Isolation and Analysis of the Cppsy Gene and Promoter from Chlorella protothecoides CS-41

Phytoene synthase (PSY) catalyzes the condensation of two molecules of geranylgeranyl pyrophosphate to form phytoene, the first colorless carotene in the carotenoid biosynthesis pathway. So it is regarded as the crucial enzyme for carotenoid production, and has unsurprisingly been involved in genetic engineering studies of carotenoid production. In this study, the psy gene from Chlorella protothecoides CS-41, designated Cppsy, was cloned using rapid amplification of cDNA ends. The full-length DNA was 2488 bp, and the corresponding cDNA was 1143 bp, which encoded 380 amino acids. Computational analysis suggested that this protein belongs to the Isoprenoid_Biosyn_C1 superfamily. It contained the consensus sequence, including three predicted substrate-Mg²⁺ binding sites. The Cppsy gene promoter was also cloned and characterized. Analysis revealed several candidate motifs for the promoter, which exhibited light- and methyl jasmonate (MeJA)-responsive characteristics, as well as some typical domains universally discovered in promoter sequences, such as the TATA-box and CAAT-box. Light- and MeJA treatment showed that the Cppsy expression level was significantly enhanced by light and MeJA. These results provide a basis for genetically modifying the carotenoid biosynthesis pathway in C. protothecoides.     

野生蕉ω-3脂肪酸去饱和酶基因FAD7密码子偏性分析

为了解FAD7的密码子使用特性,试验采用CodonW、SPSS19.0、MEGA5.0等软件和EMBOSS在线程序对野生蕉FAD7密码子偏性进行分析,同时与其他单/双子叶植物FAD7和模式生物基因组进行比较。结果显示,野生蕉FAD7密码子偏性水平较弱,密码子组成和结尾偏好使用G或C,而CUC、CAG和AGG等在该基因中具有较高的使用频率;物种间FAD7比较结果发现,单子叶植物FAD7密码子组成明显偏好G或C,而双子叶植物则完全相反;基于密码子使用频率FAD7聚类结果与CDS分类结果一致,均能将单/双子叶植物区分开来;此外,大肠杆菌原核表达受体系统可作为野生蕉FAD7基因异源表达理想试验体系。研究结果为野生蕉FAD7后续结构和功能研究提供了科学依据。     

Effects of dietary sunflower oil on growth parameters,fatty acid profiles and expression of genes regulating growth and metabolism in the pejerrey (Odontesthes bonariensis) fry

Aquaculture fish diets usually contain an addition of fish oil to improve their nutritional value. The effect of the replacement of dietary fish oil (FO) by sunflower oil (SfO) on growth, fatty acid composition and expression of genes implicated in somatic growth, feed intake and fatty acid metabolism was studied in pejerrey fry. Fry were fed per 45 days with diets containing FO/SfO ratios of 100% FO; 50% FO:50% SfO; 20% FO:80% SfO; and 100% SfO. No differences were detected in growth and in the total per cent of saturated and monounsaturated fatty acids. Gh, ghr‐I and ghr‐II showed a higher mRNA expression in head and trunk of fry fed with 100% SfO diet. Expression of igf‐II was higher in trunk of fry fed with 100% SfO diet compared with 100% FO diet. The Δ6‐desaturase gene expression was upregulated in head and trunk of fry fed with 100% SfO diet. The nucb2/nesfatin‐1 gene expression decreased in the trunk of fry with increasing dietary SfO. We conclude that the replacement of fish oil by sunflower oil in pejerrey fry feed does not affect growth and is a viable strategy to reduce production costs of this fish.     

Adiposity, fatty acid composition, and delta-9 desaturase activity during growth in beef cattle

Oleic acid (18:1n‐9) is the most abundant fatty acid in bovine adipose tissue. Because most of the lipid in bovine muscle is contributed by intramuscular adipocytes, oleic acid also is the predominant fatty acid in beef. In many species, the concentration of oleic acid in adipose tissue is dictated by the average concentration of oleic acid in the diet, but in ruminant species such as beef cattle, oleic acid is hydrogenated largely to stearic acid by ruminal microorganisms. In these species, the concentration of oleic acid in adipose tissue is dependent upon the activity of Δ⁹ desaturase, encoded by the stearoyl coenzyme A desaturase (SCD) gene. Expression of the SCD gene is essential for bovine preadipocyte differentiation, and desaturase gene expression and catalytic activity increase dramatically as adipose tissue mass increases after weaning. Feeding a hay‐based diet to American Wagyu steers to a typical Japanese bodyweight endpoint (650 kg) markedly stimulated desaturase enzyme activity as well as the accumulation of both oleic acid and intramuscular lipid, but the increase in oleic acid and intramuscular lipid was much less in hay‐fed Angus steers. Increasing the concentration of oleic acid improves the palatability and healthiness of beef, and Korean Hanwoo and Japanese Black (and American Wagyu) seem especially well adapted to accumulate oleic acid in their adipose tissue.     

