异色瓢虫丙酮酸激酶基因序列分析及其调控海藻糖代谢功能

【目的】丙酮酸激酶(pyruvate kinase,PYK)是一种调节性糖酵解酶,负责调节糖酵解和糖异生之间的平衡,具有重要的生理作用。本研究通过分析异色瓢虫(Harmonia axyridis)3条HaPYK的序列结构,并结合RNA干扰(RNAi)技术,探讨HaPYK在瓢虫体内的生物学功能。【方法】基于从异色瓢虫全基因组中获得的3条HaPYK序列(被分别命名为HaPYK1-1、HaPYK1-2、HaPYK2),通过生物信息学方法分析其理化性质和序列结构,以及与其他昆虫之间的同源性。然后采用显微注射的方法将体外合成的dsRNA导入羽化第1天成虫体内,在注射后48和72 h分别收集试验材料,提取虫体总RNA后进行反转录,随后采用实时荧光定量PCR(qRT-PCR)技术测定3个HaPYK的表达水平以评估RNAi效果,并检测与海藻糖代谢相关基因的表达水平,最后检测葡萄糖、糖原、海藻糖含量以及两种海藻糖酶的活性。【结果】HaPYK1-1、HaPYK1-2、HaPYK2的开放阅读框分别为1 350、1 569、1 656 bp;蛋白大小分别为449、522、551 aa,理论等电点分别为5.04...     

Identification of chorion genes and RNA interference-mediated functional characterization of chorion-1 in Plutella xylostella

Choriogenesis is the last step of insect oogenesis, a process by which the chorion polypeptides are produced by the follicular cells and deposited on the surface of oocytes in order to provide a highly specialized protective barrier to the embryo. The essential features of chorion genes have yet to be clearly understood in the diamondback moth, Plutellaxylostella, a worldwide Lepidoptera pest attacking cruciferous crops and wild plants. In this study, complete sequences for 15 putative chorion genes were identified, and grouped into A and B classes. Phylogenetic analysis revealed that both classes were highly conserved and within each, branches are also species-specific. Chorion genes from each class were located in pairs on scaffolds of the P. xylostella genome, some of which shared the common promoter regulatory region. All chorion genes were highly specifically expressed in the P. xylostella adult females, mostly in the ovary with full yolk, which is a crucial period to build the shells of the eggs. RNAi-based knockdown of chorion-1, which is located on the Px_scaffold 6 alone, although had no effect on yolk deposition, resulted in smaller eggs and sharply reduced hatchability. Additionally, inhibition of PxCho-1 expression caused a less dense arrangement of the columnar layers, reduced exochorion roughness and shorter microvilli. Our study provides the foundation for exploring molecular mechanisms of female reproduction in P. xylostella, and for making use of chorion genes as the potential genetic-based molecular target to better control this economically important pest.     

Pathogenicity and Gene Expression Pattern of the Exocrine Protein LtGH61A of Grape Canker Fungus

【Objective】Grape canker disease, caused by Botryosphaeria genus fungi, occurs in a wide range of grape-producing areas in China and seriously threatens the yield and quality of grape. The objective of this study is to analyze the function of a hypothetical exocrine protein, LtGH61A, in grape canker fungus Lasiodiplodia theobromae, and to lay a foundation for in-depth analysis of the pathogenic mechanism and disease control of grape canker fungus.【Method】The signal peptide of LtGH61A protein was predicted by SignalP 4.0. The function of LtGH61A protein was predicted by the homologous comparison and functional annotation. The exocrine characteristic of LtGH61A protein was analyzed by yeast complementary experiment. The quantitative real-time PCR (qRT-PCR) was used to analyze the expression of LtGH61A in vegetative hyphae and different infection processes. The expression of LtGH61A was inhibited through RNA interference (RNAi). The effect of LtGH61A protein on the pathogenicity of L. theobromae was analyzed by in vitro inoculation test of grape shoots. The effect of LtGH61A protein on the hyphal growth rate of L. theobromae was analyzed by comparing the colony diameter.【Result】Amino acid sequence analysis predicts that the N-terminal of the LtGH61A protein contains a signal peptide with a length of 18 amino acids. The gene function annotation suggests that LtGH61A belongs to glycoside hydrolase family 61 (GH61) and can degrade cellulose as a substrate. Yeast complementary experiments showed that the signal peptide of LtGH61A protein could guide the secretion of invertase of yeast YTK12. Compared with the vegetative hyphae, the expression of LtGH61A was increased significantly at the infectious stages, and the mRNA accumulation of LtGH61A at 48 h post inoculation was 19 times of that in the vegetative hyphae. Moreover, RNAi lines were constructed for LtGH61A and two lines RNAi-LtGH61A1 and RNAi-LtGH61A2 were confirmed by qRT-PCR. The results of in vitro inoculation test of wild-type and RNAi transformants on wounded grape shoots showed that the lesion length caused by both RNAi-LtGH61A1 and RNAi-LtGH61A2 was significantly shorter than that of wild type (WT) CSS-01s, which was about 55% of WT, indicating that LtGH61A affected the pathogenicity of L. theobromae. The colony diameter comparison showed that compared with WT, the colony diameter of RNAi-LtGH61A1 and RNAi-LtGH61A2 transformants became smaller, about 85% of WT, indicating that LtGH61A affected the hyphal growth rate of L. theobromae.【Conclusion】LtGH61A affects the pathogenicity and hyphal growth of grape canker pathogen. LtGH61A protein can be secreted outside the cell. The expression level of LtGH61A during infectious stages is significantly increased, suggesting that LtGH61A can destroy the host plant tissue by exerting its own enzyme activity function, thus promoting pathogen infection.     

高效抑制O型口蹄疫病毒shRNA分子的筛选与验证

针对O型12蹄疫病毒（Foot—and—mouth disease veirus,FMDV）基因组高度保守的VP1和3D区段,选取了120个干扰靶位,根据RNAi技术原理,构建了包括对照在内的82个shRNA表达质粒;通过细胞攻毒试验,提取细胞毒液RNA,然后RT-PCR,最后real—time PCR检测病毒和shRNA的表达量。结果显示,干扰质粒组基因表达抑制率为63％～96．8％,其中pSiRe—Zs—VP1—54和pSiRe-Zs-3D-12等2个表达质粒抑制效率最好,分别为96．8％和87％,表明这2个表达质粒能够明显抑制FMDV的复制。     

沉默GSTs基因影响棉铃虫对甲氧虫酰肼的敏感性

利用RNAi技术对GSTs基因进行沉默处理,明确了沉默棉铃虫GSTs基因可以影响其对甲氧虫酰肼的敏感性。结果表明,通过饲喂siRNA在1d内就可以有效干扰GSTs基因表达,进而降低了GSTs酶活性;停止饲喂siRNA后,RNA干扰效果并不会立刻消失;沉默GSTs基因后,棉铃虫对甲氧虫酰肼的敏感性显著提高。     

雄性育性基因RNA干扰载体的构建与鉴定

1材料与方法1．1材料酶和试剂：各种限制性内切酶购自Roche公司,T4DNA连接酶购自上海生工公司,Taq聚合酶、dNTP、凝胶回收试剂盒均购自Promega公司,引物由上海生工公司合成,其他试剂均为进口或国产分析纯。植物材料和质粒：棉花洞A雄性可育株由西南大学棉花教研室提供,表达载体P^BП21质粒和大肠杆菌菌株XL1-Blue由西南大学生物技术中心实验室保存。pUCm-T克隆载体购自上海生工公司。