水稻可溶性淀粉合成酶基因SSⅡa和SSⅢa的功能、等位变异及其互作研究进展

水稻淀粉合成对稻米品质的影响研究一直备受关注,是水稻基础科学研究的热点和难点之一。淀粉合成途径受众多酶催化调控,可溶性淀粉合成酶(soluble starch synthase, SSS)是其中较重要的一种,极大地影响稻米食味品质的形成。可溶性淀粉合成酶基因SSⅡa和SSⅢa是控制稻米糊化温度和胚乳支链淀粉生物合成途径中的两个关键基因。本文重点回顾并归纳了国内外关于SSⅡa和SSⅢa基因的功能、等位变异及其互作对稻米蒸煮食味品质影响的最新研究进展,同时对SSⅡa和SSⅢa基因的育种利用前景进行了展望,以期为稻米品质分子改良和育种提供参考依据。     

苎麻木质素合成酶CAD基因的cDNA克隆及表达分析

采用PCR结合RACE技术克隆苎麻栽培种湘苎3号CAD基因全长cDNA序列;再运用qRT–PCR技术对木质素含量不同的代表性苎麻品种湘苎1号、湘苎3号、湘潭大叶白和城步青麻的CAD基因在其茎秆韧皮部和木质部的表达进行定量分析;使用1%间苯三酚和25%盐酸对4种苎麻茎横切片进行特异染色,观察品种间木质素染色度。结果表明,获得的苎麻CAD基因cDNA序列全长为1 378 bp(GenBank登录号,KF758396),编码359个氨基酸,推导为苎麻肉桂醇脱氢酶(BnCAD)。该推导蛋白质与已报道的几种植物木质素合成酶CAD的同源性均达90%以上,其中与蒺藜苜蓿的同源性达97%。运用qRT–PCR方法对BnCAD基因表达的定量分析结果显示,苎麻CAD基因在湘苎1号和湘苎3号的韧皮部表达水平明显高于其在木质部的表达水平,而在城步青麻木质部的表达水平高于其在韧皮部的表达水平,该基因在湘潭大叶白的韧皮部和木质部表达量接近。4种苎麻茎横切片的木质素染色结果表明,品种间木质素染色度与CAD基因在其韧皮部的表达量成正相关。     

不同植物黄酮醇合成酶FLS的生物信息学分析

采用生物信息学方法和工具分析19种不同植物中的黄酮醇合成酶基因的核苷酸序列及编码氨基酸序列,对其理化特性、功能结构域、亲/疏水性、二级结构、信号肽、跨膜结构域以及分子系统进化关系等进行预测和推断,并构建分子进化树。结果表明,不同植物FLS基因的开放阅读框全长在1.0 kb左右,编码蛋白含330余个氨基酸,呈酸性,是一个没有任何信号肽、转运肽和跨膜结构域的亲水性蛋白。不同植物FLS氨基酸序列包含有黄酮醇合成酶功能域,而且琢-螺旋和不规则盘绕是其蛋白质二级结构的最大量结构元件。19个植物FLS在分子进化树中主要聚成3小支,来源于同科植物的FLS首先聚在一起,再与其他物种FLS聚在一起,进化分析在一定程度上反映了这些物种FLS的进化关系。     

