木薯MeSWEET10a基因启动子EBE区编辑载体的构建及验证

木薯细菌性枯萎病（cassava bacterial blight, CBB）是由地毯草黄单胞木薯萎蔫致病变种（Xanthomonas axonopodis pv. Manihotis, Xam）引起的木薯重要病害,影响木薯产量,我国主栽的木薯品种均不抗Xam。Xam分泌的TAL20效应蛋白可结合木薯MeSWEET10a基因启动子的EBE区,激活该基因表达,促进糖类运输至Xam侵染的部位,为病菌的繁殖提供碳水化合物。本研究利用在线软件CRISPR-P v2.0设计EBE区的基因编辑靶点,构建CRISPR/Cas9基因编辑质粒pCAMBIA1301-Cas9-EBE-sgRNA。将重组质粒转化LBA4404根癌农杆菌,利用农杆菌介导法侵染木薯脆性胚性愈伤组织。提取侵染和未侵染的木薯脆性胚性愈伤组织DNA,PCR扩增靶点EBE区及潜在的脱靶位点,并进行Sanger测序分析编辑效果。结果发现EBE区被成功编辑,且没有脱靶现象。本研究有助于进一步获得MeSWEET10a基因启动子EBE区域的木薯突变体,以增强木薯抗细菌性枯萎病的能力。     

Expression of matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1 and collagen type IV in lung of rats with multiple organ dysfunction syndrome

AIM: To determine the expression of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of me-talloproteinase-1 (TIMP-1) and collagen type IV (IV-C) in the lung of rats with multiple organ dysfunction syndrome (MODS) and to investigate the mechanism of lung injury in MODS. METHODS: Adult male Sprague-Dawley (SD) rats (n=40) were randomly divided into sham control group and cecal ligation and puncture (CLP) model group. The rats in CLP group were divided into 4 subgroups as different intervals (6 h, 12 h, 24 h and 48 h), and there were 8 rats in each group. The rat model of MODS was established by CLP. All rats were sacrificed at various intervals. The functions of the liver, kidney and lung were determined by blood biochemical and blood gas analysis. The morphological changes of the lung tissues were observed with HE staining. The serum levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, MMP-9 and TIMP-1 were measured by ELISA. The expression of MMP-9 and TIMP-1 in the lung tissues was detected by RT-PCR and immunohistochemistry, and the expression of IV-C in the lung tissues was detected by immunofluorescence and Western blot. RESULTS: Compared with sham control group, the functions of the liver, kidney and lung were damaged at different degrees in model groups. No histopathological change in the lung tissues of sham control group was found, and the lung injury was serious in model groups. Compared with sham control group, the serum levels of TNF-α, IL-1β, MMP-9 and TIMP-1 in model groups increased significantly (P<0.05) and peaked at the interval of 12~24 h after modeling (P<0.01). The expression of MMP-9 and TIMP-1 in the lung tissues of model groups increased, and peaked at 12 and 24 h, respectively (P<0.01). The protein level of IV-C in MODS 6 h group was not changed as compared with control group, while that at the interval of 12~48 h after modeling was significantly decreased and dropped to the lowest at 24 h (P<0.01). CONCLUSION: MMP-9 and TIMP-1 play important roles in lung injury of MODS rats by regulating the synthesis and decomposition of IV-C which is the main component of extracellular matrix.     

利用CRISPR/Cas9技术编辑粒长基因GS3改善粳稻花时

【目的】花时不遇是影响杂交水稻制种产量的直接因素之一,已有研究表明水稻粒型差异对花时有影响。本研究采用GS3功能缺失突变来研究粳稻花时差异,以期为粒型影响花时提供佐证。【方法】利用CRISPR/Cas9技术来定向编辑控制粒长基因GS3,获得13对粒型差异的近等基因系粳稻,小区种植,采用目测法来调查花时。【结果】获得转基因T₀植株并对其T₁植株测序分析,发现长白25、吉粳102、浙粳88、武运粳27和J42均发生单碱基插入移码突变,垦鉴稻6号、空育131、浙粳22、扬粳4227、南粳9108、J5933、J6167和J5938均发生部分碱基缺失突变。对T₁植株粒型考查表明,gs3突变体的粒长均显著长于野生型。对稳定的后代花时统计分析发现,粒型变长的突变体均比野生型花时有所提前,且吉粳102、空育131、浙粳88、武运粳27、扬粳4227这5个材料的gs3突变体花时均显著早于野生型,其余材料开花也提早,但不显著。【结论】长粒型粳稻GS3突变体的花时早于短粒粳稻野生型,这一研究可为粳稻粒型育种提供参考,加速长粒粳稻亲本选育,有望推动杂交粳稻发展。     

