一个CRISPR/Cas9-VQR基因编辑系统的构建

CRISPR/Cas9系统是一种广泛应用于细菌、酵母、动物和植物中的基因组定点编辑技术,但该编辑系统的使用范围受PAM (proto-spacer-motif)位点NGG的限制。本研究通过突变Streptococcus pyogenes Cas9 (SpCas9)编码氨基酸(1135位的天冬氨酸D突变成缬氨酸V, 1335位的精氨酸R突变为谷胱氨酸Q, 1337位的苏氨酸T突变为精氨酸R,命名该突变子为Cas9-VQR)改造其识别PAM为NGA的位点以扩大其使用范围。并使用玉米Ubi启动子启动Cas9-VQR基因、优化SpCas9的密码子、加入保守的核定位信号序列、增加单子叶植物中保守的3′UTR序列和使用水稻U6启动子启动gRNA来修饰该编辑系统。结果表明Cas9-VQR系统能够识别PAM为NGA的位点,并进行有效的切割。体外酶切活性检测结果表明Cas9-VQR的切割效率为5%～70%。水稻转化检测结果表明Cas9-VQR的切割效率约为27.5%～70.5%,平均切割效率为46.23%。本研究拓宽了CRISPR/Cas9系统在作物中的使用范围,特别是NGA PAM位点较高的作物。     

森林保险制度体系重塑与运行机制优化

森林保险市场存在有效需求不足、创新供给乏力、地方政府参与积极性低的供需双冷局面,财政补贴政策低效运行。其根本原因是缺乏独立的适应其特殊性的森林保险制度体系作为保障,致使现有运行机制不协同,产品体系不科学,补贴机制不合理,导致市场发展缓慢。为此,通过林业风险与林业生产的属性特征分析,确定我国森林保险属性定位应是有政府支持的商业性森林保险,在此基础上围绕运行模式、产品体系、财政补贴体系建立相应的运行机制,从而构建一套独立综合的森林保险制度体系与运行机制,以改善供需双冷市场僵局,保障财政补贴政策有效发挥,推动森林保险健康持续发展。     

柑橘轮斑病的适生区预测及风险分析

【目的】 柑橘轮斑病（citrus target spot）作为一种新发柑橘病害,造成发病果园严重的经济损失。本研究针对该病害进行适生区预测及风险分析,以便对该病采取及时、有效的管控措施,最终达到降低其流行风险等级,防止病害传播扩展的目的。【方法】 基于环境变量数据和柑橘轮斑病发生分布数据,运用MaxEnt生态位模型模拟预测柑橘轮斑病菌（Pseudofabraea citricarpa）在中国的潜在适生区分布。并通过ROC（receiver operating characteristic）曲线下面积（area under the curve,AUC）评估预测模型的精度,运用正规化训练增益刀切法（regularized training gain）获取气候因子与分布概率间的关系。同时采用有害生物风险分析理论,以有害生物风险分析的规定程序为依据探索柑橘轮斑病病害的风险分析体系和评价值的计算方法,对评价指标进行定性分析,进而量化评价值。在建立综合评价模型的基础上,计算柑橘轮斑病风险性危害值,最后对病害的风险性危害值进行评价。【结果】 柑橘轮斑病菌MaxEnt模型预测结果的平均AUC值为0.998,表明预测结果精度高。柑橘轮斑病菌的潜在适生区面积约占全国面积12.19%,高适生区、中适生区、低适生区各占全国面积约2.85%、3.99%、5.35%。高、中适生区主要集中于长江中上游柑橘优势区及其周边。其中,高适生区主要集中在四川、重庆、陕西南部,以及贵州、湖北等少量地区。中、低适生区是高适生区的外围扩展。通过MaxEnt模型正规化训练增益刀切法获取的环境变量重要性分析结果表明,最冷季度平均温度（Bio11）、最干季度平均温度（Bio9）、最冷月最低温（Bio6）是影响柑橘轮斑病菌分布的3个关键环境因子,这意味着低温、干冷季节柑橘轮斑病发生可能性大。风险分析最终创建出5个准则层、13个指标层的多指标综合评价体系,并对各指标层定量与定性分析,柑橘轮斑病在我国的风险性危害值（R值）为2.08,处于高度风险等级,对长江中上游及湖北西部-湖南西部两大柑橘产区的潜在危害最大。【结论】 柑橘轮斑病风险性较高,需要尽快建立监测体系,针对病害采取有效控制措施,阻止病害在长江中上游柑橘优势区及相邻柑橘产区传播。     

