Boru Zhou

Genetic linkage maps of white birches ( Betula platyphylla Suk. and B. pendula Roth) based on RAPD and AFLP markers

A pseudo-testcross mapping strategy was used in combination with the random amplified polymorphism DNA (RAPD) and amplified fragment length polymorphism (AFLP) genotyping methods to develop two moderately dense genetic linkage maps for Betula platyphylla Suk. (Asian white birch) and B. pendula Roth (European white birch). Eighty F1 progenies were screened with 291 RAPD markers and 451 AFLP markers. We selected 230 RAPD and 362 AFLP markers with 1:1 segregation and used them for constructing the parent-specific linkage maps. The resultant map for B. platyphylla was composed of 226 markers in 24 linkage groups (LGs), and spanned 2864.5xa0cM with an average of 14.3xa0cM between adjacent markers. The linkage map for B. pendula was composed of 226 markers in 23 LGs, covering 2489.7xa0cM. The average map distance between adjacent markers was 13.1xa0cM. Clustering of AFLP markers was observed on several LGs. The availability of these white birch linkage maps will contribute to the molecular genetics and the implementation of marker-assisted selection in these important forest species.

Differentially expressed genes in Populus simonii × Populus nigra in response to NaCl stress using cDNA-AFLP

Salinity is an important environmental factor limiting growth and productivity of plants, and affects almost every aspect of the plant physiology and biochemistry. The objective of this study was to apply cDNA-AFLP and to identify differentially expressed genes in response to NaCl stress vs. no-stress in Populus simonii x Populus nigra in order to develop genetic resources for genetic improvement. Selective amplification with 64 primer combinations allowed the visualization of 4407 transcript-derived fragments (TDFs), and 2027 were differentially expressed. Overall, 107 TDFs were re-sequenced successfully, and 86 unique sequences were identified in 10 functional categories based on their putative functions. A subset of these genes was selected for real-time PCR validation, which confirmed the differential expression patterns in the leaf tissues under NaCl stress vs. no stress. Differential expressed genes will be studied further for association with salt or drought-tolerance in P. simonii x P. nigra. This study suggests that cDNA-AFLP is a useful tool to serve as an initial step for characterizing transcriptional changes induced by NaCl salinity stress in P. simonii x P. nigra and provides resources for further study and application in genetic improvement and breeding. All unique sequences have been deposited in the Genbank as accession numbers GW672587-GW672672 for public use.

Expression Analysis of Stress-Related Genes in Kernels of Different Maize (Zea mays L.) Inbred Lines with Different Resistance to Aflatoxin Contamination

This research examined the expression patterns of 94 stress-related genes in seven maize inbred lines with differential expressions of resistance to aflatoxin contamination. The objective was to develop a set of genes/probes associated with resistance to A. flavus and/or aflatoxin contamination. Ninety four genes were selected from previous gene expression studies with abiotic stress to test the differential expression in maize lines, A638, B73, Lo964, Lo1016, Mo17, Mp313E, and Tex6, using real-time RT-PCR. Based on the relative-expression levels, the seven maize inbred lines clustered into two different groups. One group included B73, Lo1016 and Mo17, which had higher levels of aflatoxin contamination and lower levels of overall gene expression. The second group which included Tex6, Mp313E, Lo964 and A638 had lower levels of aflatoxin contamination and higher overall levels of gene expressions. A total of six “cross-talking” genes were identified between the two groups, which are highly expressed in the resistant Group 2 but down-regulated in susceptible Group 1. When further subjected to drought stress, Tex6 expressed more genes up-regulated and B73 has fewer genes up-regulated. The transcript patterns and interactions measured in these experiments indicate that the resistant mechanism is an interconnected process involving many gene products and transcriptional regulators, as well as various host interactions with environmental factors, particularly, drought and high temperature.

Over-expression of poplar transcription factor ERF76 gene confers salt tolerance in transgenic tobacco.

