黏虫CYP9A134基因的克隆及其解毒功能

昆虫体内的细胞色素P450酶系统在昆虫解毒过程中发挥着重要作用。本次试验通过转录组测序获得一条新的黏虫P450基因,经国际委员会命名为CYP9A134（登录号为MT990973）。该序列全长为1801 bp,开放序列长度为1596 bp,编码531个氨基酸,分子质量为61.42 kDa,等电点为4.90。在黏虫幼虫阶段用2.5%高效氯氟氰菊酯和20%氯虫苯甲酰胺诱导时该基因表达量会有不同程度上升,最高的可分别达对照组的2.6倍和6.5倍。RNA干扰后,该基因表达量最低下降了70%,以上2种杀虫剂LD30杀虫效果分别提高了13%和25%;在黏虫成虫阶段用20%氯虫苯甲酰胺LD30诱导时,该基因表达量最高可达7.8倍。RNA干扰后,该基因表达量最低下降了61%,LD30剂量的氯虫苯甲酰胺杀虫效果提高26%。结果表明,该基因可能在杀虫剂诱导的解毒代谢过程中发挥重要作用。     

Identifying and Characterizing a Novel Peritrophic Matrix Protein (MdPM-17) Associated With Antibacterial Response From the Housefly,Musca domestica (Diptera: Muscidae)

Peritrophic matrix/membrane (PM) critically prevents the midgut of insects from external invasion by microbes. The proteins in the peritrophic membrane are its major structural components. Additionally, they determine the formation and function of this membrane. However, the role of PM proteins in immune regulation is unclear. Herein, we isolated a novel PM protein (MdPM-17) from Musca domestica larvae. Further, the function of MdPM-17 in regulating host innate immunity was identified. Results showed that the cDNA of MdPM-17 full is 635 bp in length. Moreover, it consists of a 477-bp open reading frame encoding 158 amino acid residues. These amino acid residues are composed of two Chitin-binding type-2 domain (ChtBD2) and 19 amino acids as a signal peptide. Moreover, tissue distribution analysis indicates that MdPM-17 was enriched expressed in midgut, and moderate levels in the fat body, foregut, and malpighian tubule. Notably, MdPM-17 recombinant protein showed high chitin-binding capacity, thus belongs to the Class III PM protein group. MdPM-17 protein silencing via RNA interference resulted in the expression of antimicrobial peptide (defensin, cecropins, and diptericin) genes, and this occurred after oral inoculation with exogenous microbes Escherichia coli (Enterobacteriales:Enterobacteriaceae), Staphylococcus aureus (Bacillales:Staphylococcaceae), and Candida albicans (Endomycetales:Saccharomycetaceae)). Therefore, all the antimicrobial peptide (AMP) gene expression levels are high in MdPM-17-depleted larvae during microbial infection compared to controls. Consequently, these findings indicate that MdPM-17 protein is associated with the antibacterial response from the housefly.     

高通量测序发现河南开封、中牟西瓜病毒病由多种病毒复合侵染导致

 调查发现河南省开封市祥符区和郑州市中牟县西瓜田病毒病严重发生,症状表现为叶片黄化、蕨叶、皱缩、卷曲,枝梢翘起,果实变小和瓜纹不清。为明确其病毒种类,通过小RNA测序及生物信息学分析发现采集的西瓜样本中存在8种病毒。其中包括5种已知病毒:小西葫芦黄花叶病毒(Zucchini yellow mosaic virus, ZYMV)、甜瓜蚜传黄化病毒(Melon aphid-borne yellows virus, MABYV)、西瓜花叶病毒(Watermelon mosaic virus, WMV)、黄瓜绿斑驳花叶病毒(Cucumber green mottle mo-saic virus, CGMMV)和西瓜隐潜病毒(Citrullus lanatus cryptic virus, CiLCV);3种为新检出的RNA病毒:西瓜病毒A(Watermelon virus A, WVA)、西瓜皱叶相关病毒1号(Watermelon crinkle leaf-associated virus 1, WCLaV-1)和西瓜皱叶相关病毒2号(Watermelon crinkle leaf-associated virus 2, WCLaV-2)。RT-PCR检测田间66个西瓜标样发现,WMV、CiLCV和ZYMV是侵染两地西瓜的主要病毒,其阳性检出率分别为61%、56%和52%,两种及以上病毒复合侵染的比率达68%。     

水稻种子总RNA提取质量问题的探究

水稻种子总RNA提取在水稻分子遗传基础实验中起着非常重要的作用,然而,高质量水稻种子RNA的获取难度高,极大阻碍了cDNA文库构建、RNA-Seq和基因表达分析等相关研究工作的开展和深入。为了探讨水稻种子总RNA提取的相关影响因素,寻找合适的水稻种子总RNA提取方式,本研究通过比较Trizol法、GeneMark试剂盒法和艾德莱试剂盒法3种不同的提取方式、不同的耗材处理方法、研磨方式等不同因素改进水稻种子总RNA的提取效果。不同的检测结果显示,艾德莱多糖多酚提取试剂盒能够提取到28S、18S条带完整、降解少和纯度高的水稻种子总RNA;不同耗材处理方法中,蛋白酶K处理过的耗材提取的总RNA比另外两种方法处理过的耗材提取的总RNA降解更少,28S和18S条带更清晰和完整、总RNA质量最高、效果最好;而研磨方式上,液氮冷冻研磨法能够有效抑制RNA酶对RNA的降解。本研究对水稻种子RNA提取过程相关因素的分析和改进,为促进以水稻种子为研究对象的分子实验奠定了一定的技术基础。     

细菌表达 dsRNA 介导桔小实蝇 flightin基因的 RNA 干扰

【目的】探索饲喂细菌表达目标基因dsRNA介导桔小实蝇flightin基因RNAi的可行性。【方法】将桔小实蝇flightin基因的相应干扰片段构建至L4440干扰载体,并转化大肠杆菌HT115(DE3)菌株,利用IPTG诱导表达flightin基因对应的dsRNA,命名为flightin-dsRNA。【结果】通过饲喂表达flightin-dsRNA的大肠杆菌10倍浓缩菌液,桔小实蝇flightin基因的表达普遍出现了不同程度的上调,其中,雌虫饲喂后5、10、20 d,雄虫饲喂后5 d与对照差异显著,雄虫饲喂后15 d诱发约43%的下调;该方法对桔小实蝇飞行能力及胸部肌肉发育未产生明显影响。【结论】通过饲喂表达flightin-dsRNA的大肠杆菌对桔小实蝇flightin基因进行RNA干扰不可行或干扰效果不明显。