Yizheng Zhang

Digital Gene Expression Analysis Based on Integrated De Novo Transcriptome Assembly of Sweet Potato [Ipomoea batatas (L.) Lam.]

Background Sweet potato (Ipomoea batatas L. [Lam.]) ranks among the top six most important food crops in the world. It is widely grown throughout the world with high and stable yield, strong adaptability, rich nutrient content, and multiple uses. However, little is known about the molecular biology of this important non-model organism due to lack of genomic resources. Hence, studies based on high-throughput sequencing technologies are needed to get a comprehensive and integrated genomic resource and better understanding of gene expression patterns in different tissues and at various developmental stages. Methodology/Principal Findings Illumina paired-end (PE) RNA-Sequencing was performed, and generated 48.7 million of 75 bp PE reads. These reads were de novo assembled into 128,052 transcripts (≥100 bp), which correspond to 41.1 million base pairs, by using a combined assembly strategy. Transcripts were annotated by Blast2GO and 51,763 transcripts got BLASTX hits, in which 39,677 transcripts have GO terms and 14,117 have ECs that are associated with 147 KEGG pathways. Furthermore, transcriptome differences of seven tissues were analyzed by using Illumina digital gene expression (DGE) tag profiling and numerous differentially and specifically expressed transcripts were identified. Moreover, the expression characteristics of genes involved in viral genomes, starch metabolism and potential stress tolerance and insect resistance were also identified. Conclusions/Significance The combined de novo transcriptome assembly strategy can be applied to other organisms whose reference genomes are not available. The data provided here represent the most comprehensive and integrated genomic resources for cloning and identifying genes of interest in sweet potato. Characterization of sweet potato transcriptome provides an effective tool for better understanding the molecular mechanisms of cellular processes including development of leaves and storage roots, tissue-specific gene expression, potential biotic and abiotic stress response in sweet potato.

Brief communication: Computational detection of microRNAs targeting transcription factor genes in Arabidopsisthaliana

MicroRNAs, an abundant class of tiny non-coding RNAs, have emerged as negative regulators for translational repression or cleavage of target mRNAs by the manner of complementary base paring in plants and animals. Recent studies have demonstrated that many known microRNAs have a remarkable propensity to target genes involved in development, particularly those of transcription factor genes. Therefore, an overall detection of Arabidopsis thaliana microRNAs targeting transcription factor genes will enhance greatly our understanding of microRNA biological functions in plant development. By searching short complementary sequences between transcription factor open-reading frames and intergenic region sequences, and considering RNA secondary structures and the sequence conversation between the genomes of Arabidopsis and Oryza sativa, we detected 96 candidate Arabidopsis microRNAs. These candidate microRNAs were predicted to target 102 transcription factor genes that are classified as 28 transcription factor gene families, particularly those of DNA-binding transcription factor families, which imply that microRNAs might be involved in complex transcriptional regulatory networks for specifying individual cell types in plant development.

Analyses of the complete genome and gene expression of chloroplast of sweet potato [Ipomoea batata].

Sweet potato [Ipomoea batatas (L.) Lam] ranks among the top seven most important food crops cultivated worldwide and is hexaploid plant (2n=6x=90) in the Convolvulaceae family with a genome size between 2,200 to 3,000 Mb. The genomic resources for this crop are deficient due to its complicated genetic structure. Here, we report the complete nucleotide sequence of the chloroplast (cp) genome of sweet potato, which is a circular molecule of 161,303 bp in the typical quadripartite structure with large (LSC) and small (SSC) single-copy regions separated by a pair of inverted repeats (IRs). The chloroplast DNA contains a total of 145 genes, including 94 protein-encoding genes of which there are 72 single-copy and 11 double-copy genes. The organization and structure of the chloroplast genome (gene content and order, IR expansion/contraction, random repeating sequences, structural rearrangement) of sweet potato were compared with those of Ipomoea (L.) species and some basal important angiosperms, respectively. Some boundary gene-flow and gene gain-and-loss events were identified at intra- and inter-species levels. In addition, by comparing with the transcriptome sequences of sweet potato, the RNA editing events and differential expressions of the chloroplast functional-genes were detected. Moreover, phylogenetic analysis was conducted based on 77 protein-coding genes from 33 taxa and the result may contribute to a better understanding of the evolution progress of the genus Ipomoea (L.), including phylogenetic relationships, intraspecific differentiation and interspecific introgression.

