黄瓜叶酸合成关键基因克隆与分析

【目的】分析黄瓜基因组中与叶酸合成代谢相关的基因数量、定位以及表达特征,对关键酶基因进行生物信息学分析与克隆,旨在为黄瓜叶酸合成调控研究奠定基础。【方法】根据已报道的拟南芥叶酸合成相关基因,利用黄瓜基因组数据库中9930_V3版本进行BLAST比对。利用MapChart绘制黄瓜染色体物理图谱并对基因定位。利用qRT-PCR分析这些基因在黄瓜果实发育不同时期和不同材料中的表达量。通过MEGA、WebLOGO、ExPASy等工具对关键酶基因进行生物信息学分析。通过PCR扩增对关键酶基因进行克隆,并测序分析基因的序列差异。【结果】同源比对获得19个黄瓜叶酸代谢相关基因,这些基因不均匀分布在黄瓜7条染色体上,且以Chr.4和Chr.5上分布最多。通过对其中11个调控叶酸合成的基因在测序黄瓜9930果实发育不同时期以及果实叶酸含量高低差异显著的2份材料的表达量分析,发现CsFPGS、CsHPPK/CsDHPS和CsDHNA 3个基因与果实叶酸含量变化趋势完全一致;CsADCS、CsADCL、CsDHNA、CsHPPK/CsDHFS、CsFPGS、CsDHFS等基因的表达量在2份材料中具有显著差异。通过对2个调控叶酸合成限速步骤的关键酶基因CsGCHI和CsADCS的蛋白序列及蛋白结构域分析,发现各物种中CsGCHI的同源基因均具有2个GTP_cyclohydroI结构域;CsADCS的同源基因均具有2个GATase结构域、1个Anth_synt_I_N结构域和1个Chorismate_bind结构域。它们在不同物种中高度保守,进化树分析亲缘关系近的物种聚类到一起。分别扩增黄瓜果实低叶酸含量自交系65G和高叶酸含量自交系02245中CsGCHI和CsADCS的同源基因,序列分析表明CsaV3_1G041250全长为3 012 bp,CDS序列长度为1 413 bp,3个SNP位点的突变导致了氨基酸序列的变异;CsaV3_7G026240全长为3 047 bp,CDS长度1 407 bp,序列无变异;CsaV3_5G036360全长7 941 bp,CDS序列长度为2 706 bp,序列无变异。【结论】鉴定出19个不均匀分布在7条染色体上的黄瓜叶酸代谢相关基因,基因CsFPGS、CsHPPK/CsDHPS 、CsDHNA和CsADCS是影响黄瓜果实叶酸含量变化、导致叶酸含量高低显著差异的关键基因,调控叶酸合成限速步骤的关键酶基因GCHI及ADCS功能相对保守,CsGCHI在65G、02245中有3个SNP位点的突变导致了氨基酸序列的差异。     

母鸡叶酸缺乏对子代MTHFD2和TYMS基因甲基化和表达水平的影响

为探究母鸡叶酸缺乏对子代与叶酸代谢相关的MTHFD2和TYMS基因甲基化模式和基因表达水平的影响,利用亚硫酸盐测序和定量PCR方法进行测定。结果表明:1)亲代叶酸缺乏对子代MTHFD2基因启动子区甲基化没有影响,该基因启动子区16个CpG位点不存在甲基化;2)子代TYMS基因ATG下游8个CpG位点甲基化主要发生在第2、3、4和5位点,但这4个CpG位点的甲基化比率在对照组和叶酸缺乏组之间均未达到差异显著水平(P0.05);并且TYMS总体甲基化水平在对照组和叶酸缺乏组之间差异也不显著(P0.05);3)母鸡叶酸缺乏可引起子代肝脏组织MTHFD2和TYMS基因表达水平均显著增加(P0.05)。综上所述,亲代叶酸缺乏对子代MTHFD2和TYMS基因表达水平发生作用,但未对基因甲基化比率造成影响,表明DNA甲基化模式由多个因素决定,可能存在叶酸以外的其他调控机制。     

pH对产叶酸植物乳杆菌叶酸产量及相关基因表达的影响

叶酸是细胞代谢的重要中间体,人类由于缺乏叶酸生物合成途径中的关键酶,需要从膳食中摄取叶酸。由于谷氨酸尾数目的差别,生物合成的叶酸更容易被人体利用。一些植物乳杆菌菌株可以合成叶酸,但培养条件对叶酸合成有较大影响。选择一株高产叶酸的植物乳杆菌4_3菌株,探讨了在同一培养基中,不调节pH和使pH稳定在6.0对其生长状况和叶酸合成的影响。比起不调节pH,维持pH 6.0可以长期保持较高的活细胞密度。实时荧光定量PCR的结果显示,在pH 6.0的培养基中,叶酸合成相关基因表达量均要高于不调节pH的培养基。     

Perspectives of folate biofortification of cereal grains

One sixth of the world’s population is suffering from hidden hunger that indicates a gross malnutrition particularly among children and women of third world countries. The deficiency of micro nutrients, especially iron (Fe) causes a number of ailments such as megaloblastic anemia and neural tube defects in poor population. There is a dire need to supplement iron in the diet. Current efforts implicate fortification of wheat flour and other grains with different iron formulations such as ethylenediaminetetraacetic acid (EDTA), FeSO₄ and elemental iron. However, all such interventions are not sustainable due to logistic and quality assurance problems in resource-limited settings. For a long term solution, development of crop plants with increased micronutrients and iron bioavailability is essential. Therefore, biofortification of cereal grains using translational genomics approaches for enhancement of folate through genome editing in cereals is inevitable to mitigate the folate deficiency in poor remote population in a cost effective manner.     

