辣椒小G蛋白CaRab8基因全长cDNA的分离及表达特征的初步分析

通过对辣椒均一化cDNA文库的筛选,分离获得了一个与烟草RAB8-2基因编码的蛋白有着高度同源性的cDNA阳性克隆,命名为CaR ab8.测序结果表明,该cDNA长度为961 bp,包含有651 bp的完整的开放阅读框,推测编码一个217个氨基酸的蛋白,该蛋白具有GTP/GDP结合活性必需的保守域和包括YYRGA在内的...     

中国水仙NtMYB1基因的克隆及表达分析

为研究调控花青素合成机制,利用RT-PCR和RACE技术从中国水仙‘金盏银台’（Narcissus tazetta var. chinensis‘Jinzhan Yintai’.）的花瓣和副冠中克隆得到一个MYB基因的cDNA序列,命名为NtMYB1,在GeneBank上登录号为KC763973。序列分析表明,NtMYB1基因全长842 bp,其中开放阅读框长681 bp,编码226个氨基酸,推测的蛋白分子量为25.2 ku,理论等电点为7.94。NtMYB1具有明显的R2R3-MYB结构域,且在R3结构域的下游含有一个相对保守的PDLNLDLSIG氨基酸基序。同源性分析表明,其编码的氨基酸序列与陆地棉、甜樱桃的MYB蛋白相似性分别达到65％和63％。利用实时荧光定量PCR技术检测NtMYB1基因在中国水仙花不同发育时期和部位的表达水平,分析结果表明,NMYB1基因在中国水仙成花不同时期均有表达,但表达量具有明显差异,其中NtMYB1基因在花蕾期的相对表达量约为花苞期的40倍,且其表达量在花瓣要高于副冠。推测NtMYB1可能参与调控花青素合成基因的转录沉默。     

琯溪蜜柚CmPME1的克隆及其在果实发育过程中的表达分析

利用RT-PCR技术从‘琯溪蜜柚’中克隆得到1条PME（pectinmethylesterase）基因的ORF序列,命名为CmPME1。该序列编码一个含有582个氨基酸的蛋白质,分子量63.37 ku,该蛋白属于稳定的碱性亲水性蛋白。同源性分析表明：其编码的氨基酸序列与可可树（Theobroma cacao）、毛果杨（Populus trichocarpa）的同源性分别为76％、74％,与甜橙的氨基酸序列同源性高达99％。实时荧光定量PCR结果表明,随着果实的发育,CmPME1的表达量逐渐升高,在花后170 d达到最高,然后逐渐降低,远中柱汁胞CmPME1的表达量高于近中柱。     

青稞幼苗期叶片中 SBEⅡa基因的克隆及其表达分析

为进一步研究淀粉分支酶(Starch branching enzyme,SBE)在青稞叶片淀粉代谢中的作用,以青稞品种喜马拉雅2号和康青1号为材料,通过同源克隆技术分离 SBEⅡa基因,并对分离得到的 SBEⅡa基因进行生物信息学分析,同时利用实时荧光定量PCR(qRT-PCR)技术和淀粉分支酶活性测定试剂盒分析了在高温、低温和盐胁迫条件下 SBEⅡa基因的表达情况和SBEⅡa蛋白酶活性的变化情况。结果表明,从喜马拉雅2号和康青1号克隆得到的 SBEⅡa基因ORF区为2466 bp,编码821个氨基酸。生物信息学分析表明,该基因编码的蛋白质分子量为92.09 kDa,预测等电点(pI)为5.4,是酸性蛋白;该蛋白酶的不稳定系数为38.03,属于稳定蛋白;参试材料与大麦的亲缘关系最近,与细江蓠的亲缘关系最远。qRT-PCR和蛋白酶活性测定结果表明, SBEⅡa基因在不同环境下表达水平和SBEⅡa蛋白酶活性变异具有高度的正相关性,在不同环境和不同材料之间没有明显差异。     

