基于转录组和WGCNA的甘薯花青素合成相关基因共表达网络的构建及核心基因的挖掘*

加权基因共表达网络分析(weighted gene co-expression network analysis, WGCNA)可通过聚类鉴定共表达的基因模块来研究生物学数据与相应性状之间的关系。甘薯[Ipomoea batatas (L.)Lam.]是世界上营养丰富的块根作物之一,紫薯是甘薯的一种特殊品种,因含有大量的花青素而具有较高的营养价值。因此,培育花青素含量高的优质紫薯一直以来都是甘薯育种家所追求的目标。利用传统的育种方法已经培育了一些紫薯品种,但其周期长、工作量大、见效慢,所以急需通过分子设计育种手段来培育高产优质的紫薯新品种。花青素合成相关关键基因的挖掘对紫薯的分子育种具有重要意义。为了挖掘甘薯花青素合成相关基因,以紫薯品种‘徐紫薯3号'和白薯品种‘徐薯18号'的块根为材料进行了转录组测序(RNA-seq),并结合公共数据库中已公布的甘薯基因组信息以及43份紫薯和45份非紫薯块根的RNA-seq数据,通过分析在不同样本间表达量差异大的前50%的基因中选择了26 760个基因进行WGCNA分析。结果表明,利用WGCNA鉴定出28个共表达模块,其中4个为紫薯特异性模块(Grey60模块和Black模块与紫薯显著正相关,Brown模块和Blue模块与紫薯显著负相关)。利用GO功能富集分析发现紫薯特异性模块Grey60可以显著富集到类黄酮和花青素代谢过程。通过计算模块内基因的连通性,分析挖掘到Grey60模块中有47个核心基因,其中包括已报道的8个花青素合成相关基因MYB113、CHS、3个CHI、F3H、GST和LDOX。利用qRT-PCR验证了其中7个核心基因的表达模式。通过构建核心基因的互作网络发现:MYB不仅与已知的花青素合成相关基因bHLH、CHI、GST、F3'H、CHS等存在互作,同时也与DUF914、ABCC4等转运蛋白基因互作;WRKY3与多个核心基因存在互作,如LDOX、GST、CHS等。为高花青素含量紫薯新品种的培育和紫薯花青素生物合成机制的解析提供了理论基础和新思路。     

Transcriptome analysis reveals anthocyanin acts as a protectant in <Emphasis Type="Italic">Begonia semperflorens</Emphasis> under low temperature

Anthocyanins are natural bioactive pigments in plants that play important roles in many physiological functions. They are found in various tissues and can protect plants against different stress conditions. Anthocyanins are synthesized and accumulate in nutritional organs, which is crucial for plants to adapt to and resist adverse environmental conditions, including high exposure to light, ultraviolet light, low temperatures, drought, pests and disease. Some progress has been made in understanding the adaptability of anthocyanin to the external environment. Begonia semperflorens is an excellent model for studying the function and regulation of anthocyanin synthesis. To investigate the biosynthesis and regulation of anthocyanins, RNA sequencing techniques were employed to investigate anthocyanin biosynthesis induced by low temperature in B. semperflorens leaves. A total of 74,779 unigenes with a mean length of 1249 bp were assembled. Functional annotations were implemented using five protein databases. Differentially expressed genes involved in the process of anthocyanin biosynthesis were identified. This study represents the first report of a broad-scale gene expression study on B. semperflorens.     

水稻花色苷含量的遗传研究进展

花色苷作为水稻重要的生物活性物质,已成为当前功能性水稻研究开发的热点之一。本文从水稻花色苷的生物合成及其组成成分入手,着重介绍了水稻花色苷含量的影响因素、遗传及分子机理的研究现状以及富集花色苷水稻种质鉴定、筛选与创新现状,并探讨了今后以提高水稻花色苷含量为目标的功能性水稻研究内容和方向。     

Identification of Novel Oryza sativa miRNAs in Deep Sequencing-Based Small RNA Libraries of Rice Infected with Rice Stripe Virus