甘蓝型油菜FAD2基因调控序列的克隆及瞬时表达分析

油酸去饱和酶FAD2（fatty acid desaturase 2）是广泛存在于植物中的一种能催化油酸脱氢生成亚油酸的还原酶。根据GenBank上已知的甘蓝型油菜FAD2基因设计引物,以甘蓝型油菜（Brassica napus L.）叶片总DNA为模板,进行TAIL-PCR扩增,获得约1.4 kb片段并测序。序列比对结果说明该片段为已知的FAD2基因编码区上游序列。将该片段进行不同长度的5′端缺失,并用缺失后的序列替换pBI221-LUC质粒上的CaMV35S启动子,构建了甘蓝型油菜瞬时表达载体,进行油菜原生质体瞬时表达的初步研究,结果表明该序列5′端-669 bp至-1019 bp对报告基因表达水平有较大的影响。结合启动子预测分析结果,此片段中可能存在的赤霉素应答元件、光调控元件等顺式作用元件对FAD2基因的表达调控具有重要作用。     

Mode of bleaching phytotoxicity of herbicidal diphenylpyrrolidinones

A mode of action study of herbicidal diphenylpyrrolidinones was carried out through carotenoid analyses in intact Scenedesmus cells and by a cell‐free plant‐type phytoene desaturase assay using Escherichia coli transformants. A series of forty‐eight diphenylpyrrolidinones decreased the carotenoid content of Scenedesmus cells in the light and inhibited phytoene desaturase. The relationship between substituents at various positions and inhibition of phytoene desaturase is discussed. Using very active bleaching diphenylpyrrolidinones, a 10⁻⁵ M concentration affected neither the ζ‐carotene desaturase nor the protoporphyrinogen‐IX oxidase. Although some differences in their inhibitory activity were found between the in vivo and cell‐free assays, it is concluded that the compounds are essentially bleachers affecting carotenoid biosynthesis in plants. Enzyme kinetics studies with recombinant phytoene desaturase revealed a non‐competitive inhibition with respect to the substrate phytoene. A competition against the inhibitor was shown by the cofactor NADP⁺, suggesting an interaction of pyrrolidinones at the cofactor‐binding site of phytoene desaturase. © 2001 Society of Chemical Industry     

花生FAD2基因家族表达分析及其对低温胁迫的响应

为了探究FAD2在花生低温响应中的作用,本研究从普通油酸花生中花16 (ZH16)和高油酸花生中花413 (ZH413)中克隆得到花生AhFAD2家族的全部基因,共7个。通过分析这些基因的表达模式发现,在ZH16和ZH413中各FAD2基因表达模式相似, AhFAD2-1A/B主要在花和发育的种子中表达, AhFAD2-3A/B主要在营养组织中表达, AhFAD2-4A/B主要在根和花中表达,表明AhFAD2基因在花生不同发育阶段和不同组织中发挥各自的生物学功能。在15℃下发芽6 d发现,ZH413的发芽率未显著下降,而ZH16的发芽率显著下降。种子萌发过程中,AhFAD2-1A/B和AhFAD2-4A/B均受低温诱导表达。在ZH16中AhFAD2-1A/B在低温诱导第6天开始显著上调表达,而在ZH413中第1天显著上调表达;在ZH16中AhFAD2-4A/B在低温诱导第3天出现显著上调表达,之后表达量下降,但在ZH413中第1天就显著上调表达,且始终维持在高水平表达。基于以上研究结果推测,高油酸花生在受到低温胁迫时,AhFAD2-1A/B编码蛋白失活,但AhFAD2-4A/B的高量表达在一定程度上弥补了这部分功能。同时也说明AhFAD2-1A/B功能的缺失并不是决定花生耐寒性的主要因素。本研究的开展为培育抗寒的高油酸花生品种奠定了理论基础,为高油酸花生在高纬度、高海拔地区推广提供了理论支持。     

甘蓝型油菜油酸脱氢酶基因(fad2)多个拷贝的发现及分析

以甘蓝型油菜湘油15为材料, 随机挑选56个来自基因组的fad2基因克隆、47个来自幼苗整株的fad2基因cDNA克隆和9个授粉27 d后种子中fad2 cDNA克隆进行双向测序。基因组中56个fad2序列的碱基同源性为91.0%~99.9%, 从中得到11个差异序列, 即11个不同的fad2基因拷贝。将其翻译成氨基酸序列发现, 6个拷贝在编码区中出现多个终止密码子, 另外5个的同源性为90.60%~99.74%, 与47个来自幼苗整株的fad2基因cDNA序列进行比较, 发现fad2基因没有内含子。从种子中的9个fad2 cDNA克隆序列中找到2个有差异的cDNA, 它们的编码区中没有终止密码子, 说明在种子中有多个fad2基因表达。基因组中的11个拷贝根据同源性可分成两组, 命名为fad2I和fad2II。RT-PCR分析发现在授粉27 d的种子中fad2I有较强表达, fad2II没有表达; 但在叶片中两者都有表达。     

油茶SAD基因的全长cDNA克隆及生物信息学分析

油茶( Camellia oleifera )是原产于我国的重要木本油料树种,广泛分布于我国南方,总面积达350万hm 2,种仁含油率约为55%.所产茶油富含90%以上的油酸、亚油酸等不饱和脂肪酸,还含有少量的亚麻酸等高价不饱和脂肪酸(胡芳名等,2006),是易消化耐贮藏的优质食用植物油,其油质甚至一定程度上优于橄榄油.