Abscisic acid plays an important role in the regulation of strawberry fruit ripening

The plant hormone abscisic acid (ABA) has been suggested to play a role in fruit development, but supporting genetic evidence has been lacking. Here, we report that ABA promotes strawberry (Fragaria ananassa) fruit ripening. Using a newly established Tobacco rattle virus-induced gene silencing technique in strawberry fruit, the expression of a 9-cis-epoxycarotenoid dioxygenase gene (FaNCED1), which is key to ABA biosynthesis, was down-regulated, resulting in a significant decrease in ABA levels and uncolored fruits. Interestingly, a similar uncolored phenotype was observed in the transgenic RNA interference (RNAi) fruits, in which the expression of a putative ABA receptor gene encoding the magnesium chelatase H subunit (FaCHLH/ABAR) was down-regulated by virus-induced gene silencing. More importantly, the uncolored phenotype of the FaNCED1-down-regulated RNAi fruits could be rescued by exogenous ABA, but the ABA treatment could not reverse the uncolored phenotype of the FaCHLH/ABAR-down-regulated RNAi fruits. We observed that down-regulation of the FaCHLH/ABAR gene in the RNAi fruit altered both ABA levels and sugar content as well as a set of ABA- and/or sugar-responsive genes. Additionally, we showed that exogenous sugars, particularly sucrose, can significantly promote ripening while stimulating ABA accumulation. These data provide evidence that ABA is a signal molecule that promotes strawberry ripening and that the putative ABA receptor, FaCHLH/ABAR, is a positive regulator of ripening in response to ABA.     

寡糖的荧光标记高效液相色谱分析

 利用氨化还原的方法把高量子产率的荧光标记物——α-萘胺,接到异麦芽糖寡糖的还原端。用硅胶薄层色谱、荧光光谱及快原子轰击质谱证实反应物的完全性及其结构。高效液相色谱用Micropak si5硅胶柱,以乙腈-水(含0.05％三乙胺)为梯度洗脱液可使含有二到九个糖残基的异麦芽糖寡糖的α-萘胺衍生物全部分离。本法对异麦芽糖二糖的荧光检测灵敏测度为2.35Pmol。     

促葡萄糖转运蛋白基因家族的分子进化研究

葡萄糖转运蛋白是一个在结构上相似功能上不同的多基因家族（ＧＬＵＴ１－ＧＬＵＴ５）。由于这一组蛋白和体内的葡萄糖利用有关,因此被认为是糖尿病胰岛素抵抗（抗性）的一个候选基因。本文比较了不同种生物这一基因家族的氨基酸和核苷酸顺序;推测了亲水性和疏水性分布;计算了蛋白质和核苷酸的进化距离,并在此基础上构建了分子进化树。研究表明：这一基因家族具有高度的同源性、极为相似的亲水性和疏水性分布以及结构的对称性。提示这一基因家族起源于一个共同的祖先并可能通过基因的重复而形成。这一进化机制可能有利于氨基酸结构的稳定及抵抗突变的作用。由于邻元法构建的进化树其分支长度存在差异,提示在这一基因家族的进化过程中,各分支上的进化速率并不相同。蛋白质进化距离和核苷酸进化距离所构建进化树的差异提示了在基因组中可能存在隐匿替换。两种方法构建的进化树都提示了ＧＬＵＴ１、３、４在结构和功能上要更为保守。     

假单胞菌YL11对扩展青霉的抑制作用及其机理初探

【背景】苹果青霉病是由扩展青霉引起的一种重要的果实采后病害,影响果实品质导致苹果腐烂从而造成经济损失。【目的】研究假单胞菌YL11对扩展青霉的抑制作用和苹果采后青霉病的防治效果,并对抑菌机理进行初步探讨。【方法】以扩展青霉为供试菌株,研究不同浓度的假单胞菌YL11无菌发酵液对扩展青霉菌落直径、孢子萌发率、菌丝体干重、苹果损伤接种病斑直径扩展的影响,利用对电导率、核酸及蛋白释放量、AKP含量、SDH活性、ATP酶活性和ATP含量的影响对抑菌机理进行探究。【结果】假单胞菌YL11无菌发酵液能有效抑制扩展青霉生长,抑菌圈直径为22.33±0.27 mm,抑菌效价为71.67 mm/mL;能有效抑制孢子萌发,100%无菌发酵液对孢子萌发抑制率达到80.2%;对扩展青霉的生物量也有一定抑制作用,体积分数为100%时,菌丝体干重为4.7mg/mL,抑制率达到39.74%;无菌发酵液处理能有效抑制苹果青霉病病斑的扩展,3d时对病斑扩展的抑制率最大,达到47.1%;无菌发酵液处理均能引起电导率升高、胞内核酸和蛋白释放量增大、胞外AKP含量升高、SDH活性降低、ATP酶活性和ATP含量均降低,且随着发酵液浓度的增加效果越明显。【结论】假单胞菌YL11能显著抑制扩展青霉的生长,破坏细胞膜结构、降低能量代谢酶活性,从而扰乱扩展青霉的正常生长,对苹果青霉病有较好的生防效果,具有潜在的开发价值。     

