RNAi技术在作物品质改良中的应用

RNA干涉(RNAi)是由同源性内源或外源dsRNA引起的序列特异性基因沉默现象,在动、植物和真菌中广泛存在并被证实。目前已应用RNAi技术在改善油脂的品质、改良淀粉品质、提高营养物质或降低有害物质含量、提高抗褐化能力、提高果实耐贮性、进行代谢调控以获得目的次生代谢物等方面进行了作物品质改良研究。作为一种下调表达技术,该技术在研究植物基因功能和改良作物品质等领域有良好的应用前景。本文重点从上述六个方面对近年来应用RNAi技术在作物品质改良研究方面进行了回顾并对其存在的问题作了初步探讨。     

Secreted proteins induced by epidermal growth factor and transforming growth factor beta in EL2 rat fibroblasts. Role in the mitogenic response

Most growth active hormones and peptides are mitogenic only in the presence of other growth factors [e.g., Platelet Derived Growth Factor (PDGF) and Epidermal Growth Factor (EGF) in "competence-progression" fibroblast model]. We have previously described that EGF alone is able to induce the signals which appear necessary for the mitogenic stimulation of EL2 rat embryo fibroblast line. Recently, we have demonstrated that Transforming Growth Factor beta (TGF beta) slightly stimulates the mitogenic response in EL2 cells. Here, we show that in EGF-treated EL2 cells the induction of at least four inducible-secreted proteins (ISPs, range from 29,000 to 68,000 Mr) is accompanied by a marked increase in DNA synthesis. In contrast, TGF beta or different concentrations of EGF induce a slow increase of the ISPs proportional to slow induction in DNA synthesis. Our results suggest that the mitogenic response in EL2 cell line may be connected with the qualitative and quantitative induction of these secreted proteins.     

Circulating exosome‐derived bona fide long non‐coding RNAs predicting the occurrence and metastasis of hepatocellular carcinoma

Although the diagnosis and therapy approach developed, techniques for the early diagnosis of HCC remain insufficient which results in poor prognosis of patients. The traditional biomarker AFP, however, has been proved with low specificity. Circulating exosomal ncRNAs revealed different profiles reflecting the characteristics of tumour. In this study, we mainly focused on circulating exosomal ncRNAs which might be the fingerprint for HCC, especially for the diagnosis or metastasis prediction. A high throughput lncRNA microarray in exosomes extracted from cell‐free plasma was applied. The risk score analysis was employed to screen the potential exosome‐derived lncRNAs in two independent sets based on different clinical parameters in 200 paired HCC patients. After a multi‐stage validation, we finally revealed three lncRNAs, ENSG00000248932.1, ENST00000440688.1 and ENST00000457302.2, increased in HCC comparing with the both chronic hepatitis (CH) patients and cancer‐free controls. ROC curve revealed a higher sensitivity and specificity in predicting the occurrence of HCC from cancer‐free controls and CH patients with the area under curve (AUC) of 0.905 and 0.879 by combining AFP. The three lncRNA panel combined with AFP also indicted a fingerprint function in predicting the metastasis of HCC with the AUC of 0.870. In conclusion, ENSG00000248932.1, ENST00000440688.1 and ENST00000457302.2 might be the potential biomarker for the tumorigenesis prediction from CH patients or healthy controls and may also be applied for dynamic monitoring the metastasis of HCC.     

The demonstration of a novel sulfur cycle-based wastewater treatment process: Sulfate reduction, autotrophic denitrification, and nitrification integrated (SANI®) biological nitrogen removal process

Saline water supply has been successfully practiced for toilet flushing in Hong Kong since 1950s, which saves 22% of freshwater in Hong Kong. In order to extend the benefits of saline water supply into saline sewage management, we have recently developed a novel biological organics and nitrogen removal process: the Sulfate reduction, Autotrophic denitrification, and Nitrification Integrated (SANI®) process. The key features of this novel process include elimination of oxygen demand in organic matter removal and production of minimal sludge. Following the success of a 500‐day lab‐scale trial, this study reports a pilot scale evaluation of this novel process treating 10 m³/day of 6‐mm screened saline sewage in Hong Kong. The SANI® pilot plant consisted of a sulfate reduction up‐flow sludge bed (SRUSB) reactor, an anoxic bioreactor for autotrophic denitrification and an aerobic bioreactor for nitrification. The plant was operated at a steady state for 225 days, during which the average removal efficiencies of both chemical oxygen demand (COD) and total suspended solids (TSS) at 87% and no excess sludge was purposefully withdrawn. Furthermore, a tracer test revealed 5% short circuit flow and a 34.6% dead zone in the SRUSB, indicating a good possibility to further optimize the treatment capacity of the process for full‐scale application. Compared with conventional biological nitrogen removal processes, the SANI® process reduces 90% of waste sludge, which saves 35% of the energy and reduces 36% of fossil CO₂ emission. The SANI® process not only eliminates the major odor sources originating from primary treatment and subsequent sludge treatment and disposal during secondary saline sewage treatment, but also promotes saline water supply as an economic and sustainable solution for water scarcity and sewage treatment in water‐scarce coastal areas. Biotechnol. Bioeng. 2012; 109: 2778–2789. © 2012 Wiley Periodicals, Inc.     