茶树P5CS基因克隆及其在渗透和高盐胁迫下的表达分析

△1-吡咯琳-5-羧酸合成酶是植物脯氨酸合成过程中的关键酶。应用同源克隆方法获得茶树P5CS,序列长为1 316 bp,编码323个氨基酸;其编码蛋白分子量为34.7 ku,p I为7.62;N端有1个氨基酸激酶超家族[Amino Acid Kinases(AAK)superfamily]功能区,C端有1个醛脱氢酶超家族[Aldehyde Dehydrogenas(ALDH)superfamily]功能区,预测为亲水性跨膜蛋白;对18个物种的P5CS进行聚类分析,结果生物学分类及进化关系吻合。并与美味猕猴桃高度同源,相似度达89%。应用实时荧光定量PCR分析表明,该基因转录本在水分胁迫24 h内升至对照组水平的2.6倍,而高盐胁迫48 h后才升至9.9倍的最高值;水分胁迫应答速度快,但相对表达量较高盐胁迫低。由此推测该基因被诱导参与了渗透胁迫应答响应,并且对渗透胁迫中的旱害脱水更为敏感。     

花生油质蛋白基因的克隆与表达分析

油质蛋白作为贮藏蛋白的一种,特异性表达在油料种子中。本研究从花生中克隆得到3个Oleosin22基因,分别命名为AhOleosin22a,AhOleosin22b,AhOleosin22c。AhOleosin22a基因全长为630bp,编码210个氨基酸;AhOleosin22b基因全长为636bp,编码212个氨基酸;AhOleosin22c基因全长为582bp,编码194个氨基酸,均属于Oleosin蛋白质家族。通过荧光定量PCR对Oleosin22基因在花生中的表达模式进行分析。结果显示,AhOleosin22基因在种子中的表达量最高。本研究为阐明Oleosin22基因在花生油脂合成的生理生化机制中的功能奠定了理论基础,丰富了花生品质改良育种的基因资源。     

Real-time PCR检测大豆CONSTANS基因在不同光照条件下的表达

采用实时荧光定量PCR方法检测了不同感光大豆品种的CO(CONSTANS) 基因在不同光照条件下的mRNA水平变化.结果表明:早熟品种东农49和晚熟品种东农42各自在短日照(8h/16h 光/暗)下会比长日照(16 h/8 h 光/暗)下表达有所增强,同时东农49中CO基因的表达要高于东农42,它们的表达峰值均出现在光暗交界处.东农42由长日照或短日照移入完全光或完全暗条件时,暗下CO基因的表达量剧增,并且大致维持之前长日照和短日照的表达规律.CO基因的表达有很强的昼夜节律性,暗下利于其表达,并且在不同感光品种中表达有所不同.     

龙眼GPX基因的克隆、原核表达及其在体胚发生过程中的表达分析

采用RT-PCR 与RACE相结合的方法,从龙眼胚性愈伤组织中克隆了长947 bp含有完整开放阅读框的龙眼DlGPX cDNA序列（GenBank登录号为EU364813）和长度为1 736 bp的DNA序列（GenBank登录号为EU680970）。其编码1个含有168个氨基酸的蛋白质。DlGPX基因中含有5个内含子,均符合真核生物内含子通用的GT-AG 法则。生物信息学分析显示：该蛋白为亲水的酸性蛋白质,定位于叶绿体;与其他植物的GPX有较高的同源性。将该基因构建成原核表达载体,经IPTG诱导表达了１个分子量约为23 ku的蛋白。利用实时荧光定量PCR（qPCR）技术研究该基因在龙眼体胚发生发育过程中的表达情况,结果显示,从松散型胚性愈伤组织到不完全胚性紧实球形结构阶段,DlGPX mRNA的转录水平逐渐升高,在胚性紧实球形结构阶段降到最低水平,子叶形胚阶段又上升到与不完全胚性紧实球形结构阶段相当的水平。     