MicroRNAs (miRNAs) play essential regulatory roles in the development of eukaryotes. Methods based on deep-sequencing have provided a powerful high-throughput strategy for identifying novel miRNAs and have previously been used to identify over 100 novel miRNAs from rice. Most of these reports are related to studies of rice development, tissue differentiation, or abiotic stress, but novel rice miRNAs related to viral infection have rarely been identified. In previous work, we constructed and pyrosequenced the small RNA (sRNA) libraries of rice infected with Rice stripe virus and described the character of the small interfering RNAs (siRNA) derived from the RSV RNA genome. We now report the identification of novel miRNAs from the abundant sRNAs (with a minimum of 100 sequencing reads) in the sRNA library of RSV-infected rice. 7 putative novel miRNAs (pn-miRNAs) whose precursor sequences have not previously been described were identified and could be detected by Northern blot or RT-PCR, and were recognized as novel miRNAs (n-miRNAs). Further analysis showed that 5 of the 7 n-miRNAs were up-expressed while the other 2 n-miRNAs were down-expressed in RSV-infected rice. In addition, 23 pn-miRNAs that were newly produced from 19 known miRNA precursors were also identified. This is first report of novel rice miRNAs produced from new precursors related to RSV infection.     

Different localization patterns of anthocyanin species in the pericarp of black rice revealed by imaging mass spectrometry

Black rice (Oryza sativa L. Japonica) contains high levels of anthocyanins in the pericarp and is considered an effective health-promoting food. Several studies have identified the molecular species of anthocyanins in black rice, but information about the localization of each anthocyanin species is limited because methodologies for investigating the localization such as determining specific antibodies to anthocyanin, have not yet been developed Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is a suitable tool for investigating the localization of metabolites. In this study, we identified 7 species of anthocyanin monoglycosides and 2 species of anthocyanin diglycosides in crude extracts from black rice by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. We also analyzed black rice sections by MALDI-IMS and found 2 additional species of anthocyanin pentosides and revealed different localization patterns of anthocyanin species composed of different sugar moieties. Anthocyanin species composed of a pentose moiety (cyanidin-3-O-pentoside and petunidin-3-O-pentoside) were localized in the entire pericarp, whereas anthocyanin species composed of a hexose moiety (cyanidin-3-O-hexoside and peonidin-3-O-hexoside) were focally localized in the dorsal pericarp. These results indicate that anthocyanin species composed of different sugar moieties exhibit different localization patterns in the pericarp of black rice. This is the first detailed investigation into the localization of molecular species of anthocyanins by MALDI-IMS.     

Integrated small RNA profiling and degradome analysis of Anthurium andraeanum cultivars with different-colored spathes

MicroRNAs (miRNAs) are known to play vital roles in coloration of leaves, flowers, and fruits in plants. However, their functions in spathe coloration are poorly known. Anthurium andraeanum is a popular ornamental plant with various spathe colors. In this study, small RNA and degradome libraries from three A. andraeanum cultivars with different-colored spathes were constructed and sequenced. Illumina sequencing resulted in 94 conserved miRNAs, and 34 novel miRNAs in total were then identified based on precursor sequences and hairpin structures. Differential expression analysis showed that 52, 51, and 49 miRNAs were differentially expressed in comparisons of orange- versus white-colored spathe, purple- versus white-colored spathe, and purple- versus orange-colored spathe, respectively. The expression patterns of miRNAs and their corresponding targets involved in spathe coloration were further analyzed, and displayed that miR156b and miR529 were highly abundant in the spathes with higher anthocyanin content. These two miRNAs co-targeted a gene encoding SPL17, which may function as a negative regulator in anthocyanin accumulation. In addition, miR408 was also abundantly expressed in purple- and orange-colored spathes, and its typical targets were also identified. This comprehensive integrated analysis provides insight into the miRNA-mediated genetic regulation in spathe coloration of A. andraeanum.

