Bird flu (H7N9) outbreak and its implications on the supply chain of poultry meat in China

During the past two decades, food safety issues in China not only posed serious threats to Chinese consumers but also damaged the image of Chinese products internationally. In China, food safety is not only about scientific discoveries, advanced laboratories, and sanitation equipment; it is more about the role of different players in the food supply chain. The poultry meat supply chain is instrumental in the spread of the avian influenza A virus (H7N9), raising questions about how policymakers respond to such threats and whether industries need to be restructured to manage and control this epidemic so that it does not recur. As a short-term measure, to prevent the spread of this disease, government authorities enforced the closure of live bird markets (LBM) in disease-affected areas of China. However, in the long term, the poultry meat supply chain needs to be restructured. The aim of the current study was to analyze distribution channels for chicken meat in China and then describe arrangements in poultry meat sectors that incorporate small- and medium-scale producers into the supply chain while responding to shifts in LBMs. We also assessed the role of LBMs in spreading H7N9 and how these interventions affect the poultry meat supply chain in the Chinese market.     

Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR/Cas9-Edited Rice Plants

Elimination of the CRISPR/Cas9 constructs in edited plants is a prerequisite for assessing genetic stability, conducting phenotypic characterization, and applying for commercialization of the plants. However, removal of the CRISPR/Cas9 transgenes by genetic segregation and by backcross is laborious and time consuming. We previously reported the development of the transgene killer CRISPR (TKC) technology that uses a pair of suicide genes to trigger self-elimination of the transgenes without compromising gene editing efficiency. The TKC technology enables isolation of transgene-free CRISPR-edited plants within a single generation, greatly accelerating crop improvements. Here, we presented two new TKC vectors that show great efficiency in both editing the target gene and in undergoing self-elimination of the transgenes. The new vectors replaced the CaMV35S promoter used in our previous TKC vector with two rice promoters to drive one of the suicide genes, providing advantages over our previous TKC vector under certain conditions. The vectors reported here offered more options and flexibility to conduct gene editing experiments in rice.     

利用CRISPR/Cas9系统编辑日本结缕草ZjSGR基因技术研究

日本结缕草(Zoysia japonica)是一种常见的具有众多优良性状的暖季型草种,秋冬季节在我国北方地区会较早出现叶片脱绿现象,在一定程度上限制了日本结缕草在北方的大面积使用。ZjSGR是从结缕草中分离出来的一种衰老诱导基因,负调控叶片滞绿性状。本研究利用CRISPR/Cas9系统,通过基因枪介导的遗传转化方法实现对日本结缕草ZjSGR基因的编辑,经测序验证获得1株单碱基缺失突变植株。突变植株叶片深绿,在正常生长温度(28~30℃)条件下,和对照相比,叶绿素含量更高(P<0.05)。本研究为进一步阐述ZjSGR基因在日本结缕草滞绿性状调控中的作用机理提供了基础研究材料,也为培育日本结缕草滞绿品种育种工作奠定了基础。     

Reduction in cadmium accumulation in japonica rice grains by CRISPR/Cas9-mediated editing of OsNRAMP5

Cadmium (Cd) intake is harmful to human health and Cd contamination in rice grains represents a severe threat to those consuming rice as a staple food. Knockout of Cd transporters is a promising strategy to reduce Cd accumulation in rice grains. OsNRAMP5 is the major transporter for Cd and manganese (Mn) uptake in rice. Nevertheless, it is uncertain whether knockout of OsNRAMP5 is applicable to produce low Cd rice without affecting plant growth and grain yield. In this study, we adopted CRISPR/Cas9-based gene editing technology to knock out OsNRAMP5 in two japonica varieties. We generated three independent transgene-free osnramp5 mutants and investigated the effect of osnramp5 mutations on Cd accumulation and plant growth. Hydroponic experiments showed that plant growth and chlorophyll content were significantly reduced in osnramp5 mutants at low Mn conditions, and this defective growth in the mutants could be fully rescued by supply of high levels of Mn. Cd and Mn accumulation in both roots and shoots was markedly reduced in the mutants compared to that in wild-type plants. In paddy field experiments, although Cd in flag leaves and grains was greatly reduced in osnramp5 mutants, some agronomic traits including plant height, seed setting rate, and grain number per panicle were affected in the mutants, which ultimately caused a mild reduction in grain yield. The reduced plant growth in the mutants can be attributed to a marked decrease in Mn accumulation. Our results reveal that the manipulation of OsNRAMP5 should be treated with caution: When assessing the applicability of osnramp5 mutants, soil pH and soil water content in paddy fields need to be taken into consideration, since they might affect the levels of available Mn in the soil and consequently determine the effect of the mutation on grain yield.     

