本研究比较了分别摄取了dsHvβ′COPI（外壳蛋白复合物I，β′亚基）和dsGFP的茄二十八星瓢虫（Henosepilachna viginitoctopunctata）转录组，以期了解外源dsRNA在茄二十八星瓢虫体内引起的脱靶效应。RNA-Seq结果显示，与dsGFP对照组相比，dsHvβ′COPI处理组中分别有63个上调和44个下调的差异表达基因（DEG）。通过荧光定量PCR（RT-qPCR）分析验证选取的DEG的差异表达，证实了转录组分析结果是可靠的。进一步的分析表明，在茄二十八星瓢虫转录组中没有与Hvβ'COPI同源的基因。此外，在转录组中未发现与dsHvβ'COPI连续匹配>11 bp的基因。我们选择了6个可能参与茄二十八星瓢虫中Hvβ'COPI所参与的代谢路径基因（Hvcitron、Hvhelicase、Hvtransportsase、Hvserine、Hvdynein和HvE3 ubiquitin）来检测dsHvβ'COPI可能引起的脱靶效应。利用RNAi评估这6个脱靶基因的沉默效率。然而，RNAi结果表明这6个基因的表达量虽下调，但对茄二十八星瓢虫无明显的致死作用。此外，本研究的结果将有助于将来RNAi介导的害虫防治的风险分析。

植物病原菌侵染植物过程中分泌大量的外泌蛋白调控植物免疫反应，这些外泌蛋白中包含一类富含半胱氨酸(small cysteine-rich, SCR)的小分泌蛋白，它能帮助病原菌在寄主植物中定殖。烟草疫霉中同样存在富含半胱氨酸外泌蛋白，但其功能尚不清楚。本研究通过生物信息学分析，发现烟草疫霉PnSCR82同源于恶疫霉的植物毒性蛋白PcF (Phytophthora cactorum Fragaria)，其具有外泌信号肽，同时富含半胱氨酸。为研究烟草疫霉PnSCR82功能，将PnSCR82构建于马铃薯X病毒（PVX）表达载体(pGR106)，利用农杆菌介导的转化方法注射本氏烟草叶片和番叶片茄，发现在烟草叶片瞬时表达PnSCR82，能够诱导烟草叶片和番茄叶片出现过敏性坏死，并且能增加胼胝质和活性氧在叶片的积累，接种辣椒疫霉后发现其增强对辣椒疫霉的抗性。通过构建其亚细胞定位载体PnSCR82-GFP，利用农杆菌介导的转化注射烟草叶片发现其定位于烟草细胞膜。同时RT-PCR分析发现在烟草叶片中瞬时表达PnSCR82能够激活烟草水杨酸，茉莉酸和乙烯途径的部分相关基因上调表达。烟草疫霉PnSCR82作为激发子一员，能够诱导烟草细胞过敏性坏死同时也能激活不同免疫信号通路相关基因上调表达，能够增强植物对辣椒疫霉的抗性，具有潜在的提高植物免疫应用价值。本文首次验证了烟草疫霉富含半胱氨酸蛋白PnSCR82功能，作为激发子，PnSCR82能够诱导植物产生过敏性坏死反应，能够激活部分免疫相关基因的上调表达，具有潜在的应用价值。

RNAi介导的有害生物防控策略是农业生物技术领域的最新突破之一。但是，目前对RNAi防控策略可能产生的脱靶效应仍未完全了解。本文中，我们研究了两种昆虫致死基因siRNA在靶标和非标靶昆虫中的脱靶效应。结果表明，致死基因siRNA的脱靶效应广泛存在于靶标昆虫和非靶标昆虫中。我们根据基因的同源性、相关KEGG途径以及与siRNA序列的连续匹配度，对所有表达量受影响的基因进行了分类。出人意料的是，非靶标基因表达量受影响的程度与序列的连续匹配度并不一直。而少部分同源基因和KEGG相关基因的表达量正如预期的一样发生了显著变化。通过计算转录组熵值，结果表明尽管在siRNA处理后数百个基因的表达受到影响，但转录组的熵值保持不变，这表明转录组的表达模式在整体上是平衡的。本文的结果表明，siRNA与非靶标生物中的单个基因发生交叉反应，但在基因组水平上对靶标和非靶标生物中转录组完整性并没有显着影响。同时，本文提出了一种评估昆虫致死基因siRNA脱靶效应的体系，将有助于评估RNAi害虫防控策略的安全性。