     

Cloning and characterization of miRNAs from maize seedling roots under low phosphorus stress

MicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition in plants and animals. In this study, a small RNA library was constructed to identify conserved miRNAs as well as novel miRNAs in maize seedling roots under low level phosphorus stress. Twelve miRNAs were identified by high throughput sequencing of the library and subsequent analysis, two belong to conserved miRNA families (miRNA399b and miRNA156), and the remaining ten are novel and one of latter is conserved in gramineous species. Based on sequence homology, we predicted 125 potential target genes of these miRNAs and then expression patterns of 7 miRNAs were validated by semi-RT-PCR analysis. MiRNA399b, Zma-miR3, and their target genes (Zmpt1 and Zmpt2) were analyzed by real-time PCR. It is shown that both miRNA399b and Zma-miR3 are induced by low phosphorus stress and regulated by their target genes (Zmpt1 and Zmpt2). Moreover, Zma-miR3, regulated by two maize inorganic phosphate transporters as a newly identified miRNAs, would likely be directly involved in phosphate homeostasis, so was miRNA399b in Arabidopsis and rice. These results indicate that both conserved and maize-specific miRNAs play important roles in stress responses and other physiological processes correlated with phosphate starvation, regulated by their target genes. Identification of these differentially expressed miRNAs will facilitate us to uncover the molecular mechanisms underlying the progression of maize seedling roots development under low level phosphorus stress.     

Massive analysis of rice small RNAs: mechanistic implications of regulated microRNAs and variants for differential target RNA cleavage

Small RNAs have a variety of important roles in plant development, stress responses, and other processes. They exert their influence by guiding mRNA cleavage, translational repression, and chromatin modification. To identify previously unknown rice (Oryza sativa) microRNAs (miRNAs) and those regulated by environmental stress, 62 small RNA libraries were constructed from rice plants and used for deep sequencing with Illumina technology. The libraries represent several tissues from control plants and plants subjected to different environmental stress treatments. More than 94 million genome-matched reads were obtained, resulting in more than 16 million distinct small RNA sequences. This allowed an evaluation of ~400 annotated miRNAs with current criteria and the finding that among these, ~150 had small interfering RNA-like characteristics. Seventy-six new miRNAs were found, and miRNAs regulated in response to water stress, nutrient stress, or temperature stress were identified. Among the new examples of miRNA regulation were members of the same miRNA family that were differentially regulated in different organs and had distinct sequences Some of these distinct family members result in differential target cleavage and provide new insight about how an agriculturally important rice phenotype could be regulated in the panicle. This high-resolution analysis of rice miRNAs should be relevant to plant miRNAs in general, particularly in the Poaceae.     

Viral infection induces expression of novel phased microRNAs from conserved cellular microRNA precursors

RNA silencing, mediated by small RNAs including microRNAs (miRNAs) and small interfering RNAs (siRNAs), is a potent antiviral or antibacterial mechanism, besides regulating normal cellular gene expression critical for development and physiology. To gain insights into host small RNA metabolism under infections by different viruses, we used Solexa/Illumina deep sequencing to characterize the small RNA profiles of rice plants infected by two distinct viruses, Rice dwarf virus (RDV, dsRNA virus) and Rice stripe virus (RSV, a negative sense and ambisense RNA virus), respectively, as compared with those from non-infected plants. Our analyses showed that RSV infection enhanced the accumulation of some rice miRNA*s, but not their corresponding miRNAs, as well as accumulation of phased siRNAs from a particular precursor. Furthermore, RSV infection also induced the expression of novel miRNAs in a phased pattern from several conserved miRNA precursors. In comparison, no such changes in host small RNA expression was observed in RDV-infected rice plants. Significantly RSV infection elevated the expression levels of selective OsDCLs and OsAGOs, whereas RDV infection only affected the expression of certain OsRDRs. Our results provide a comparative analysis, via deep sequencing, of changes in the small RNA profiles and in the genes of RNA silencing machinery induced by different viruses in a natural and economically important crop host plant. They uncover new mechanisms and complexity of virus-host interactions that may have important implications for further studies on the evolution of cellular small RNA biogenesis that impact pathogen infection, pathogenesis, as well as organismal development.