Review of cobalamin status and disorders of cobalamin metabolism in dogs

Disorders of cobalamin (vitamin B₁₂) metabolism are increasingly recognized in small animal medicine and have a variety of causes ranging from chronic gastrointestinal disease to hereditary defects in cobalamin metabolism. Measurement of serum cobalamin concentration, often in combination with serum folate concentration, is routinely performed as a diagnostic test in clinical practice. While the detection of hypocobalaminemia has therapeutic implications, interpretation of cobalamin status in dogs can be challenging. The aim of this review is to define hypocobalaminemia and cobalamin deficiency, normocobalaminemia, and hypercobalaminemia in dogs, describe known cobalamin deficiency states, breed predispositions in dogs, discuss the different biomarkers of importance for evaluating cobalamin status in dogs, and discuss the management of dogs with hypocobalaminemia.     

叶酸的研究进展

叶酸的开发与应用是当今世界强化食品研究与开发的新热点。介绍了叶酸的结构、特性、制备及其检测方法。重点讲述了叶酸在人体内的吸收、代谢和需要量,以及与人体健康的关系,并综述了叶酸在食品、饲料和医药等领域的应用进展。     

Effect of dietary folate level on organ weight,digesta pH,short-chain fatty acid concentration,and intestinal microbiota of weaned piglets

Folate is increasingly thought to promote gastrointestinal health and regulate the diversity of gut microbiota to alleviate weaning stress in piglets. The present study was conducted to investigate the effects of folate on organ weight, digesta pH, short-chain fatty acids (SCFAs) concentration, and intestinal microbiota in weaned piglets. A total of 28 piglets (6.73 ± 0.62 kg) were allocated to four dietary treatments consisting of a control group, 3, 9, and 18 mg/kg of folate supplementation in a 14-d feeding trial. The results showed that piglets fed with 9 and 18 mg/kg of folate supplementation had greater (P < 0.05) average liver and spleen weight than the control group. Folate supplementation (9 and 18 mg/kg) can significantly increase (P < 0.05) the stomach pH and tend (P < 0.10) to decrease the cecum pH. Folate treatment (9 and 18 mg/kg) had a positive effect on the metabolism of SCFAs in piglets, in particular, compared with the control group, and the content of acetic acid (AA) and valeric acid was markedly increased (P < 0.05) in the cecum and colon, respectively. Moreover, isobutyric acid, butyric acid, and isovaleric acid were tended (P < 0.10) to increase in the colon. Cecum contents samples were used to determine bacterial community diversity by 16S rRNA gene amplicon sequencing. At the genus level, in the cecum, there was a higher (P < 0.05) relative abundance of Lactobacillus reuteri, Lactobacillus salivarius, and Lactobacillus mucosae in the 9 mg/kg folate supplementation group. The functional pathways analysis predicted that folate may modify nutrient metabolism by changing the gut microbiota function of weaned piglets. Furthermore, the data showed that Lactobacillus was positively correlated with AA in the cecum. Overall, these findings suggested that folate treatment could increase the organ weight and the stomach pH of weaned piglets and had beneficial effects on gut health, which might be attributed to the alteration in intestinal microbiota induced by folate and the interaction of the intestinal microbiota with SCFAs.     

Folate in barley grain and fractions

Wholegrain cereals are good sources of folate. However, little is known about folate in barley. In this study, total folate was analysed in five hulled Finnish barley cultivars from three harvesting years (2006–2008). In addition, different fractions were produced from two hulled cultivars by scarification and from mixes of hulled cultivars by industrial milling. Total folate was determined in all samples with a microbiological method and vitamer distribution in scarification and industrial milling fractions, using HPLC or UPLC. Limited variation was observed in the total folate content of the five cultivars. The average contents of the three harvest years ranged from 563 to 773 ng/g dm. Fractions containing germ and outer layers were the richest in folate. The highest total folate content in the scarification fractions was up to 1710 ng/g dm. The total folate content of the hull fraction obtained by dehulling 10% of the grains with an industrial pearler was almost 4-fold compared to the corresponding dehulled grain. This hull fraction is normally not used as food. The main folate vitamers in the fractions were 5-HCO-H₄folate, 5-CH₃-H₄folate, 5,10-CH⁺-H₄folate, and 10-HCO-PGA. Folate content could be enhanced naturally by introducing folate-rich fractions of barley into cereal products.     

过表达蝶呤还原酶PTR1基因促进植物叶酸合成的研究

叶酸是生物体维持正常生命活动所必需的小分子代谢物,还原态的蝶呤参与叶酸的合成。通过利用组成型启动子驱动依赖NADPH的蝶呤还原酶基因（NADPH-dependent pterin reductase gene, PTR1）在模式植物拟南芥和烟草中进行过表达,探讨了来源于利什曼原虫的PTR1基因对植物叶酸合成代谢的影响。结果表明,在过表达PTR1的转基因拟南芥和烟草植株中,叶酸含量有不同程度的升高,其中转基因拟南芥的5-甲酰四氢叶酸有显著提高,而转基因烟草中5-甲基四氢叶酸有显著提高。说明将来源于原生动物的蝶呤还原酶引入植物,可以促进转基因植物中叶酸的合成代谢,为深入了解植物叶酸合成代谢的规律奠定了基础。