外来入侵物种是指被引入到其原产地范围以外的地区，并对当地的经济、生物多样性和生态环境造成了严重危害的物种。明确入侵性的遗传机制对于开发生态友好型的方法来防控外来入侵物种至关重要，同时，揭示外来物种的基因组特征有利于准确预测其入侵性潜能。然而，尽管已经有大量外来入侵物种的基因组被测序，但这些数据存放零散，缺少一个综合的外来入侵物种基因组数据管理与分析平台。因此，我们通过文献调研和数据库检索，收集了已完成基因组测序的外来入侵物种的组学相关数据，构建了InvasionDB数据库。该数据库包含131个外来入侵物种（100种入侵动物和31种入侵植物）的基因组和转录组数据，其中，76个物种的基因组被详细注释，并提供基因功能（包括Pfam、KEGG和NR注释信息）查询、序列比对和基因组可视化，以及全部数据下载功能。为了提供更多与入侵性相关的信息，我们进一步分析了19个与入侵昆虫的入侵性相关的基因家族，以及非编码RNAs（包括135494 个miRNAs, 89294 个rRNAs和2671941 个tRNAs）。因此，InvasionDB对于从基因组水平来研究外来入侵物种的入侵性，以及发展新的防控技术具有重要指导意义。

Methylation of the N6 position of adenine, termed N6-methyladenine, protects DNA from restriction endonucleases via the host-specific restriction-modification system. N6-methyladenine was discovered and has been well studied in bacteria. N6-adenine-specific DNA methyltransferase (N6AMT) is the main enzyme catalyzing the methylation of the adenine base and knowledge of this enzyme was mainly derived from work in prokaryotic models. However, large-scale gene discovery at the genome level in many model organisms indicated that the N6AMT gene also exists in eukaryotes, such as humans, mice, fruit flies and plants. Here, we cloned a N6AMT gene from Nilaparvata lugens (Nlu-N6AMT) and amplified its fulllength transcript. Then, we carried out a systematic investigation of N6AMT in 33 publically available insect genomes, indicating that all studied insects had N6AMT. Genomic structure analysis showed that insect N6AMT has short introns compared with the mammalian homologs. Domain and phylogenetic analysis indicated that insect N6AMT had a conserved N6-adenineMlase domain that is specific to catalyze the adenine methylation. Nlu-N6AMT was highly expressed in the adult female. We knocked down Nlu-N6AMT by feeding dsRNA from the second instar nymph to adult female, inducing retard development of adult female. In all, we provide the first genome-wide analysis of N6AMT in insects and presented the experimental evidence that N6AMT might have important functions in reproductive development and ovary maturation.

The normalization of quantitative real-time PCR (qPCR) is important to obtain accurate gene expression data, and the most common method for qPCR normalization is to use reference genes. However, reference genes can be regulated under different conditions. qPCR has recently been used for gene expression study in Laodelphax striatellus, but there is no study on validation of the reference genes. In this study, five new housekeeping genes (LstrTUB1, LstrTUB2, LstrTUB3, LstrARF and LstrRPL9) in L. striatellus were cloned and deposited in the GenBank with accession numbers of JF728809, JF728810, JF728811, JF728807 and JF728806, respectively. Furthermore, mRNA expressions of the five genes and β-actin were measured by qPCR with insect samples of different instar at nymph stage, and the expression stabilities were determined by the software geNorm and NormFinder. As a result, ARF and RPL9 were consistently more stable than β-actin, while three TUB genes were less stable than β-actin. To determine the optimal number of reference genes used in qPCR, a pairwise variations analysis by geNorm indicated that two references ARF and RPL9 were required to obtain the accurate quantification. These results were further confirmed by the validation qPCR experiment with chitinase gene as the target gene, in which the standard error of the mRNA quantification by using binary reference ARF-RPL9 was much lower than those by ARF, RPL9 or β-actin alone. Taken together, our study suggested that the combination of ARF-RPL9 could replace β-actin as the reference genes for qPCR in L. striatellus.

In order to determine the potential for haploid induction via in vitro gynogenesis in tomato, the ovules and protoplasts of embryo sacs from the hybrids Zhongza 101 and Zhongza 105 were cultured. An efficient method of ovule isolation was established in this study. Using this method, 100-150 ovules could be isolated from one ovary. Isolated ovules were cultured on three induction media to induce gynogenesis in vitro. During culture, ovules were enlarged markedly, with opaque white color. When observed microscopically, there were cell divisions and cell clumps in embryo sacs. Subsequently, the cell clumps in embryo sacs ceased growth, likely because the integument grew faster than embryo sacs did and hindered the further development of embryo sacs. Therefore, subsequent callus morphogenesis might be originated from the integument. Thousands of calli from the two tomato varieties were obtained. Five diploid plants were regenerated after 15 months of subculturing. To eliminate the hindering effect of integument on embryo sac cells, the protoplasts of embryo sacs were prepared and cultured. After 48 hours of culture, the protoplasts of embryo sacs doubled in size and gradually formed clusters of cells. These results suggested that gynogenesis might be a potential way for haploid induction in tomato.