不同O/L值花生品种油酸脱氢酶基因的时空表达特征

本研究以不同油酸/亚油酸比值（O/L比）花生品种为材料,利用实时荧光定量PCR技术,对四个不同O/L花生品种不同组织中油酸脱氢酶基因（FAD2）的表达进行相对定量分析。结果表明：FAD2基因在不同O/L基因型中组成型表达,其在根、茎、叶中表达量低,在花中表达量最高,显著高于荚果等其它组织中的表达量,且品种间差异达显著水平;花U606和花U12在花、幼荚、成熟荚果中的表达量呈依次下降趋势;花育17和狮油红4号在花、幼荚、成熟荚果中的表达量则呈现高-低-次高的表达趋势,差异达显著水平。       

辣椒非特异性脂质转移蛋白基因CaLTP1的克隆及表达分析

非特异性脂质转移蛋白(nsLTPs)是植物中大量存在的小分子脂类结合蛋白,具有多种生物学功能。从辣椒cDNA文库中筛选得到1个nsLTPs基因,命名为CaLTP1;序列分析结果表明:该基因全长cDNA序列为1 211 bp,推测编码1个包含129个氨基酸残基的前体蛋白。CaLTP1蛋白含有典型的脂质转移蛋白N-端信号肽,保守的AAI结构域和半胱氨酸位点。预测CaLTP1蛋白含有多种功能位点,包括3个CK2磷酸化位点,3个N-豆蔻酰化位点和1个PKC磷酸化位点。实时荧光定量分析组织特异性表达模式表明,CaLTP1在辣椒茎、叶、花中表达量升高。     

A study on soil solarization and combined with fumigant application to control Phytophthora crown blight (Phytophthora capsici Leonian) on peppers in the East Mediterranean region of Turkey

Field trials conducted in the pepper growing area ( çel/Turkey) showed that solarization alone and combined with a reduced dose of methyl bromide (40 g/m²), were effective in controlling crown blight disease caused by Phytophthora capsici. Solarization was achieved by covering plots with a clear polyethylene sheet, 0.03 mm thick for 8 weeks. The temperature reached 47 °C and 35 °C at 5 cm and 30 cm soil depth, respectively, an average of 3–8 °C higher than those at equivalent depths in the untreated plots. The average incidence of the disease in solarization plus a reduced dose of methyl bromide, the recommended dose of methyl bromide, solarized and untreated plots in 1991 and 1992 were 17.6 and 13.3%; 20.8 and 16.8%; 24.1 and 19.7%; 39.8 and 42.9%, respectively. All treatments significantly reduced disease incidence.     

Identification and Fine Mapping of a Gene Related to Pale Green Leaf Phenotype near the Centromere Region in Rice （Oryza sativa）

A thermo-insensitive pale green leaf mutant (pgl2) was isolated from T-DNA inserted transgenic lines of rice (Oryza sativa L. subsp. japonica cv. Nipponbare). Genetic analysis indicated that the phenotype was caused by a recessive mutation in a single nuclear-encoded gene. To map the PGL2 gene, an F2 population was constructed by crossing the mutant with Longtefu (Oryza sativa L. subsp. indica). The PGL2 locus was roughly linked to SSR marker RM331 on chromosome 8. To finely map the gene, 14 new InDel markers were developed around the marker, and PGL2 was further mapped to a 2.37 Mb centromeric region. Analysis on chlorophyll contents of leaves showed that there was no obvious difference between the mutant and the wild type in total chlorophyll (Chl) content, while the ratio of Chl a / Chl b in the mutant was only about 1, which was distinctly lower than that in the wild type, suggesting that the PGL2 gene was related to the conversion between Chl a and Chl b. Moreover, the method of primer design around the centromeric region was discussed, which would provide insight into fine mapping of the functional genes in plant centromeres.     

