂肪氧化酶基因双价RNAi表达载体的改造及转化

【目的】应用RNA干扰技术同时抑制大豆脂肪氧化酶(Lox)和胰蛋白酶抑制剂(KTi)基因的表达,改良大豆品质,培育缺失Lox和KTi的大豆新材料。【方法】应用RNAi原理,以双价RNAi植物表达载体pCAM-BIA1301-KtiRi-LoxRi为基础,以植物表达载体pCAMBIA3301的质粒DNA为模板,利用CaMV35S启动子的上游引物和CaMV35S终止子的下游引物,PCR扩增含有除草剂抗性基因bar的整个表达原件,然后将其插入到pCAM-BIA1301-KtiRi-LoxRi中,构建以除草剂为筛选标记的双价RNAi表达载体,并通过花粉管通道法转化至大豆吉农18、吉农28和吉农27 3个品种中,对转基因植株进行PCR、Southern杂交、RT-PCR分子水平检测和除草剂抗性检测。【结果】质粒PCR和酶切鉴定以及测序结果表明,双价RNAi植物表达载体pCAMBIA1301-KtiRi-LoxRi-bar构建成功。将其转入到大豆中,分别获得吉农18、吉农28、吉农27T1代籽粒47,25和86粒,以及T2代转基因植株50株。RT-PCR结果表明,转基因株系中脂肪氧化酶和胰蛋白酶抑制剂mRNA积累受到明显抑制。【结论】获得了转pCAMBIA1301-KtiRi-LoxRi-bar的T2代转基因大豆。     

俄引春小麦脂肪氧化酶活性基因的标记分析

为了改良黑龙江省小麦品种,加强其种质创新,利用与脂肪氧化酶基因(LOX)活性有关的2个遗传位点相连锁的3对分子标记,对247份俄罗斯引进的春小麦品种进行研究。结果表明:对于SSR标记Xwmc312,142份品种扩增出247bp特异条带,即Xwmc312-247基因型比例为57.49%,85份品种扩增出235bp特异条带,Xwmc312-235基因型比例为34.41%,20份品种扩增出227bp特异条带,Xwmc312-227基因型比例为8.10%。TaLOX-B1位点上,16份品种扩增出标记LOX16的特异条带,即高活性等位基因型TaLoxB1a分布比例是6.48%,230份品种扩增出标记LOX18特异条带,即低活性的等位基因型TaLOX-B1b分布比例是93.12%,有一个品种在此位点表现杂合。表明以TaLox-B1b基因型为主。2个位点不同等位基因组合共有6种,Xwmc312-247/TaLOX-B1b分布比例最高(53.85%),Xwmc312-22 7/TaLOX-B1a分布比例最低(0.41%),高活性组合Xwmc312-235/TaLOX-B1a(分布比例2.43%)等其它4种组合型介于二者之间,表明俄罗斯引进小麦品种以LOX低活性组合型为主。     

68份小麦脂肪氧化酶活性和戊聚糖含量的测定和聚类分析

脂肪氧化酶(LOX)活性和戊聚糖是影响小麦营养和加工品质的重要因素。选用68个小麦品种(系)作为试验材料,测定其籽粒LOX活性、总戊聚糖、水溶性和非水溶性戊聚糖含量,并进行方差分析。结果表明,LOX活性、总戊聚糖、水溶性和非水溶性戊聚糖含量在品种间差异均达到极显著,LOX活性和3种戊聚糖含量的平均值分别为10.35 nkat·g-1、4.49%、0.90%和3.59%,变幅分别为4.50~19.40 nkat·g-1、2.93%~5.95%、0.49%~1.35%和1.92%~5.01%。采用最长距离法对参试的68个小麦品种籽粒的LOX活性、总戊聚糖、水溶性以及非水溶性戊聚糖含量进行聚类分析,结果聚为4类。     

厚朴酚对大鼠白细胞5-脂氧合酶活性和细胞内钙离子浓度的影响

目的：探讨厚朴酚对5—脂氧合酶活性的影响．以阐明其抗炎机理。方法：以大鼠胸腔白细胞为材料，分别用高效液相色谱法和荧光分光光谱法测定白三烯B4(LTB4)，5—羟二十碳四烯酸(5—HETE)的生成和细胞内钙离子水平。结果：厚朴酚对白细胞LTB4和5—HETE的生物合成有较强的抑制作用，其IC50值分别为8．5μmol/L和3．1μmol/L；厚朴酚还可以抑制趋化三肽(fMLP)刺激的白细胞内钙升高．但对静息状态的白细胞内钙离子水平没有明显的影响。结论：厚朴酚可以明显影响白细胞的功能，抑制炎性介质LTB4和5—HETE的生成，提示此功能与其抗炎作用机理有关。     

Compositions of fatty acid hydroperoxide positional isomers in sweet smelt, yellowtail and rainbow trout livers phosphatidylcholine