Oriented immobilization of the tobacco etch virus protease for the cleavage of fusion proteins

The tobacco etch virus (TEV) protease is a useful tool for the removal of fusion tags from recombinant proteins. The difficulty in obtaining this enzyme led us to look for an optimal method for its use. In this work, we produced both the wild-type and the S219V mutant TEV proteases fused to the Streptag II affinity sequence (Streptag II-TEV(WT), and Streptag II-TEV(S219V), respectively). The two enzymes were affinity immobilized on a streptavidin-agarose matrix and compared to their respective free forms. Both immobilized Streptag II-TEV(WT) and Streptag II-TEV(S219V) were active on the 74-kDa Streptag II substrate with a retained activity of 83.5% and 81%, respectively compared to their free corresponding forms. The slight enzyme activity decrease caused by the immobilization was balanced by the enhanced stability and the successful repetitive use of the proteolytic columns. Thus, the wild-type and the mutant immobilized proteases were used, during a period of 18 months, for nine batch reactions with retention of 38% and 51% of their initial activities, respectively. The present results demonstrate that immobilized TEV protease on streptavidin-agarose is an attractive and efficient tool for fusion protein cleavage, especially when the target protein is fused to a streptagged fusion partner. Using this strategy, the total process can be shortened by performing the cleavage and the recovery of the purified target protein in one step.     

In-depth molecular and phenotypic characterization in a rice insertion line library facilitates gene identification through reverse and forward genetics approaches

We report here the molecular and phenotypic features of a library of 31,562 insertion lines generated in the model japonica cultivar Nipponbare of rice (Oryza sativa L.), called Oryza Tag Line (OTL). Sixteen thousand eight hundred and fourteen T-DNA and 12,410 Tos17 discrete insertion sites have been characterized in these lines. We estimate that 8686 predicted gene intervals--i.e. one-fourth to one-fifth of the estimated rice nontransposable element gene complement--are interrupted by sequence-indexed T-DNA (6563 genes) and/or Tos17 (2755 genes) inserts. Six hundred and forty-three genes are interrupted by both T-DNA and Tos17 inserts. High quality of the sequence indexation of the T2 seed samples was ascertained by several approaches. Field evaluation under agronomic conditions of 27,832 OTL has revealed that 18.2% exhibit at least one morphophysiological alteration in the T1 progeny plants. Screening 10,000 lines for altered response to inoculation by the fungal pathogen Magnaporthe oryzae allowed to observe 71 lines (0.7%) developing spontaneous lesions simulating disease mutants and 43 lines (0.4%) exhibiting an enhanced disease resistance or susceptibility. We show here that at least 3.5% (four of 114) of these alterations are tagged by the mutagens. The presence of allelic series of sequence-indexed mutations in a gene among OTL that exhibit a convergent phenotype clearly increases the chance of establishing a linkage between alterations and inserts. This convergence approach is illustrated by the identification of the rice ortholog of AtPHO2, the disruption of which causes a lesion-mimic phenotype owing to an over-accumulation of phosphate, in nine lines bearing allelic insertions.     

尿苷二磷酸葡萄糖焦磷酸化酶

介绍了尿苷二磷酸葡萄糠噍磷酸化酶定位、酶学特征及其的克隆、儿调控的研究近况。     

Mechano-biology in the thoracic aortic aneurysm: a review and case study

An aortic aneurysm is a permanent and localized dilatation of the aorta resulting from an irreversible loss of structural integrity of the aortic wall. The infrarenal segment of the abdominal aorta is the most common site of aneurysms; however, they are also common in the ascending and descending thoracic aorta. Many cases remain undetected because thoracic aortic aneurysms (TAAs) are usually asymptomatic until complications such as aortic dissection or rupture occurs. Clinical estimates of rupture potential and dissection risk, and thus interventional planning for TAAs, are currently based primarily on the maximum diameter and growth rate. The growth rate is calculated from maximum diameter measurements at two subsequent time points; however, this measure cannot reflect the complex changes of vessel wall morphology and local areas of weakening that underline the strong regional heterogeneity of TAA. Due to the high risks associated with both open and endovascular repair, an intervention is only justified if the risk for aortic rupture or dissection exceeds the interventional risks. Consequently, TAAs clinical management remains a challenge, and new methods are needed to better identify patients for elective repair. We reviewed the pathophysiology of TAAs and the role of mechanical stresses and mathematical growth models in TAA management; as a proof of concept, we applied a multiscale biomechanical analysis to a case study of TAA.     

十字花科栽培蔬菜及野生近缘种资源对小菜蛾的抗性分析

小菜蛾是危害十字花科蔬菜最严重的害虫,造成蔬菜产业巨大经济损失。现有育种材料中抗小菜蛾的资源非常稀缺,制约了抗虫新品种的选育。本研究利用国家蔬菜种质资源中期库中近年收集的来自10个国家,42个种,218份十字花科栽培蔬菜及野生近缘种资源进行了小菜蛾抗性鉴定。这些资源的抗性水平从极抗到极感皆有分布,除极端抗/感材料较少外,总体上呈均匀分布。属间、种间、变种间的平均抗性差异显著。同种植物的不同种质间抗性分布亦广。栽培资源比野生资源具有相对更高的平均抗虫水平,但是后者高抗材料更多。北部欧洲等地是抗性资源分布相对集中的地理区域。本研究获得抗虫资源19份,其中高抗资源4份,这些材料对蔬菜抗虫育种和抗虫基因发掘具有重要应用价值。