不同植物LDOX/ANS基因的生物信息学分析

花青素是一类重要的植物次生代谢产物。本文采用生物信息学的方法对已在GenBank上登录的拟南芥、西洋梨、苹果、桃、葡萄、芥菜、菊花新品种和水稻等植物的无色花青素双加氧酶/花青素合成酶(LDOX/ANS)基因的核酸及氨基酸序列、组成成分、导肽、信号肽、跨膜结构域、疏水性/亲水性、蛋白质二级结构、三级结构及功能域等进行预测和推断。结果表明,ORF除了桃和菊花新品种为500bp左右之外,其它几种植物都在1070bp左右,分子量均为4kD左右,理论等电点均低于7,说明LDOX/ANS呈酸性。Glu、Leu和Val是这些植物共有的主要氨基酸。核苷酸同源性比对结果显示,拟南芥LDOX/ANS与其它植物LDOX/ANS的同源性较高;8个物种的LDOX/ANS基因被分为两个大类,单子叶植物水稻LDOX/ANS基因被单独分为一类,其它的均聚成一大类。研究还发现这些植物的LDOX/ANSN端不存在导肽和信号肽,无跨膜结构域,肽链表现为亲水性。蛋白质二级结构中最主要的结构元件是α-螺旋和无规则卷曲,包含一个20G-FeⅡOxy功能结构域。通过此次研究,希望为今后深入研究该类酶的功能和结构特征提供依据。     

马铃薯野生种CHS基因cDNA的克隆与表达分析

设计简并引物,用RT-PCR法从马铃薯野生种(Solanum pinnatisectum)克隆到了CHS(Chalcone synthase,查尔酮合酶)基因的全长cDNA,并用半定量RT-PCR法进行了表达分析.序列分析表明,CHS基因编码一个389个氨基酸残基的多肽,在氨基酸水平上与其他茄科植物的同源性达到89%～93%,多重比较和系统发育分析均表明该基因为CHS家族中的一个新成员;检测了CHS基因的空间表达模式,该基因在花和匍匐茎中表达量最高,在根和块茎中没有检测到,这与花和匍匐茎呈淡紫色有花色苷合成,而根和块茎呈白色无花色苷合成是相一致的;发现在块茎中CHS受光诱导表达,光照后的第3天表达量最高,而在光照前检测不到,光照前块茎为白色,随着光照时间的延长,其表层颜色因积累花色苷而变成紫红色.     

Evaluation of Root Sink Ability of Sweet Potato (Ipomoea batatas Lam.) Cultivars on the Basis of Enzymatic Activity in the Starch Synthesis Pathway

Activities of five enzymes, sucrose synthase (SUS), uridine 5'-diphosphate glucose pyrophosphorylase (UDPGPPase), fructose-1,6 bisphosphatase (FBPase), adenosine 5'diphosphate glucose pyrophosphorylase (ADPGPPase) and starch synthase (STS), in the metabolic pathway of starch synthesis, were compared between two sweet potato ( Ipomoea batatas Lam.) cultivars, Koganesengan (C.V.KOG, a recently released high yield cultivar) and Tsurunasigenji (C.V.TSU, an old, local cultivar with poor yield). The measurements were carried out using the root samples (tuberous, thick and fibrous roots) harvested at the fast tubering stage. Of the five enzymes, SUS, ADPGPPase and STS showed high activities in the tuberous root, particularly in that of C.V.KOG, and a similar trend was observed for activities of these enzymes on a protein basis. The increased activity of the three enzymes is considered to be one of the characteristics in a high yield cultivar, allowing the root to function effectively as a starch synthesis and storage organ.     

Astragalus polysaccharides protects against free fatty acid-induced human vascular endothelial cell dysfunction via AMPK-eNOS pathway

