菠萝酸性转化酶基因家族初步生物信息学及表达分析

酸性转化酶（acid invertase, AIN）在菠萝采后蔗糖降解过程中起着重要作用,基于菠萝全基因组数据库,预测菠萝AIN家族基因并进行生物信息学分析,解析其在采后菠萝不同贮藏温度下的表达变化情况,为阐明AIN基因在采后菠萝果实贮藏特性中的作用奠定基础。以水稻AIN家族基因为探针,在菠萝全基因组中鉴定到2个菠萝细胞壁酸性转化酶基因（cell wall acid invertase, CWIN）和2个液泡酸性转化酶基因（vacuolar acid invertase, VIN）,分别命名为AcCWIN1、AcCWIN2、AcVIN1、AcVIN2,设计编码区引物进行测序验证,并进行生物信息学分析。进化分析结果表明,AcCWIN1、AcCWIN2和AcVIN1、AcVIN2蛋白分别归于细胞壁酸性转化酶和液泡酸性转化酶2个进化支上,且均属于糖基水解酶家族GH32,基因结构、保守域和保守基序均一致。荧光定量分析结果表明,菠萝果肉中AcVIN1和AcVIN2在果实采后贮藏过程中表达量升高,且AcVIN1在发生黑心病的部位大量表达,而AcCWIN1和AcCWIN2在采后贮藏过程中表达量逐渐降低,且随着贮藏温度的升高其表达量降低,预示AcVIN1、AcVIN2较AcCWIN1、AcCWIN2在菠萝采后蔗糖降解和黑心病的发生方面发挥着更为重要的作用。     

Roles of protein phosphatase 4 in down-regulation of eNOS Ser633 phosphorylation induced by palmitic acid in human umbilical vein endotheli?al cells

AIMTo investigate the roles of protein phosphatase 4 (PP4) in down-regulation of endothelial nitric oxide synthase (eNOS) Ser633 phosphorylation induced by palmitic acid (PA). METHODSHuman umbilical vein endothelial cells (HUVECs) were treated with PA at 25 μmol/L, 50 μmol/L, 100 μmol/L and 200μmol/L for 36 h, or treated with PA at 100 μmol/L for 12 h, 24 h, 36 h and 48 h. Protein phosphatase 2A (PP2A) family inhibitor fostriecin (FST, 20 nmol/L) or okadaic acid (OA, 5 nmol/L) was selected to pretreat the HUVECs for 30 min. Protein phosphatase 4 catalytic subunit (PP4c) siRNA or protein phosphatase 2A catalytic subunit (PP2Ac) siRNA was transfected into the HUVECs. The protein expression levels of of eNOS, PP4c and PP2Ac, as well as the level of eNOS Ser633 phosphorylation, were detected by Western blot. The intracellular nitric oxide (NO) content was measured by DAF-FM DA. RESULTS(1) Compared with control group, the levels of eNOS Ser633 phosphorylation were decreased in PA groups in which the HUVECs were treated with 25 μmol/L, 50 μmol/L, 100 μmol/L and 200 μmol/L PA for 36 h (P<0.05) and 100 μmol/L PA for 24 h, 36 h and 48 h (P<0.05). No significant difference in the level of total eNOS protein expression among all the groups was observed. (2) Compared with control group, both FST and OA pretreatment reversed the reduction of eNOS Ser633 phosphorylation (P<0.05) and the decrease in intracellular NO content (P<0.05) induced by PA. No significant difference in the level of total eNOS protein expression among all the groups was observed. (3) Compared with si-Control group, the PP4c protein expression was significantly reduced (P<0.05), while the level of eNOS Ser633 phosphorylation was significantly increased in si-PP4c group (P<0.05). Although the levels of PP2Ac protein expression declined significantly (P<0.05), the level of eNOS Ser633 phosphorylation remained unchanged in si-PP2Ac group. No significant differencein the level of total eNOS protein expression among all the groups was found. CONCLUSION PA significantly reduces the level of eNOS Ser633 phosphorylation and the content of NO in the HUVECs, which may be due to PA inducing the activation of the PP2A family member PP4 rather than PP2A.     