ABSTRACT: Hydroxy fatty acid isomers derived from phosphatidylcholine hydroperoxides (PC-OOH) in the livers of three fish species, including sweet smelt, yellowtail and rainbow trout, were determined by selected ion monitoring of gas chromatography/mass spectrometry. Total molar amounts of all hydroxy fatty acids determined in the present study coincided well with those of PC-OOH reported previously, suggesting that hydroxy fatty acid composition reflects hydroperoxide composition. The total amount of hydroperoxide isomers accumulated in the livers of sweet smelt was much higher than those of yellowtail and rainbow trout. The amounts of certain isomers, including 10-hydroperoxy octadecanoic acid, 12-hydroperoxy eicosanoic acid and 14-hydroperoxy docosanoic acid, were significantly higher than those of rainbow trout and yellowtail. These results suggest that differences in the contents and compositions of certain hydroperoxide isomers, which are possible precursors of a watermelon-like or cucumber-like aroma, result in differences of fresh fish aroma between aromatic fish and non-aromatic fish.     

Oxylipin Diversity in the Diatom Family Leptocylindraceae Reveals DHA Derivatives in Marine Diatoms

Marine planktonic organisms, such as diatoms, are prospective sources of novel bioactive metabolites. Oxygenated derivatives of fatty acids, generally referred to as oxylipins, in diatoms comprise a highly diverse and complex family of secondary metabolites. These molecules have recently been implicated in several biological processes including intra- and inter-cellular signaling as well as in defense against biotic stressors and grazers. Here, we analyze the production and diversity of C₂₀ and C₂₂ non-volatile oxylipins in five species of the family Leptocylindraceae, which constitute a basal clade in the diatom phylogeny. We report the presence of species-specific lipoxygenase activity and oxylipin patterns, providing the first demonstration of enzymatic production of docosahexaenoic acid derivatives in marine diatoms. The differences observed in lipoxygenase pathways among the species investigated broadly reflected the relationships observed with phylogenetic markers, thus providing functional support to the taxonomic diversity of the individual species.     

Screening soybean (grain and vegetable) genotypes for nutrients and anti-nutritional factors

Fifty six genotypes of grain-type soybean and 17 genotypes of vegetable-type soybean collections were analyzed for protein and oil content, trypsin inhibitor, and lipoxygenase activities. The protein and oil content ranged from 36.9 to 47.9% and from 13.3 to 23.0% for different accessions in grain- and vegetable-type soybeans, respectively. Trypsin inhibitor and lipoxygenase activities ranged from 22.0 to 47.0 trypsin inhibitor units/mg meal and from 482 to 6265 lipoxygenase units/min/mg meal for grain- and vegetable-type soybeans, respectively. Significant correlations (r=–0.62 and –0.52,P<0.05) were found between protein and oil, and between protein and trypsin inhibitor. A significant positive correlation (r=0.42,P<0.05) was also calculated for oil and lipoxygenase activity. Several genotypes of soybean and vegetable soybean (plant introductions 423905, 417330, 417223, 171451, 200506, 200523, 417124, 227687, 203402, 445842, 203399, 423852, 416771, FC31927, Avoyelles, and Sooty) showed good nutritional potential and may be useful in a breeding program to improve the nutritional quality of soybean. Screening for essential amino acids, fatty acids, and trace minerals for selected genotypes is underway.     

水稻种胚脂肪氧化酶Lox1基因的遗传分析及定位

为研究水稻种胚脂肪氧化酶Lox1的遗传规律及分子机制,以Lox1缺失突变体1297分别与Lox1活性正常的9311、日本晴杂交,构建2个F2群体,对2个F2群体种子的种胚Lox1进行定量测定和遗传学分析,结果表明低Lox1是受1对单基因控制的隐性性状。以1297与日本晴组配的F2分离群体300个单株为定位群体,将水稻种胚低Lox1基因定位于水稻第3染色体的RM4512和RM282之间,距两侧标记的遗传距离分别为13.0cM和9.1cM,为进一步的分子标记辅助育种和图位克隆打下了基础。通过人工加速老化对种子进行了耐储性评价,表明水稻种胚Lox1与种子储藏特性关系密切。     

绿叶挥发物代谢调控及分子机理研究进展

植物处于复杂的自然环境中,常受到植食性昆虫、病原菌及机械损伤等生物或非生物的侵害并造成植物的局部伤害,从而启动复杂的防御反应。释放绿叶挥发物（green leaf volatiles,GLVs）是植物防御性反应之一。绿叶挥发物是植物不饱和脂肪酸经过Oxylinpins生化途径中的脂氧合酶（LOX ）和脂氢过氧化物裂解酶（HPL）催化而形成的一类C6和C9醛、醇及其相应的酯类。作为启动植物防御机制的信号分子,这类挥发性物质通过长距离的气态传输在植物与病原菌、植物与植食性昆虫、植物自身和相邻的植物之间起作用,从而介导防御性反应。研究其生化途径及其调节机理,对探索其对病虫害的直接、间接防御、改善作物风味品质及抗病新种质创制起着非常重要的作用,且对园艺植物甚至农林生态系统中作物病虫害综合治理策略的制定具有重要的指导意义。该文综述了绿叶挥发物合成的生化途径及其作为信号转导物质的作用机制,并阐述了绿叶挥发物在植物抗逆方面的生理作用及其应用前景。