转基因抗草甘膦油菜内源特异参照基因BnACCg8和外源E9 3''终止子的检测研究

对转基因抗草甘膦油菜内源特异参照基因BnACCg8和外源基因E9 3’终止子进行了普通PCR和。TaqMan实时荧光PCR检测，建立了相应的检测方法。     

夏谷新品种秦谷九号及其栽培技术

秦谷九号是陕西省渭南市农业科学研究所选育的夏谷新品种。该品种抗例抗旱，优质高产，抗病性强，综合性状全面，是陕西省渭北旱原粮食结构调整中的首选优质良种。     

甘蓝花粉特异表达的多聚半乳糖醛酸酶基因BoMF9的克隆与特征分析

根据白菜花粉特异的多聚半乳糖醛酸酶基因BcMF9的全长序列设计引物，通过PCR直接扩增的方法从结球甘蓝（Brassica. oleracea var. capitata）和芥蓝（B. oleracea var. alboglabra）中克隆得到BcMF9的同源基因BoMF9（BoMF9c和BoMF9l）。生物信息学分析和系统树构建发现BoMF9c和BoMF9l具有花粉特异表达的多聚半乳糖醛酸酶基因的序列特征。不同组织的表达特征分析发现它们分别在结球甘蓝和芥蓝的花蕾中特异表达。推测BoMF9c和BoMF9l可能在花粉萌发和花粉管伸长中起作用。     

杂交稻甬优9号在泉州市种植表现及高产栽培技术研究

甬优9号是浙江省宁波市农业科学院作物所和宁波市种子公司合作育成的籼粳杂交水稻新组合。该组合具有丰产稳产性好、抗逆性较强、增产潜力大、米质较优等特点,适宜作中稻和双季晚稻栽培。介绍了甬优9号在泉州市的种植表现及高产栽培技术。     

Identification and Expression of Two Novel Cytochrome P450 Genes,CYP6CV1 and CYP9A38, in Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

Cnaphalocrocis medinalis Güenée can cause severe losses in rice. Cytochrome P450s play crucial roles in the metabolism of allelochemicals in herbivorous insects. Two novel P450 cDNAs, CYP6CV1 and CYP9A38, were cloned from the midgut of C. medinalis. CYP6CV1 encodes a protein of 500 amino acid residues, while CYP9A38-predicted protein has 531 amino acid residues. Both cDNA-predicted proteins contain the conserved functional domains for all P450s. Phylogenetic analyses showed that CYP6CV1 is grouped in the cluster containing CYP6B members, while CYP9A38 is in the cluster including CYP9 members. However, both clusters are contained in the same higher lineage. Homologous analysis revealed that CYP6CV1 is most similar to CYP6B8, CYP6B7, CYP6B6, CYP6B2, and CYP6B4 with the highest amino acid identity of 41%. CYP9A38 is closest to CYP9A17, CYP9A21, CYP9A20, and CYP9A19 with the highest amino acid identity of 66%. Studies of temporal expression profiles revealed that CYP9A38 showed a steady increase in mRNA level during the five instar stages, but a low-expression level in pupae, and then presented at a high-expression level again in adults. Similar expression patterns were obtained with CYP6CV1. In the fifth instar larvae, CYP6CV1 was mainly expressed in midgut and fat bodies, whereas CYP9A38 was mainly expressed in midgut. Expression studies also revealed a 3.20-fold over-expression of CYP6CV1 and 3.54-fold over-expression of CYP9A38 after larval exposure to host rice resistance. Our results suggest that both CYP6CV1 and CYP9A38 may be involved in detoxification of rice phytochemicals.     

Fucoidan from Laminaria japonica Inhibits Expression of GLUT9 and URAT1 via PI3K/Akt,JNK and NF-κB Pathways in Uric Acid-Exposed HK-2 Cells

This work aimed to investigate the effect of fucoidan (FPS) on urate transporters induced by uric acid (UA). The results showed that UA stimulated the expression of glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1) in HK-2 cells, and FPS could reverse the effect. Moreover, UA could activate NF-κB, JNK and PI3K/Akt pathways, but both pathway inhibitors and FPS inhibited the UA-induced activation of these three pathways. These data suggested that FPS effectively inhibited the expression induction of reabsorption transporters URAT1 and GLUT9 by UA, through repressing the activation of NF-κB, JNK and PI3K/Akt signal pathways in HK-2 cells. The in vitro research findings support the in vivo results that FPS reduces serum uric acid content in hyperuricemia mice and rats through inhibiting the expression of URAT1 and GLUT9 in renal tubular epithelial cells. This study provides a theoretical basis for the application of FPS in the treatment of hyperuricemia.