Transcriptome Analysis to Identify Putative Floral-Specific Genes and Flowering Regulatory-Related Genes of Sweet Potato

Sweet potato flowers were collected for a transcriptome analysis to identify the putative floral-specific and flowering regulatory-related genes by using the RNA-sequencing technique. Pair-end short reads were de novo assembled by an integrated strategy, and then the floral transcriptome was carefully compared with several published vegetative transcriptomes. A total of 2595 putative floral-specific and 2928 putative vegetative-specific transcripts were detected. We also identified a large number of transcripts similar to the key genes in the flowering regulation network of Arabidopsis thaliana.

Brief Communication: Tracing the evolution of the mitochondrial protein import machinery

Mitochondria are eukaryotic organelles originated from a single bacterial endosymbiosis about 2 billion years ago. One of the earliest events in the evolution of mitochondria was the acquisition of a mechanism that facilitated the import of proteins from cytosol. The mitochondrial protein import machinery consists of dozens of subunits, and they are of modular design. However, to date, it is not clear when certain component was added to the machinery. Using extensive homology searches, the evolutionary history of the mitochondrial protein import machinery was reconstructed. The results indicated that 6 of the 35 subunits have homologs in prokaryote, suggesting that they were prokaryotic origin; the major subunit gains were occurred in the earliest stage of eukaryotic evolution; subsequent to the gain of these conserved set of subunits, the mitochondrial protein import machinery components diversified along the eukaryotic lineages and a number of lineage-specific subunits can be observed. Furthermore, protein import systems of mitochondria-like organelles (hydrogenosomes and mitosomes) have dramatically reduced their subunit contents, however, they share most of the prokaryotic origin components with mitochondrion.

Draft Genome Sequence of Bacillus pumilus BA06, a Producer of Alkaline Serine Protease with Leather-Dehairing Function

Bacillus pumilus BA06 was isolated from the proteinaceous soil and produced an extracellular alkaline protease with leather-dehairing function. The genome of BA06 was sequenced. The comparative genome analysis indicated that strain BA06 is different in genome from the other B. pumilus strains, with limited insertions, deletions, and rearrangements.

Transcriptomic Analysis and the Expression of Disease-Resistant Genes in Oryza meyeriana under Native Condition.

Oryza meyeriana (O. meyeriana), with a GG genome type (2n = 24), accumulated plentiful excellent characteristics with respect to resistance to many diseases such as rice shade and blast, even immunity to bacterial blight. It is very important to know if the diseases-resistant genes exist and express in this wild rice under native conditions. However, limited genomic or transcriptomic data of O. meyeriana are currently available. In this study, we present the first comprehensive characterization of the O. meyeriana transcriptome using RNA-seq and obtained 185,323 contigs with an average length of 1,692 bp and an N50 of 2,391 bp. Through differential expression analysis, it was found that there were most tissue-specifically expressed genes in roots, and next to stems and leaves. By similarity search against protein databases, 146,450 had at least a significant alignment to existed gene models. Comparison with the Oryza sativa (japonica-type Nipponbare and indica-type 93–11) genomes revealed that 13% of the O. meyeriana contigs had not been detected in O. sativa. Many diseases-resistant genes, such as bacterial blight resistant, blast resistant, rust resistant, fusarium resistant, cyst nematode resistant and downy mildew gene, were mined from the transcriptomic database. There are two kinds of rice bacterial blight-resistant genes (Xa1 and Xa26) differentially or specifically expressed in O. meyeriana. The 4 Xa1 contigs were all only expressed in root, while three of Xa26 contigs have the highest expression level in leaves, two of Xa26 contigs have the highest expression profile in stems and one of Xa26 contigs was expressed dominantly in roots. The transcriptomic database of O. meyeriana has been constructed and many diseases-resistant genes were found to express under native condition, which provides a foundation for future discovery of a number of novel genes and provides a basis for studying the molecular mechanisms associated with disease resistance in O. meyeriana.