AIM: To study the protective effect of Astragalus polysaccharides (APS) on free fatty acid-induced injury in human umbilical vein endothelial cells (HUVECs). METHODS: Cultured HUVECs were divided into control group, APS group [APS (200 mg/L) treated for 24 h], free fatty acid group [free fatty acid (0.25 mmol/L) treated for 24 h], free fatty acid plus APS group [free fatty acid (0.25 mmol/L) and APS (200 mg/L) treated for 24 h], and compound C group [free fatty acid (0.25 mmol/L) and APS (200 mg/L) and AMPK inhibitor compound C (10 μmol/L) treated for 24 h]. The cell viability was detected by MTT assay. Nitric oxide (NO) content in the medium was determined by nitrate reductase assay. The protein levels of total adenosine monophosphate-activated protein kinase (AMPK), phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK), endothelial nitric oxide synthase (eNOS) and phosphorylated endothelial nitric oxide synthase (p-eNOS) were measured by Western blot. RESULTS: No significant difference of all indexes between APS group and control group was observed. The cell viability in free fatty acid group decreased significantly compared with control group. The cell viability in free fatty acid plus APS group was significantly improved as compared with free fatty acid group. The cell viability in compound C group was almost the same as that in free fatty acid group. The No content and protein levels of p-AMPK and p-eNOS in free fatty acid group decreased obviously as compared with control group, while the NO content and protein levels of p-AMPK and p-eNOS in free fatty acid plus APS group increased obviously compared with free fatty acid group. No significant difference of the p-AMPK and p-eNOS protein levels between free fatty acid plus APS group and free fatty acid group was observed. No significant difference of the AMPK and eNOS protein levels in all groups was found. CONCLUSION: APS attenuates the free fatty acid-induced injury, and its mechanism is related to the AMPK-eNOS signal pathway.     

Effects of eplerenone on expression and activity of aortic endothelial nitric oxide synthase in hypertensive rats induced by high-salt intake

AIM: To explore the effects of eplerenone on the expression and activity of aortic endothelial nitric oxide synthase(eNOS) in high salt-induced hypertensive rats.METHODS: Male Wistar rats(4 week old, weighting 50～60 g) were randomly divided into control group, high-salt diet group and eplerenone group. The rats in control group were fed with ordinary rodent animal diet, the rats in high-salt group and eplerenone group were exposed to 5% salt diet for 16 weeks and administrated with the same dosage of saline or eplerenone(40 mg·kg^-1·d^-1) by gavage for 4 weeks, respectively. Systolic blood pressure(SBP) was measured by tail-cuff every 2 weeks. The rats were sacrificed after 16 weeks and the thoracic aorta was collected. The aldosterone content in the aorta was measured by ELISA. The protein levels of mineralocorticoid receptor(MR) and eNOS were determined by Western blot. The activitie of constitutive NOS(cNOS) was measured by chemocolorimetry. The protein localization of eNOS, neuronal nitric oxide synthase(nNOS) and MR was observed by immunohistochemistry.RESULTS: A process of 8-week high-salt diet increased SBP gradually. SBP in the rats exposure to high salt for 16 weeks was significantly higher than that in control group(P<0.05). After 4 weeks of eplerenone treatment, SBP in the rats was significantly lower than that before treatment(P<0.05). Compared with control group, the aldosterone content in the aorta were significantly increased in high-salt diet group and eplerenone group(P<0.05), the expression level of MR also increased significantly(P<0.05). Compared with control group, both eNOS protein expression(P<0.05) and cNOS activity in high-salt diet group were significantly decreased(P<0.05). The protein expression of eNOS as well as cNOS activity in aorta increased significantly in eplerenone group compared with high-salt diet group(P<0.05).CONCLUSION: Aldosterone content in aorta of high-salt-induced hypertensive rats increases significantly. Aldosterone attenuates the protein expression of eNOS and reduces the enzyme activity through the activation of mineralocorticoid receptor. The selective mineralocorticoid receptor antagonist eplerenone enhances the protein expression of eNOS and its activity, thereby improves eNOS function.     

水稻灌浆期籽粒中3个与淀粉合成有关的酶活性变化

在水培和盆栽条件下，研究了6个水稻品种(含籼/粳杂交组合、新株型品系)灌浆期强、弱势粒中ADPG焦磷酸酶(EC2.7.7.21)、淀粉合成酶(EC2.4.1.21)和淀粉分枝酶或Q-酶(EC2.4.1.18)的活性变化及其与灌浆充实的关系。3个酶的活性变化与籽粒灌浆动态相关联：淀粉酶最高活性出现的时间稍前或同步于最大灌浆速率的时间；ADPG焦磷酸酶