AtNHX8, a member of the monovalent cation: proton antiporter-1 family in Arabidopsis thaliana, encodes a putative Li/H antiporter

The Arabidopsis monovalent cation:proton antiporter-1 (CPA1) family includes eight members, AtNHX1-8. AtNHX1 and AtNHX7/SOS1 have been well characterized as tonoplast and plasma membrane Na+/H+ antiporters, respectively. The proteins AtNHX2-6 have been phylogenetically linked to AtNHX1, while AtNHX8 appears to be related to AtNHX7/SOS1. Here we report functional characterization of AtNHX8. AtNHX8 T-DNA insertion mutants are hypersensitive to lithium ions (Li+) relative to wild-type plants, but not to the other metal ions such as sodium (Na+), potassium (K+) and caesium (Cs+). AtNHX8 overexpression in a triple-deletion yeast mutant AXT3 that exhibits defective Na+/Li+ transport specifically suppresses sensitivity to Li+, but does not affect Na+ sensitivity. Likewise, AtNHX8 overexpression complemented sensitivity to Li+, but not Na+, in sos1-1 mutant seedlings, and increased Li+ tolerance of both the sos1-1 mutant and wild-type seedlings. Results of Li+ and K+ measurement of loss-of-function and gain-of-function mutants indicate that AtNHX8 may be responsible for Li+ extrusion, and may be able to maintain K+ acquisition and intracellular ion homeostasis. Subcellular localization of the AtNHX8-enhanced green fluorescent protein (EGFP) fusion protein suggested that AtNHX8 protein is targeted to the plasma membrane. Taken together, our findings suggest that AtNHX8 encodes a putative plasma membrane Li+/H+ antiporter that functions in Li detoxification and ion homeostasis in Arabidopsis.     

Ubc9 mediates nuclear localization and growth suppression of BRCA1 and BRCA1a proteins

BRCA1 gene mutations are responsible for hereditary breast and ovarian cancers. In sporadic breast tumors, BRCA1 dysfunction or aberrant subcellular localization is thought to be common. BRCA1 is a nuclear-cytoplasm shuttling protein and the reason for cytoplasmic localization of BRCA1 in young breast cancer patients is not yet known. We have previously reported BRCA1 proteins unlike K109R and cancer-predisposing mutant C61G to bind Ubc9 and modulate ER-α turnover. In the present study, we have examined the consequences of altered Ubc9 binding and knockdown on the subcellular localization and growth inhibitory function of BRCA1 proteins. Our results using live imaging of YFP, GFP, RFP-tagged BRCA1, BRCA1a and BRCA1b proteins show enhanced cytoplasmic localization of K109 R and C61G mutant BRCA1 proteins in normal and cancer cells. Furthermore, down-regulation of Ubc9 in MCF-7 cells using Ubc9 siRNA resulted in enhanced cytoplasmic localization of BRCA1 protein and exclusive cytoplasmic retention of BRCA1a and BRCA1b proteins. These mutant BRCA1 proteins were transforming and impaired in their capacity to inhibit growth of MCF-7 and CAL51 breast cancer cells. Interestingly, cytoplasmic BRCA1a mutants showed more clonogenicity in soft agar and higher levels of expression of Ubc9 than parental MCF7 cells. This is the first report demonstrating the physiological link between cytoplasmic mislocalization of mutant BRCA1 proteins, loss of ER-α repression, loss of ubiquitin ligase activity and loss of growth suppression of BRCA1 proteins. Thus, binding of BRCA1 proteins to nuclear chaperone Ubc9 provides a novel mechanism for nuclear import and control of tumor growth.     