稻瘟菌糖原合成酶激酶MoGSK3功能探究

【目的】观测稻瘟菌（Magnaporthe oryzae）糖原合成酶激酶基因MoGSK3敲除突变体表型,明确MoGSK3在稻瘟菌中的潜在生物学功能,为挖掘防治稻瘟菌新型药剂的潜在靶标提供参考。【方法】基于同源重组原理,用split-PCR方法获得稻瘟菌MoGSK3敲除突变体菌株,将MoGSK3基因克隆到pFL2载体上得到MoGSK3-C融合载体,并将其通过PEG介导的原生质体转化法导入MoGSK3突变体中得到回补菌株。培养观察野生型菌株Guy11、突变体菌株G3-9及回补菌株GC-1的菌落形态和生长状况,采用实时荧光定量PCR(qRT-PCR)检测稻瘟菌产孢相关基因的表达量;观察稻瘟菌不同菌株的分生孢子形态,通过附着胞洋葱表皮穿透试验及接种水稻叶片,研究其致病力;通过KI-I-2染色对野生型菌株Guy11及突变体菌株G3-9分生孢子和附着胞中的糖原运输能力进行观测。【结果】稻瘟菌MoGSK3突变体存在多个表型缺陷,与野生型菌株Guy11相比,MoGSK3突变体菌株G3-9菌落直径显著变小,生长缓慢,产孢相关基因表达量下降且分生孢子出现末端伸长畸形的状态。MoGSK3基因缺失还会导致稻瘟菌菌丝末端无法形成正常的分生孢子梗,附着胞无法穿透洋葱表皮形成侵染菌丝,接种划伤水稻叶片也无法形成褐色病斑。对MoGSK3突变体菌株G3-9分生孢子及附着胞进行糖原染色后发现,突变体菌株G3-9在糖原转运能力方面存在明显缺陷。【结论】MoGSK3基因参与稻瘟菌的生长、分生孢子形成及形态建成、侵染、糖原转运等过程,是稻瘟菌重要的毒力因子。     

Challenges and opportunities of unmanned aerial vehicles as a new tool for crop pest control

The application of pesticides is not simply delivering chemicals to the target area. It also involves considering the negative aspects and developing strategies to deal with them during the application process, to ensure the maximization of pesticides use efficiency and the maintenance of the ecosystem. Unmanned aerial vehicle (UAV) sprayers demonstrate unique advantages compared to traditional ground sprayers, particularly in terms of maneuverability and labor intensity reduction, showed great potential for chemical application in pest control. It is undeniable that there exist challenges in the practice of UAV spraying, such as higher potential risks of pesticide drift or pathogen transmission, uncertainty canopy deposition for different crops, and unexpected leaf breakage induced by downwash flow. Maximizing the utilization of downwash flow while avoiding lateral air movement outside the intended target crop area is a major issue for chemical application with UAV sprayers, particularly in light of the increasingly apparent consensus on the need for enhanced environmental protection during the chemical application process. It must be considered that the operation strategy in different scenarios and for different crop targets is not the same, unique requirements should be given on nozzle atomization, flight parameters, adjuvants and aircraft types in specific working situations. In future, the implementation of spray drift prediction, technical procedures development, and other solutions aimed at reducing pesticide drift and improving deposition quality, is expected to promote the adoption of UAV sprayers by more farmers. © 2023 Society of Chemical Industry.     

