苹果柠檬酸合酶3基因家族成员鉴定及表达分析

柠檬酸合酶(citrate synthase 3, CS3)是细胞代谢途径中的关键酶之一,其活性调节着生物体的物质和能量代谢过程。本研究旨在从苹果全基因组中鉴定CS3基因家族成员,并进行生物信息学和表达模式分析,为研究苹果CS3基因的潜在功能提供理论基础。利用BLASTp基于GDR数据库鉴定苹果CS3家族成员,通过Pfam、SMART、MEGA5.0、clustalx.exe、ExPASy Proteomics Server、MEGAX、SOPMA、MEME和WoLF PSORT等软件分析CS3蛋白序列基本信息、亚细胞定位情况、结构域组成、系统进化关系以及染色体定位情况。利用酸含量的测定和实时荧光定量PCR (real-time fluorescence quantitative polymerase chain reaction, qRT-PCR)技术检测苹果6个CS3的组织表达和诱导表达特性。苹果CS3基因家族包含6个成员,这些CS3蛋白包括473−608个不等的氨基酸残基,等电点分布在7.21−8.82。亚细胞定位结果显示CS3蛋白分别定位在线粒体和叶绿体。系统进化分析可将其分为3类,各亚家族基因数量分别为2个。染色体定位结果显示,CS3基因分布在苹果不同的染色体上。蛋白二级结构以a-螺旋为主,其次是无规则卷曲,b-转角所占比例最小。筛选的6个家族成员在不同苹果组织中均有表达,整体表达趋势从高到低依次为MdCS3.4相对表达含量最高,MdCS3.6次之,其他家族成员相对表达量依次为MdCS3.3>MdCS3.2>MdCS3.1>MdCS3.5。qRT-PCR结果显示,MdCS3.1和MdCS3.3基因在酸含量较低的‘成纪1号’果肉中相对表达量最高,酸含量较高的‘艾斯达’果肉中MdCS3.2和MdCS3.3基因相对表达量最高。因此,本研究对不同苹果品种中CS3基因相对表达量进行了检测,并分析了其在苹果果实酸合成过程中的作用。结果表明,CS3基因在不同苹果品种中的相对表达量存在差异,为后续研究苹果品质形成机制提供了参考。     

Caveolin-1 Expression Level in Cancer Associated Fibroblasts Predicts Outcome in Gastric Cancer

Aims

Altered expression of epithelial or stromal caveolin-1 (Cav-1) is observed in various types of human cancers. However, the clinical significance of Cav-1 expression in gastric cancer (GC) remains largely unknown. The present study aims to explore the clinicopathological significance and prognostic value of both tumor cells and cancer associated fibroblasts (CAFs) Cav-1 in GC.

Methods and Results

Quantum dots immunofluorescence histochemistry was performed to examine the expression of Cav-1 in 20 cases of gastritis without intestinal metaplasia (IM), 20 cases of gastritis with IM and 286 cases of GC. Positive rates of epithelial Cav-1 in gastritis without IM, gastritis with IM and GC showed a decreasing trend (P = 0.012). Low expression of Cav-1 in CAFs but not in tumor cells was an independent predictor of poor prognosis in GC patients (P = 0.034 and 0.005 respectively in disease free survival and overall survival). Cav-1 level in tumor cells and CAFs showed no significant correlation with classic clinicopathological features.

Conclusions

Loss of epithelial Cav-1 may promote malignant progression and low CAFs Cav-1 level herald worse outcome of GC patient, suggesting CAFs Cav-1 may be a candidate therapeutic target and a useful prognostic marker of GC.     

Genome-Wide Analysis of the Apple (Malus domestica) Cysteine-Rich Receptor-Like Kinase (CRK) Family: Annotation,Genomic Organization,and Expression Profiles in Response to Fungal Infection

Plant Molecular Biology Reporter - Cysteine-rich receptor-like kinases (CRKs) took crucial roles in plant cell growth and development, as well as environmental adaption. Apple (Malus domestica) had...     