内标校准液相色谱荧光法测定食用油中苯并(a)芘

采用内标苯并（b）屈校正,建立了正相固相净化－反相液相色谱－荧光分光光度法测定食用油中苯并(a)芘的方法。正相Plus Silica固相萃取柱作为净化柱,反相C18柱作为分离柱。以苯并（b）屈作为内标,测定了苯并(a)芘的校正因子。使用本方法,苯并(a)芘在液态食用油和固态食用油中的检出限分别为0.01和0.03μg/kg。苯并(a)芘在不同食用油回收率在83%～108%之间,方法的日内精密度和日间精密度分别小于7.7%和13.2%。     

晋麦47幼苗中一个水分胁迫应答蛋白的SDS-PAGE和Nano LC-MS/MS鉴定

水分胁迫应答蛋白的表达与小麦品种的抗旱性密切相关。为了明确与抗旱相关的水分胁迫D-应答蛋白的表达特点及蛋白组成,对小麦品种晋麦47幼苗进行不同供水量处理,应用SDS-PAGE、纳升级液相色谱-电喷雾串联质谱联用技术(Nano LC-MS/MS),分析了水分胁迫D-应答蛋白条带表达量的变化和蛋白组成。SDS-PAGE检测结果表明,水分胁迫D-应答蛋白条带在-0.5 MPa PEG-6000水溶液胁迫下,6 h出现可见表达,胁迫至48 h时表达量最大,之后,此蛋白条带的表达量又逐渐下降;幼苗胁迫48 h后恢复正常供水,复水72 h后该蛋白消失。切取正常供水和胁迫处理两个条件下SDS-PAGE胶差异表达蛋白条带,经质谱分析分别获得两个阳性结果,且正常供水和PEG-6000胁迫两种处理条件下D-应答蛋白组成一致,均由核酮糖-1,5-二磷酸羧化酶大亚基和磷酸甘油酸变位酶组成(P<0.05);PEG-6000胁迫条件下两个蛋白(亚基)的鉴定得分分别为1 632和88,覆盖率分别为30%和21%;在鉴定的肽段中,有65个肽段同属于核酮糖-1,5-二磷酸羧化酶大亚基,其中35个肽段可信度均超过阈值分数;有5个肽段同属于磷酸甘油酸变位酶,其中4个肽段的可信度超过阈值分数。结果表明该蛋白条带可能主要由核酮糖-1,5-二磷酸羧化酶大亚基等组成。     

β-Carotene from the Alga Dunaliella bardawil Decreases Gene Expression of Adipose Tissue Macrophage Recruitment Markers and Plasma Lipid Concentrations in Mice Fed a High-Fat Diet

Vitamin A and provitamin A carotenoids are involved in the regulation of adipose tissue metabolism and inflammation. We examined the effect of dietary supplementation using all-trans and 9-cis β-carotene-rich Dunaliella bardawil alga as the sole source of vitamin A on obesity-associated comorbidities and adipose tissue dysfunction in a diet-induced obesity mouse model. Three-week-old male mice (C57BL/6) were randomly allocated into two groups and fed a high-fat, vitamin A-deficient diet supplemented with either vitamin A (HFD) or β-carotene (BC) (HFD-BC). Vitamin A levels in the liver, WATs, and BAT of the HFD-BC group were 1.5–2.4-fold higher than of the HFD group. BC concentrations were 5–6-fold greater in BAT compared to WAT in the HFD-BC group. The eWAT mRNA levels of the Mcp-1 and Cd68 were 1.6- and 2.1-fold lower, respectively, and the plasma cholesterol and triglyceride concentrations were 30% and 28% lower in the HFD-BC group compared with the HFD group. Dietary BC can be the exclusive vitamin A source in mice fed a high-fat diet, as shown by the vitamin A concentration in the plasma and tissues. Feeding BC rather than vitamin A reduces adipose tissue macrophage recruitment markers and plasma lipid concentrations.     