Effects of apelin-13 on oxidative stress induced by high uric acid in adipocytes

AIM: To study the effects of apelin-13 on oxidative stress induced by high uric acid in 3T3-L1 adipocytes and its underlying mechanisms. METHODS: 3T3-L1 adipocytes were stimulated with uric acid at 10 mg/dL for 48 h. Some of the adipocytes were administered with 1 μmol/L apelin-13 in the presence of uric acid at 10 mg/dL. The adipocytes stimulated with 100 μmol/L H₂O₂ were served as positive controls. The intracellular reactive oxygen species (ROS) concentrations were detected by flow cytometry. The biochemical kits were used to measure the activities of superotide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and NADPH oxidase (NOX) activity, and the content of malondialdehyde (MDA) in the cell lysate and the supernatant. The mRNA levels of renin-angiotensin system (RAS) components, including angiotensinogen (AGT), angiotensin-converting enzyrne1 (ACE1), angiotensin II type 1 receptor (AT1R) and AT2R, as well as angiotensin II receptor -like 1 (APJ) were measured by real-time PCR. The concentrations of angiotensin II (AngⅡ) in the cell lysate and the supernatant were measured by ELISA. RESULTS: Adipocytes stimulated with uric acid at 10 mg/dL had lower activities of antioxidant enzymes (SOD, GSH-PX and CAT) and higher levels of NOX activity and MDA content (P < 0.05). Accordingly, the intracellular ROS levels were found to be dramatically increased. However, apelin-13 administration attenuated uric acid-induced oxidative stress in the 3T3-L1 adipocytes. Uric acid at 10 mg/dL upregulated the mRNA expression of local RAS, enhanced AngⅡ concentrations both in the cell lysate and the supernatant, and down-regulated the mRNA level of APJ in the adipocytes (P < 0.05). Conversely, apelin-13 partially reversed these parameters. CONCLUSION: Apelin-13 attenuates oxidative stress induced by uric acid, may be via down-regulation of local RAS expression in the 3T3-L1 adipocytes.     

刺萼龙葵种子中适宜内参基因的筛选

入侵杂草刺萼龙葵Solanum rostratum Dunal传播扩散的主要载体是种子,研究其种子休眠萌发基因的激素调控对于其防除具有重要意义,而选择合适的内参基因可以提高相关基因表达分析的准确性。本研究以赤霉素、脱落酸和水处理的刺萼龙葵种子为材料,利用geNorm、NormFinder、BestKeeper和RefFinder 4种软件对15个候选内参基因进行表达稳定性评价,并通过检测ABI5(abscisic acid-insensitive 5)的表达验证所筛选的内参基因的适用性。结果表明,对于赤霉素、脱落酸和水处理过的种子,最稳定的内参基因分别为eIF(eukaryotic initiation factor)、SAND(SAND protein family)和ACT(β-actin);对所有种子样本而言,PP2Acs(a catalytic subunit of protein phosphatase 2A)是最稳定的内参基因。研究结果将为刺萼龙葵种子休眠萌发的遗传调控研究提供重要参考。     

光氮互作对芹菜幼苗生长及生理特性的影响

光质和施氮量是影响芹菜生长发育的关键因素,适宜的光氮组合能有效提升芹菜幼苗质量。为优化芹菜工厂化育苗,本试验设置2种光质(白光,W;蓝光,B)和2种施氮量(8mmol/L KNO_3,高氮,H;4mmol/L KNO_3,低氮,L),以WH为对照,研究光氮互作对芹菜幼苗生长、生理代谢和元素积累的影响。结果表明:与WH相比,WL和BH处理的芹菜全株干重分别显著减少43.18%和55.07%,WL处理的叶片和叶柄中硝酸盐、可溶性蛋白质和总游离氨基酸质量分数均显著降低,BH处理的叶片和叶柄中硝酸盐、可溶性蛋白质和矿质元素质量分数显著增加,而叶片中可溶性糖、丙氨酸族和丝氨酸族氨基酸质量分数均显著降低。然而BL处理的芹菜全株干重比WH显著增加32.18%,叶片可溶性蛋白质、叶柄组氨酸(His)和脯氨酸(Pro)质量分数均显著增加。利用隶属函数分析对芹菜幼苗生长发育进行综合评价发现,BL处理表现最优。综上所述,蓝光和低氮组合能促进芹菜干物质积累,提高可溶性蛋白质和游离氨基酸质量分数,进而促进芹菜幼苗生长发育。     