TTC染色鉴定葡萄抗寒性方法的优化与应用北大核心CSCD

以‘左山1号’、101-14、3309C、SO4、140R和‘黑比诺’等葡萄砧木和品种直径为0.5~0.7 cm的一年生枝条为材料,经低温梯度(0℃、-14℃、-19℃、-24℃、-29℃和-34℃)处理后,对TTC染色的温度和时间进行优化并观察统计葡萄枝条不同组织的存活情况,测定枝条的相对电导率,以及韧皮部和木质部的可溶性蛋白、游离脯氨酸、可溶性糖、淀粉含量和束缚水自由水比例(束自比)5个生理指标,分别用枝条纵切面染色面积、相对电导率拟合Logistic方程计算枝条的半致死温度(LT_(50))来评价枝条的抗寒性,同时通过隶属函数法综合评价枝条韧皮部和木质部抗寒性,并将3种方法的评价结果进行比较,以建立一种直观高效鉴定葡萄品种抗寒性的方法。结果表明:(1)依据低温胁迫下各品种葡萄砧木扦插枝条的萌芽率和生根率表现,其抗寒性由强到弱依次为‘左山1号’、101-14、3309C、SO4、140R和‘黑比诺’。(2)葡萄枝条TTC活力染色的最佳条件为pH=7.0,0.5%TTC-0.1 mol/L磷酸缓冲液在35℃下避光染色36 h。随着胁迫温度的降低,各品种枝条纵切面染色面积均逐渐降低,根据优化TTC法获得‘左山1号’、101-14、3309C、SO4、140R和‘黑比诺’枝条的LT_(50)分别为-31.38℃、-26.51℃、-26.10℃、-23.60℃、-23.33℃和-19.26℃。(3)随着胁迫温度的降低,各品种枝条的相对电导率逐渐增加,且‘黑比诺’的相对电导率基本保持最高、增幅最大(51.93%),而‘左山1号’的相对电导率始终最低、增幅最小(44.07%);根据相对电导率获得‘左山1号’、101-14、3309C、SO4、140R和‘黑比诺’枝条的LT_(50)分别为-30.02℃、-26.40℃、-25.75℃、-23.16℃、-21.13℃和-17.72℃。(4)通过5种生理指标进行枝条抗寒性隶属函数综合性评价结果显示,同一品种中韧皮部抗寒性强于木质部,同一部位不同品种的抗寒性表现为:‘左山1号’>101-14>3309C>SO4>140R>‘黑比诺’。研究发现,TTC染色法与电导法和综合隶属函数法获得的葡萄枝条抗寒力评价结果一致,也与枝条生长恢复鉴定结果一致;但与其他两种方法相比,TTC染色法能更加直观和有效地预测评估葡萄枝条的抗寒性。     

葡萄BRX基因家族生物信息学分析

BRX基因家族是一类植物特有的转录因子家族,在拟南芥中参与调节根细胞的增殖与伸长。利用生物信息学方法对葡萄基因组中存在的BRX基因家族进行了电子克隆,并对其进行了基因组的定位、蛋白质的结构、理化性质、二级结构及亚细胞定位的预测与分析,并对其与其它植物进化的亲缘关系进行了研究。基因组定位结果发现:葡萄基因组中6个BRX基因集中分布在3条染色体上,其中Vv BRX1和Vv BRX2分布在第2条染色体上,Vv BRX3和Vv BRX4分布在第9条染色体上,Vv BRX5和Vv BRX6分布在第11条染色体上;编码蛋白的氨基酸数目为360~560个,Vv BRX5的相对分子量(61 884.4)和理论等电点(9.38)均最大,而Vv BRX1的相对分子量(40 239.1)和理论等电点(6.23)均最小。研究显示,不同成员间氨基酸数目、氨基酸序列间存在一定的差异,但都为疏水性蛋白;α-螺旋和无规则卷曲为6个BRX氨基酸序列的主要组成部分;均不存在跨膜域及信号肽。基因结构分析表明,6个BRX基因都含有外显子和内含子结构。亚细胞定位分析表明:6个Vv BRX基因均定位于细胞核。系统进化分析结果表明,Vv BRX1、Vv BRX2基因与胡杨的亲缘关系最近,相似性达96%;Vv BRX3、Vv BRX4与蓖麻、麻疯树、柑橘、可可、大豆聚为一类,说明其进化关系较近;Vv BRX5与其它Vv BRX基因明显分开;Vv BRX6基因与莲的亲缘关系最近。试验结果为葡萄BRX基因家族的克隆和功能分析奠定了一定的研究基础。     

Negative pressure wound therapy promotes muscle‐derived stem cell osteogenic differentiation through MAPK pathway

Negative pressure wound therapy (NPWT) has been revealed to be effective in the treatment of open fractures, although the underlying mechanism is not clear. This article aimed to investigate the effects of NPWT on muscle‐derived stem cell (MDSC) osteoblastic differentiation and the related potential mechanism. The cell proliferation rate was substantially increased in NPWT‐treated MDSCs in comparison with a static group for 3 days. There was no observable effect on the apoptosis of MDSC treated with NPWT compared with the control group for 3 days. The expression levels of HIF‐1α, BMP‐2, COL‐I, OST and OPN were increased on days 3, 7 and 14, but the expression level of Runx2 was increased on days 3 and 7 in the NPWT group. Pre‐treatment, the specific inhibitors were added into the MDSCs treated with NPWT and the control group. ALP activity and mineralization were reduced by inhibiting the ERK1/2, p38 and JNK pathways. The expression levels of Runx2, COL‐I, OST and OPN genes and proteins were also decreased using the specific MAPK pathway inhibitors on days 3, 7 and 14. There were no significant effects on the expression of BMP‐2 except on day 3. However, the expressions of the HIF‐1α gene and protein slightly increased when the JNK pathway was inhibited. Therefore, NPWT promotes the proliferation and osteogenic differentiation of MDSCs through the MAPK pathway.     