凸加夫蛤的藻类滤食能力及其对海水氮磷含量的影响

研究热带亚热带双壳贝类凸加夫蛤(Gafrarium tumidum)对富营养化水体中藻类的滤食能力及其对海水氮磷含量的影响。结果表明,凸加夫蛤能有效地清除水体中的叶绿素a,在海水N、P含量分别为0.5 mg/L和0.04 mg/L,初始藻密度为5×106 cell/L的情况下,72 h就能起到显著效果,144 h(6 d)之后对藻类密度的控制达到理想的效果。当贝密度在1~2 kg/m3范围内时效果最佳,且不会引起水体中N、P含量的增加。因此,凸加夫蛤是热带海域控制藻类密度、改善水质和预防赤潮的经济双壳贝类之一。     

Seasonal changes in the crude protein concentration of mixed swards of white clover/perennial ryegrass grown without fertilizer N in an organic farming system in the United Kingdom

Changes in the crude protein (CP) concentration of white clover and perennial ryegrass herbage from a mixed sward were determined on six sampling dates from May to October in each of 2 years. The swards were grown without fertilizer N in an organic farming system and continuously grazed by dairy cows during the grazing season. The annual mean contents of white clover in the dry matter (DM) of the sward were 272·3 and 307·0 g kg⁻¹ in Years 1 and 2. The mean CP concentrations of the white clover and perennial ryegrass herbage were 251·6 and 151·9 g kg⁻¹ DM in Year 1 and 271·9 and 174·0 g kg⁻¹ DM in Year 2 respectively. The CP concentration of the white clover increased significantly during the grazing season from 220·0 to 284·1 g kg⁻¹ DM in Year 1 and from 269·0 to 315·5 g kg⁻¹ DM in Year 2. In the perennial ryegrass herbage the CP concentration increased from 112·2 to 172·6 g kg⁻¹ DM in Year 1 and from 142·7 to 239·5 g kg⁻¹ DM in Year 2. The rate of increase during the season in the CP concentration of the perennial ryegrass herbage was similar to the rate of increase recorded in the white clover herbage.     

A single amino acid substitution in puroindoline b impacts its self-assembly and the formation of heteromeric assemblies with puroindoline a

Puroindolines (PINs) A and B were purified from soft (Paledor) and hard (Recital, Courtot) wheat cultivars. Their purity and heterogeneity due to post-translational processing were characterized by SDS- and acid-PAGE, reversed-phase HPLC and mass spectrometry. By using dynamic light scattering (DLS), asymmetrical flow field-flow fractionation (AF4) and size exclusion chromatography (SEC), we showed that the size distributions of PINA are similar for the three varieties and that, in solution, they self-assembled into small aggregates, mainly dimers. Conversely, PINB isolated from hard varieties (PINB-D1b and PINB-D1d) are assembled into large aggregates while PINB-D1a formed small aggregates, mainly monomers. Mixed solutions of PINA and PINB formed heteromeric aggregates. The large PINB-D1b aggregates were retained even at a high (4:1) PINA/PINB weight ratio. Reversible dissociation of large aggregates into small aggregates suggested that weak interactions control the self-assembly of PINs. The aggregative properties of PINs have now to be taken into account when studying their interactions with other components to decipher the causal relationships between these proteins and grain hardness.     

Effects of post-anthesis fertilizer on the protein composition of the gluten polymer in a US bread wheat

Both genetic and environmental factors influence the types and amounts of wheat proteins that link together to form polymers essential for flour quality. To understand how plant growth conditions might influence gluten polymer formation, protein fractions containing small and large polymers were separated from flour from the US wheat Butte 86 grown in the absence or presence of post-anthesis fertilizer. Proteins in the polymer fractions were analyzed by quantitative two-dimensional gel electrophoresis (2-DE). The ratio of high molecular weight glutenin subunits (HMW-GS) to low molecular weight glutenin subunits (LMW-GS) increased in both fractions in response to fertilizer, due in part to small increases in the proportions of individual HMW-GS. There were also changes within the LMW-GS. In particular, omega and alpha chain terminators increased in proportion in both polymer fractions, but changes were more pronounced in the large polymer fractions. Serpins also increased in both polymer fractions. Additionally, the study revealed differences in the proportions of traditional LMW-GS in small and large polymer fractions. LMW-s type proteins were more abundant in the large polymers while LMW-i type proteins were more prevalent in the small polymers, suggesting that these proteins may play different roles in the gluten polymer.