Sublytic C5b-9 induces protective autophagy in cultured podocytes

AIM: In podocytes, autophagy occurs at a high basal level and dysregulated autophagy is associa-ted with a variety of podocytopathies. This paper is to investigate the role of autophagy in sublytic C5b-9-induced podocyte injury. METHODS: Sublytic complement C5b-9 stimulation was used as an in vitro model. Autophagosomes were confirmed using monodansylcadaverine (MDC) staining. Immunoblotting was used to measure the change of autophagy-related markers. Cellular morphological changes were observed by Wright-Giemsa staining. Immunofluorescence staining and confocal microscopy were used to detect the expression and distribution of nephrin. The cell viability was assessed by methylthiazol tetrazolium (MTT) assay. The cell apoptosis was assessed by Annexin V-fluorescein isothiocyanate/PI staining. RESULTS: For ensuring sublytic complement injury, the maximal amounts of anti-podocyte antiserum and 160×-diluted normal human serum were used without inducing cell lysis (defined as >5% LDH release). Sublytic C5b-9 promoted autophagy of podocytes in vitro. The proautophagic effect of sublytic C5b-9 manifested in the form of accumulated MDC-labeled vesicles and enhanced the expression of LC3-Ⅱ. Autophagy inhibitor 3-methyladenosine (3-MA) promoted sublytic C5b-9-induced podocyte morphological abnormalities. Compared with the sublytic C5b-9-injured podocytes, 3-MA exposure further decreased the expression of nephrin. 3-MA enhanced sublytic C5b-9-induced podocyte apoptosis. CONCLUSION: Sublytic C5b-9 attack induces autophagy, which may play a protective role against complement-mediated podocyte injury.     

利用CRISPR/Cas9和λ-Red级联技术构建产肠毒素大肠杆菌LT敲除菌株

本研究旨在利用CRISPR/Cas9和λ-Red级联的技术对产肠毒素大肠杆菌（enterotoxigenic Escherichia coli，ETEC）K88的热不稳定性肠毒素（heat-labile toxin，LT）基因进行无痕敲除并获得K88 LT^-缺陷菌株。通过序列比对获取LT两端同源序列，并构建包含LT边界、氯霉素筛选标记、sgRNA和LT同源臂的供体片段；将供体片段转化至ETEC K88，同时分别利用λ-Red同源重组系统和CRISPR/Cas9基因编辑系统，对LT基因进行敲除；通过PCR验证获得了K88 LT^-缺陷菌株，并通过试验测定了敲除菌株的溶血能力和生长曲线。结果显示，λ-Red同源重组系统可成功地将LT基因替换为相应的供体片段，CRISPR/Cas9基因编辑系统可高效地对筛选标记进行删除，最终通过λ-Red和CRISPR/Cas9结合的基因编辑系统可成功对ETEC K88的LT基因进行无痕敲除。体外试验结果表明，K88 LT^-缺陷菌株的溶血能力丧失，并且生长速度比野生型菌株减缓，LT可能和ETEC K88的致病能力和生长性能有关。表明λ-Red和CRISPR/Cas9级联的基因敲除方法可用于LT毒素基因及其他一些大肠杆菌基因的敲除。K88 LT^-缺陷菌株的构建为下一步研究LT毒素的致病机制奠定基础。     

茶树咖啡碱合成酶CRISPR/Cas9基因组编辑载体的构建

CRISPR/Cas9技术是一门新兴的基因组定点编辑技术,具有操作简单、高效的优点,可轻松实现对目标基因的敲除、替换和定点突变等操作。该技术刚诞生,就受到了全球生命科学领域研究者的关注,不到3年的时间就已经成功应用于多种动、植物当中。然而CRISPR/Cas9技术在茶树中的应用面临载体构建问题,本文以茶树咖啡碱合成酶为例,联合采用常规PCR、Overlapping PCR和Golden Gate Cloning技术,构建了包含茶树咖啡碱合成酶双靶点的CRISPR/Cas9基因编辑载体,为CRISPR/Cas9介导的基因组编辑技术在茶树中的应用奠定了坚实基础。     

黑花生“中花9号”的施肥效应研究

在露地栽培条件下,设置5个复合肥施用量处理,探讨使用万植牌有机无机复合肥(N-P-K=15-15-15)栽培黑花生"中花9号"的适宜施肥量。结果表明:三峡库区采用万植牌有机无机复合肥露地栽培"中花9号"的适宜施肥量为40kg/667m~2,荚果产量可达249.5 kg/667m~2,饱果产量218.0 kg/667m~2,籽仁产量170.3 kg/667m~2,饱满籽仁产量可达126.6 kg/667m~2。     

Preliminary study of endocytosis-related gene mRSD-9

AIM: To study the function of the gene mRSD-9 . METHODS: The techniques of immunoprecipitation and immunofluorescence were applied to verify the interaction between mRSD-9 and endophlin 3. The ATP/GTP combined experiment and the clathrin releasing experiment were conducted to investigate the effect of mRSD-9 on endocytosis. RESULTS: Expressed Myc-mRSD-9 was precipitated by Flag-endophilin 3. Conversely, the expressed Flag-endophilin 3 was also precipitated by Myc-mRSD-9. Under laser scanning confocal microscope, mRSD-9-GFP fusion protein and endophilin 3-RFP fusion protein were observed to co-localize in CHO cells. The combination of mRSD-9 protein with ATP/GTP was found and was specific because no combination was detected using mutant ΔmRSD-9. In empty vector transfected group, the quantity of transferrin in red fluorescent expressed cells was roughly the same as the untransfected cells. In pDsRed1-N1-mRSD-9 transfected group, the quantity of transferrin in mRSD-9 protein expressed cells was obviously reduced. In pDsRed1-N1-ΔmRSD-9 transfected group, the quantity of transferrin in ΔmRSD-9 protein expressed cells was significantly increased. CONCLUSION: The protein coded by mRSD-9 gene can suppress endocytosis of transferrin.