Molecular basis of dysfunctional Kv channels in small coronary artery smooth muscle cells of streptozotocin-induced diabetic rats

We have previously shown that diabetes impaired cAMP-mediated endothelium independent vasodilation of rat small coronary arteries. Inhibition of Kv channel activity plays an important role in the decrease of cAMP mediated vasodilation. The present study investigated the effect of streptozotocin (STZ)-induced diabetes on mRNA and protein expressions of Kv1.2 and Kv1.5 channels in vascular smooth muscle cells of rat small coronary artery using RT-PCR, Western blot and immunohistochemistry methods. STZ-induced diabetes obviously impaired mRNA expression of Kv1.2 and Kv1.5 channel. The mRNA levels of Kv1.2 channel were 0.65 +/- 0.08 and 1.02 +/- 0.17 in STZ rats and control rats, respectively (n = 7, P < 0.05). Whereas the levels of Kv1.5 channel were 0.58 +/- 0.05 and 0.94 +/- 0.13 in STZ rats and control rats, respectively (n = 7, P < 0.05). Western blotting analysis showed that protein expression of Kv1.2 channel was decreased significantly but not Kv1.5 channel. Protein expressions of Kv1.2 channel were 0.49 +/- 0.04 and 0.70 +/- 0.06 in STZ rats and control rats, respectively (n = 5, P < 0.05), but those of Kv1.5 channel were 0.61 +/- 0.12 and 0.59 +/- 0.14 in STZ rats and control rats, respectively (n = 5, P > 0.05). Immunohistochemistry identification indicated that immunological reaction of Kv1.2 channel protein was attenuated, but Kv1.5 channel protein was not altered. Positive staining intensity normalized by gray values of Kv1.2 channel were 173 +/- 13 and 131 +/- 11 in STZ rats and control rats, respectively (n = 5, P < 0.05), but those of Kv1.5 channel were 139 +/- 16 and 141 +/- 12 in STZ rats and control rats, respectively (n = 5, P > 0.05). These results suggested that impairment of cAMP-mediated endothelium independent vasodilation of rat small coronary artery by STZ-induced diabetes was resulted from decrease of mRNA and protein expressions of Kv channels, and which eventually leads to a reduced current from Kv channels.     

The N-terminal tail of hERG contains an amphipathic α-helix that regulates channel deactivation

The cytoplasmic N-terminal domain of the human ether-a-go-go related gene (hERG) K+ channel is critical for the slow deactivation kinetics of the channel. However, the mechanism(s) by which the N-terminal domain regulates deactivation remains to be determined. Here we show that the solution NMR structure of the N-terminal 135 residues of hERG contains a previously described Per-Arnt-Sim (PAS) domain (residues 26-135) as well as an amphipathic α-helix (residues 13-23) and an initial unstructured segment (residues 2-9). Deletion of residues 2-25, only the unstructured segment (residues 2-9) or replacement of the α-helix with a flexible linker all result in enhanced rates of deactivation. Thus, both the initial flexible segment and the α-helix are required but neither is sufficient to confer slow deactivation kinetics. Alanine scanning mutagenesis identified R5 and G6 in the initial flexible segment as critical for slow deactivation. Alanine mutants in the helical region had less dramatic phenotypes. We propose that the PAS domain is bound close to the central core of the channel and that the N-terminal α-helix ensures that the flexible tail is correctly orientated for interaction with the activation gating machinery to stabilize the open state of the channel.     

Maternal serum-derived exosomal lactoferrin as a marker in detecting and predicting ventricular septal defect in fetuses

Among different types of congenital heart diseases, ventricular septal defect is the most frequently diagnosed type and is frequently missed in early prenatal screening programs. Herein, we explored the role of maternal serum-derived exosomes in detecting and predicting ventricular septal defect in fetuses in the early stage of pregnancy. A total of 104 pregnant women consisting of 52 ventricular septal defect cases and 52 healthy controls were recruited. TMT/iTRAQ proteomic analysis uncovered 15 maternal serum exosomal proteins, which showed differential expression between ventricular septal defect and control groups. Among these, four down-regulated proteins, lactoferrin, SBSN, DCD, and MBD3, were validated by Western blot. The protein lactoferrin was additionally verified by ELISA which was able to distinguish ventricular septal defects from controls with area under the ROC curve (AUC) 0.804 (p < 0.001). Our findings reveal that lactoferrin in maternal serum-derived exosomes may be a potential biomarker for non-invasive prenatal diagnosis of fetal ventricular septal defects.