Ethylene response factors (ERFs) belong to a large plant-specific transcription factor family, which play a significant role in plant development and stress responses. Poplar ERF76 gene, a member of ERF TF family, can be up-regulated in response to salt stress, osmotic stress, and ABA treatment. The ERF76 protein was confirmed to be targeted preferentially in the nucleus of onion cell by particle bombardment. In order to understand the functions of ERF76 gene in salt stress response, we conducted temporal and spatial expression analysis of ERF76 gene in poplar. Then the ERF76 cDNA fragment containing an ORF was cloned from di-haploid Populus simonii×P. nigra and transferred into tobacco (Nicotiana tobacum) genome by Agrobacterium-mediated leaf disc method. Under salt stress, transgenic tobacco over-expressing ERF76 gene showed a significant increase in seed germination rate, plant height, root length, and fresh weight, as well as in relative water content (RWC), superoxide dismutase (SOD) activity, peroxidase (POD) activity, and proline content, compared to control tobacco lines. In contrast, transgenic tobacco lines displayed a decrease in malondialdehyde (MDA) accumulation, relative electrical conductivity (REC) and reactive oxygen species (ROS) accumulation in response to salt stress, compared to control tobacco lines. Over all, the results indicated that ERF76 gene plays a critical role in salt tolerance in transgenic tobacco.

Expression Pattern of ERF Gene Family under Multiple Abiotic Stresses in Populus simonii × P. nigra

Identification of gene expression patterns of key genes across multiple abiotic stresses is critical for mechanistic understanding of stress resistance in plant. In the present study, we identified differentially expressed genes (DEGs) in di-haploid Populus simonii × P. nigra under respective stresses of NaCl, KCl, CdCl2, and PEG. On the basis of RNA-Seq, we detected 247 DEGs that are shared by the four stresses in wild type poplar, and mRNA abundance of the DEGs were validated in transgenic poplar overexpressing ERF76 gene by RNA-Seq and RT-qPCR. Results from gene ontology analysis indicated that these genes are enriched in significant pathways, such as phenylpropanoid biosynthesis, phenylalanine metabolism, starch and sucrose metabolism, and plant hormone signal transduction. Ethylene response factor (ERF) gene family plays significant role in plant abiotic stress responses. We also investigated expression pattern of ERF gene family under the four stresses. The ERFs and DEGs share similar expression pattern across the four stresses. The transgenic poplar is superior to WT in morphologic, physiological and biochemical traits, which demonstrated the ERF76 gene plays a significant role in stress resistance. These studies will give a rise in understanding the stress response mechanisms in poplar.

Characterization of ERF76 promoter cloned from Populus simonii × P. nigra

The poplar transcription factor ERF76 gene is salt-inducible and plays an important role in regulating stress-related genes. To understand the regulatory mechanisms of ERF76 gene, the 1537-bp upstream DNA sequence of ERF76 gene was cloned from di-haploid Populus simoniixa0×xa0P. nigra. Sequence analysis indicated that the fragment contains a series of regulatory elements related to defense and stress responsiveness. The protein–DNA interaction assay indicated that the sequence interacted with proteins involved in oxidoreductase activity and plant defense and stress response. Biological evidence from promoter segmentation and deletion analysis demonstrated that the promoter segments in different length possess different transcriptional activity and respond to salt stress differentially in transgenic Arabidopsis. The characterization of ERF76 promoter provides mechanistic understanding of ERF76 gene in salt stress responses.

Characterization of the basic helix–loop–helix gene family and its tissue-differential expression in response to salt stress in poplar

The basic helix–loop–helix (bHLH) transcription factor gene family is one of the largest gene families and extensively involved in plant growth, development, and stress responses. However, limited studies are available on the gene family in poplar. In this study, we focused on 202 bHLH genes, exploring their DNA and protein sequences and physicochemical properties. According to their protein sequence similarities, we classified the genes into 25 groups with specific motif structures. In order to explore their expressions, we performed gene expression profiling using RNA-Seq and identified 19 genes that display tissue-differential expression patterns without treatment. Furthermore, we also performed gene expression profiling under salt stress. We found 74 differentially expressed genes (DEGs), which are responsive to the treatment. A total of 18 of the 19 genes correspond well to the DEGs. We validated the results using reverse transcription quantitative real-time PCR. This study lays the foundation for future studies on gene cloning, transgenes, and biological mechanisms.