De Novo Transcriptome Sequencing of Oryza officinalis Wall ex Watt to Identify Disease-Resistance Genes

Oryza officinalis Wall ex Watt is one of the most important wild relatives of cultivated rice and exhibits high resistance to many diseases. It has been used as a source of genes for introgression into cultivated rice. However, there are limited genomic resources and little genetic information publicly reported for this species. To better understand the pathways and factors involved in disease resistance and accelerating the process of rice breeding, we carried out a de novo transcriptome sequencing of O. officinalis. In this research, 137,229 contigs were obtained ranging from 200 to 19,214 bp with an N50 of 2331 bp through de novo assembly of leaves, stems and roots in O. officinalis using an Illumina HiSeq 2000 platform. Based on sequence similarity searches against a non-redundant protein database, a total of 88,249 contigs were annotated with gene descriptions and 75,589 transcripts were further assigned to GO terms. Candidate genes for plant–pathogen interaction and plant hormones regulation pathways involved in disease-resistance were identified. Further analyses of gene expression profiles showed that the majority of genes related to disease resistance were all expressed in the three tissues. In addition, there are two kinds of rice bacterial blight-resistant genes in O. officinalis, including two Xa1 genes and three Xa26 genes. All 2 Xa1 genes showed the highest expression level in stem, whereas one of Xa26 was expressed dominantly in leaf and other 2 Xa26 genes displayed low expression level in all three tissues. This transcriptomic database provides an opportunity for identifying the genes involved in disease-resistance and will provide a basis for studying functional genomics of O. officinalis and genetic improvement of cultivated rice in the future.

Cloning and characterization of ATP synthase CF1 α gene from sweet potato

ATP synthase CF1 α subunit protein is a key enzyme for energy metabolism in plant kingdom, and plays an important role in multiple cell processes. In this study, the complete atpA gene (accession no. JN247444) was cloned from sweet potato ( Ipomoea batatas L. Lam) by reverse transcriptasepolymerase chain reaction (RT-PCR). This atpA gene contains an open reading frame (ORF) of 1524 bp coding for a peptide of 507 amino acids with a molecular mass of 55.36 kD. Sequence analysis showed that atpA gene from sweet potato has high homology with the other plant chloroplast atpA. The transcript levels of the atpA gene in young leaves, mature leaves, stems and tuberous roots were examined by the digital gene expression profiling (DGE), and then confirmed by semi-quantitative RTPCR. The results demonstrate that the highest transcription of atpA gene was found in young leaves, but it was relatively lower in other three tissues. In addition, the atpA gene was successfully expressed in Escherichia coli. Key words: Sweet potato, atpA gene, gene expression, digital gene expression profiling, quantitative analysis

Using the CRISPR/Cas9 system to eliminate native plasmids of Zymomonas mobilis ZM4

The CRISPR/Cas system can be used to simply and efficiently edit the genomes of various species, including animals, plants, and microbes. Zymomonas mobilis ZM4 is a highly efficient, ethanol-producing bacterium that contains five native plasmids. Here, we constructed the pSUZM2a-Cas9 plasmid and a single-guide RNA expression plasmid. The pSUZM2a-Cas9 plasmid was used to express the Cas9 gene cloned from Streptococcus pyogenes CICC 10464. The single-guide RNA expression plasmid pUC-T7sgRNA, with a T7 promoter, can be used for the in vitro synthesis of single-guide RNAs. This system was successfully employed to knockout the upp gene of Escherichia coli and the replicase genes of native Z. mobilis plasmids. This is the first study to apply the CRISPR/Cas9 system of S. pyogenes to eliminate native plasmids in Z. mobilis. It provides a new method for plasmid curing and paves the way for the genomic engineering of Z. mobilis. Graphical abstract The CRISPR/Cas9 system was employed to knockout the replicase genes of Z. mobilis to eliminate native plasmids. It provides a new method of plasmid curing.