蒜和葱上二斑叶螨的发生为害及其抗药性监测

二斑叶螨寄主植物范围极广, 但其对百合科蔬菜造成危害的研究报道极少?本研究采集了来自北京海淀百合科蔬菜蒜和葱上的2个二斑叶螨种群, 生物测定结果显示, 2个种群对阿维菌素呈现出极高水平抗性, 对哒螨灵为中等以上抗性(LC50>5 000 mg/L), 对新型药剂联苯肼酯?虫螨腈和腈吡螨酯也表现出中等到高水平抗性?抗性基因突变频率检测发现, 与阿维菌素抗性相关基因G314D和G326E位点以及和哒螨灵抗性相关基因H92R位点的突变频率均高达100%, 为纯合抗性种群?因此, 本研究所测试的2个二斑叶螨种群对阿维菌素和哒螨灵单剂的敏感性极低, 需慎重选用这2种药剂, 而3种新型杀螨剂联苯肼酯?虫螨腈和腈吡螨酯在田间需轮换使用?     

Analysis of genomic mutation and immunohistochemistry of platelet‐derived growth factor receptors in canine vascular tumours

We examined whether mutation of the platelet‐derived growth factor receptor protein tyrosine kinase (PDGFR)‐α and PDGFR‐β genes contributes to their overexpression in canine vascular tumours. Genomic sequences of trans‐ or juxtamembrane regions of PDGFR‐α and PDGFR‐β were analysed with immunohistochemical staining and polymerase chain reaction‐direct sequencing using DNA from paraffin‐embedded neoplastic tissues of 27 hemangiosarcomas (HSAs) and 20 hemangiomas (HAs). Immunohistochemically, 75% of the HA cases were positive for PDGFR‐α and almost most of the HA cases were negative for PDGFR‐β. Of the HSA cases, 55.6% were negative for PDGFR‐α and 63% were strongly positive for PDGFR‐β. Among the HA cases, 1 missense mutation was detected in PDGFR‐α exon 18 and 1 in PDGFR‐β exon 17. Two HSA cases had missense mutations in exon 14 and 1 in exon 17 of PDGFR‐β. Thus, genomic mutation of trans‐ or juxtamembrane regions of PDGFRs was not the main mechanism driving the activation of receptors in HSA and HA.     

牧草的航天诱变研究

航天诱变育种是近十几年来快速发展的农业高科技新领域,将高科技的空间技术与常规农业育种相结合,是一项高创新性育种方法,其目的是利用空间诱变技术选育植物新种质、新材料、培育新品种.我国的航天育种技术走在世界前列,已在农作物、蔬菜及花卉上广泛应用,且已通过国家或省级审定、鉴定了70多个品种,牧草航天育种尚处于初始阶段,目前主要集中于紫花苜蓿(Medicago sativa)方面的相关研究.为此,简要阐述了航天诱变育种的定义、机理和特点,介绍了紫花苜蓿、燕麦、猫尾草、红三叶、白三叶、黄花矶松、红豆草和沙打旺等几种牧草航天诱变研究的自身特点,以及目前我国牧草航天诱变研究进展,并提出存在的问题和今后的研究内容.     

Highly recurrent BRAF p.V595E mutation in canine papillary oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the most common oral epithelial malignancy in dogs. It exhibits locally aggressive biological behaviour with the potential to metastasize, and a reported 1-year survival rate of 0% when left untreated. Expression studies suggest that aberrant MAPK signalling plays a key role in canine OSCC tumorigenesis, which is consistent with BRAF and HRAS MAPK-activating mutations reported in some tumours. Several morphological subtypes of canine OSCC have been described, with papillary, conventional, and basaloid as the most common patterns. We hypothesized that mutational differences may underlie these phenotypic variations. In this study, targeted Sanger sequencing and restriction fragment length polymorphism assays demonstrate that up to 85.7% of canine papillary OSCC (n = 14) harbour a BRAF p.V595E mutation. Assessment of neoplastic epithelial cell proliferation using Ki67 immunolabelling (n = 10) confirmed a relatively high proliferation activity, consistent with their known aggressive clinical behaviour. These findings underscore a consistent genetic feature of canine papillary OSCC and provide a basis for the development of novel diagnostic and targeted therapeutic approaches that can improve the quality of veterinary care.