Structure analysis and expression pattern of the ERF transcription factor family in poplar

The ethylene response factor (ERF) family is one of the largest families of plant-specific transcription factors, and plays significant roles in plant growth and development, and stress tolerance processes. To understand the different structure and expression pattern of the ERF family, a total of 209 poplar ERF transcription factor genes were identified in silico. Based on the alignment of AP2/ERF domains, 209 PtERF proteins were classified into ten phylogenetic groups (I–X), and amino acid residues Gly-5 (G), Arg-7 (R), Glu-17 (E), Trp-36 (W), Leu-37 (L), Gly-38 (G), and Ala-46 (A) are conserved among these proteins. Protein structure analysis revealed that all poplar ERF family members contained the WLG motif (motif-1), but motif-6 was mainly conserved in the ERF subfamily. In addition, 209 genes were classified into three clusters under the salt stress condition: the lowly expressed Cluster (L), intermediately expressed Cluster (M1 and M2), and highly expressed Cluster (H1 and H2), in which some genes from highly expressed cluster were involved in salt stress response. Moreover, quantitative RT-PCR was used to analyze the tissue-specific expression pattern of 71 highly expressed genes. All of these results are of interest to better understand how and where each ERF functions in poplar.

PsnERF75 Transcription Factor from Populus simonii × P. nigra Confers Salt Tolerance in Transgenic Arabidopsis

ERF transcription factor involves in many aspects of plant development and response to both biotic and abiotic environmental stimuli. In this study, we provided evidence that PsnERF75 can improve the salt tolerance ability of transgenic Arabidopsis. RT-qPCR analysis described the spatio-temporal expression patterns of PsnERF75 in different poplar tissues. Under salt stress condition, root and leaf tissues were more sensitive than stem tissue. In addition, seed germination rates of PsnERF75 transgenic Arabidopsis were significantly enhanced. Growth status of PsnERF75 transgenic Arabidopsis seedlings was much better than that of the controls. Physiological and histochemical assays indicated PsnERF75 transgenic Arabidopsis have a greater capacity to eliminate reactive oxygen species and lighten the damages to plants than the controls. Using RNA-seq analysis, 100 DEGs that were significant differentially expressed between PsnERF75 transgenic Arabidopsis and the controls under salt stress conditions were identified. Seventeen upregulated and 10 down-regulated DEGs participate in plant salt stress response process. In addition, subcellular localization showed that PsnERF75 is a nuclear-localized protein. All of the results will provide novel insights into the functions of ERF75 in plants under abiotic stress condition.

Over expression of TaFer gene from Tamarix androssowii improves iron and drought tolerance in transgenic Populus tomentosa

Ferritin, a universal intracellular protein, can store large amounts of iron and improve plant resistance to abiotic and biotic stress. In this study, a ferritin gene (TaFer) from Tamarix androssowii Litv. was transferred into Populus tomentosa Carr. cv ‘BJR01’ via Agrobacterium. Six independent transgenic lines were obtained with a tolerance to kanamycin and three were randomly selected for further analysis. The PCR and RT-PCR results indicate that the TaFer gene had been integrated into the poplar genome. The effect of the gene on abiotic stress tolerance was tested, and the results show that transgenic plants improve growth, had higher chlorophyll and lower MDA contents, and higher relative electrical conductivity, fewer changes of SOD and POD activities, higher iron content, higher root ferric reductase activity and lower levels of ROS accumulation and cell death in response to drought, Fe-insufficient or Fe-excess tolerance. These results indicate that the TaFer gene can improve abiotic stress tolerance in transgenic Populus tomentosa.