L-肉碱对眼点拟微绿球藻种群增长及生化组成的影响

【目的】探究L-肉碱对眼点拟微绿球藻(Nannochloropsis oculata)种群增长及生化组成的影响,为L-肉碱对眼点拟微绿球藻营养调控作用和调控机理研究提供参考。【方法】分别采用不同质量浓度(0(对照组),5,50,100 mg/L)的L-肉碱强化培养眼点拟微绿球藻6 d,每组3个重复。每天检测眼点拟微绿球的种群密度,试验结束时收集样品并检测眼点拟微绿球的生化组成。【结果】5 mg/L L-肉碱组眼点拟微绿球的种群密度显著高于其他组(P0.05)。5和50 mg/L L-肉碱组眼点拟微绿球藻总糖含量无显著差异,但均显著高于对照组(P0.05),而显著低于100 mg/L L-肉碱组(P0.05)。5 mg/L L-肉碱组眼点拟微绿球藻的总脂含量显著低于其他3组(P0.05),而其他3组间无显著差异,但以100 mg/L L-肉碱组最高。5 mg/L L-肉碱组眼点拟微绿球藻的可溶性蛋白含量最高,显著高于其他3组(P0.05)。5 mg/L L-肉碱组眼点拟微绿球藻的叶绿素a含量最高,虽与对照组无显著差异(P0.05),但均显著高于50和100 mg/L L-肉碱组(P0.05),而50 mg/L L-肉碱组显著高于100 mg/L L-肉碱组(P0.05)。5和50 mg/L L-肉碱组眼点拟微绿球藻的∑SFA含量显著低于对照组和100 mg/L L-肉碱组(P0.05)。5 mg/L L-肉碱组眼点拟微绿球藻的∑MUFA含量显著高于其他3组(P0.05),其他3组间无显著差异(P0.05)。5和50 mg/L L-肉碱组眼点拟微绿球藻的∑PUFA、∑EPA+DHA和∑n-3含量均显著高于对照组和100 mg/L L-肉碱组(P0.05)。5和100 mg/L L-肉碱组眼点拟微绿球藻的∑n-6含量显著低于对照组和50 mg/L L-肉碱组(P0.05)。【结论】在本试验条件下,添加5 mg/L L-肉碱能显著促进眼点拟微绿球藻的种群增长,提高其可溶性蛋白和总糖含量,并能显著改善其脂肪酸组成。     

台湾青枣乳酸发酵饮料的研制

通过单因素试验(青枣果肉添加量、蔗糖添加量、奶粉添加量、接种量、发酵温度和发酵时间)研究青枣发酵饮料的感官品质,通过正交试验,确定台湾青枣发酵饮料的最佳配方为青枣果肉添加量30%,蔗糖添加量10%,奶粉添加量6%;青枣发酵乳酸饮料的最佳发酵工艺为发酵时间35 h,发酵温度30℃,接种量0.25%。     

胡麻脂肪酸去饱和酶基因(fads)差异表达分析

为了了解fad基因在胡麻蒴果发育过程中对不饱和脂肪酸的调控,对高、中、低三个不同亚麻酸(C18:3)含量的胡麻品种(’CDC Gold’,‘内亚7号’,’Linola’)进行了不同时期的品质测定,以及脂肪酸去饱和酶2a基因(fad2a)、脂肪酸去饱和酶2b基因(fad2b)、脂肪酸去饱和酶2c基因(fad2c)、脂肪酸去饱和酶3a基因(fad3a)、脂肪酸去饱和酶3b基因(fad3b)的qRT-PCR定量分析。结果表明,随着蒴果成熟,可溶性糖含量呈降低趋势,粗脂肪与粗蛋白不断积累,且差异显著(p<0.05)。fad2a基因、fad3a基因以及fad3b基因在各个时期中的表达符合正态分布。以0 d的蒴果为对照,在胡麻种子形成过程中,‘内亚7号’的三个基因在5 d和15 d的表达量迅速增加,到30 d时急剧减少,15 d的fad2a基因表达量为5.23倍,fad3a基因表达量是fad2a基因表达量的14.52倍,fad3b基因的表达量是fad2a基因表达量的16.14倍,表明这三个基因参与不饱和脂肪酸积累过程。在低亚麻酸含量品种‘Linola’30 d中,fad3a基因是fad2a基因表达量的52.71倍,fad3b呈下调趋势;在高亚麻酸含量品种‘CDCGold’30d中,fad3a基因与fad2a基因的表达量均呈下调趋势,fad3b的表达量为3.92倍;在中等亚麻酸含量品种‘内亚7号’中,fad2a基因表达量降低了0.31倍,fad3a基因是fad3b基因表达量的1.87倍。fad2b基因、fad2c基因熔解曲线不稳定,峰值低,可以在后续试验中继续探索。     

苜蓿中草酸含量的测定及煮制对其含量影响研究

紫花苜蓿营养丰富,越来越受到大家的青睐,但苜蓿中的草酸会对人体健康产生间接影响.本文通过对比实验研究了煮制对苜蓿中草酸含量的影响情况.结果显示:新鲜苜蓿中含有一定量的草酸,但煮制后能去除约75％的草酸,故水煮苜蓿是有效去除草酸并保留营养成分的首选方法.