外源ALA对干旱胁迫下山定子叶片叶绿素荧光特性及抗性生理指标的影响

以两年生山定子(Malus baccata(L.)Borkh)盆栽苗为材料,正常浇水为对照,控水条件下喷施不同浓度(0、25、50、75、100mg/L)5-氨基乙酰丙酸(ALA),测定各持续干旱胁迫时期(第0、4、8、12天)叶片的抗旱生理指标及光合荧光参数,研究ALA对干旱胁迫下山定子叶绿素荧光和抗逆生理生化特性的影响及其调节机制。结果显示:(1)持续干旱胁迫条件下,外源ALA处理使得山定子叶片渗透调节物质(SS、SP、Pro)含量增加,抗氧化酶(SOD、POD、APX)活性升高,丙二醛(MDA)积累减少。(2)ALA处理增大了干旱胁迫下山定子叶片的气孔开度,促进叶绿素的合成,并对干旱引起的最大光化学效率(F_v/F_m)、实际光量子效率(YⅡ)、光化学淬灭系数(qP)及光合电子传递速率(ETR)的减小,以及PSⅡ调节性能量耗散的量子产额(Y_(NPQ))、非调节性能量耗散的量子产额(Y_(NO))和非光化学淬灭系数(NPQ/4)的增大有明显的缓解作用。研究认为,外喷ALA能够有效缓解干旱胁迫对山定子生理代谢功能的损伤,但干旱胁迫条件下山定子对不同浓度ALA响应程度不尽相同,其中以75mg/L的ALA处理效果较为理想。     

Genome-Wide Identification and Expression Analysis of the CrRLK1L Gene Family in Apple (Malus domestica)

It has been reported that members of the Catharanthus roseus receptor-like kinase1-like kinase (CrRLK1L) gene family detect cell wall integrity, cell-to-cell communication, and biotic and abiotic stress. We performed a comprehensive study including the genome-wide identification, characterization, and gene expression analysis of CrRLK1Ls in apple (Malus domestica). Sixty-seven M. domestica CrRLK1Ls (MdCrRLK1Ls) were identified based on their domain structure. Molecular weight and pI ranged from 52.36–141 kDa and 5.05–8.9, respectively. They were distributed across 16 of the 18 chromosomes and classified into five phylogenetic branches. Exon-intron structural analysis indicated a wide range of exon numbers. Collinearity analysis showed that both segmental-and tandem-duplication contributed to the expansion of this family. Cis-elements in the MdCrRLK1L promoter region responded mainly to light, circadian rhythm, phytohormones, and biotic or abiotic stress. Many members exhibited tissue-specific expression patterns and differentially expressed under biotic stresses, which may contribute to the different functional roles of MdCrRLK1Ls under physiological stress and/or pathological conditions. This study provides new insights into the CrRLK1Ls in Malus spp.

     

Monolithic microfluidic mixing–spraying devices for time-resolved cryo-electron microscopy

The goal of time-resolved cryo-electron microscopy is to determine structural models for transient functional states of large macromolecular complexes such as ribosomes and viruses. The challenge of time-resolved cryo-electron microscopy is to rapidly mix reactants, and then, following a defined time interval, to rapidly deposit them as a thin film and freeze the sample to the vitreous state. Here we describe a methodology in which reaction components are mixed and allowed to react, and are then sprayed onto an EM grid as it is being plunged into cryogen. All steps are accomplished by a monolithic, microfabricated silicon device that incorporates a mixer, reaction channel, and pneumatic sprayer in a single chip. We have found that microdroplets produced by air atomization spread to sufficiently thin films on a millisecond time scale provided that the carbon supporting film is made suitably hydrophilic. The device incorporates two T-mixers flowing into a single channel of four butterfly-shaped mixing elements that ensure effective mixing, followed by a microfluidic reaction channel whose length can be varied to achieve the desired reaction time. The reaction channel is flanked by two ports connected to compressed humidified nitrogen gas (at 50 psi) to generate the spray. The monolithic mixer-sprayer is incorporated into a computer-controlled plunging apparatus. To test the mixing performance and the suitability of the device for preparation of biological macromolecules for cryo-EM, ribosomes and ferritin were mixed in the device and sprayed onto grids. Three-dimensional reconstructions of the ribosomes demonstrated retention of native structure, and 30S and 50S subunits were shown to be capable of reassociation into ribosomes after passage through the device.