[Show abstract][Hide abstract] ABSTRACT: Abiotic stresses such as drought and high salinity inhibit plant growth and productivity. NAC transcription factors play a variety of important roles in plant development and abiotic stress responses. To date, the transformation of NAC genes into poplar plants has not been reported. In this study, we introduced the CarNAC3 and CarNAC6 salinity and drought tolerant genes from Cicer arietinum (chickpea) into Populus deltoides × P. euramericana ‘Nanlin895’ (poplar) plants using Agrobacterium tumefaciens-mediated transformation. We verified the integration of the two genes into the poplar genome using PCR and their stable expression was confirmed at the transcript level using real-time (RT)-PCR. The growth rates of roots and shoots and the clawed root rate increased in transformed plants grown in agar medium or in soil and increased in response to drought and salt stress conditions. Proline and photoprotectant pigment accumulation and antioxidant enzyme activities increased in response to abiotic stress, but the accumulation of photosynthetic pigments and malondialdehyde (MDA) decreased in transgenic lines relative to wild-type control plants. The CarNAC3 transgene was expressed at higher levels than the CarNAC6 transgene. Our results showed that the CarNAC3 and CarNAC6 genes enhanced the capacity for osmotic adjustment and increased antioxidant enzyme activity and suggest that these genes could play a significant role in improving drought and salt tolerance when expressed in poplar plants
[Show abstract][Hide abstract] ABSTRACT: The increasing resistance of bacteria and fungi to currently available antibiotics is a major concern worldwide, leading to enormous efforts to develop new antibiotics with new modes of actions. In this paper, cDNA encoding cecropin A was amplified from drury (Hyphantria cunea) (dHC) pupa fatbody total RNA using RT-PCR. The full-length dHC-cecropin A cDNA encoded a protein of 63 amino acids with a predicted 26-amino acid signal peptide and a 37-amino acid functional domain. We synthesized the antibacterial peptide (ABP) from the 37-amino acid functional domain (ABP-dHC-cecropin A), and amidated it via the C-terminus. Time-of-flight mass spectrometry showed its molecular weight to be 4,058.94. The ABP-dHC-cecropin A was assessed in terms of its protein structure using bioinformatics and CD spectroscopy. The protein's secondary structure was predicted to be α-helical. In an antibacterial activity analysis, the ABP-dHC-cecropin A exhibited strong antibacterial activity against E. coli K12D31 and Agrobacterium EHA105.
[Show abstract][Hide abstract] ABSTRACT: CarNAC3 contains 285 amino acids and a conserved NAC domain. NAC genes, including NAM,
ATAF1, ATAF2, and CUC2, are members of one of the largest transcription factor families in plants. CarNAC3, a member of the NAP group, plays an important role in plant development and responses to abiotic stresses. In this study, CarNAC3 was transformed into hybrid poplar plants (Populus deltoides 9 P. euramericana ‘Nanlin895’) using Agrobacterium tumefaciens. PCR analysis confirmed integration of the introduced T-DNA into the target genome. Reverse transcription PCR confirmed the transformation,
and Southern and northern blotting verified the transgene copy number and gene expression, respectively. Fourteen lines of positive transformants were transplanted into a greenhouse to verify their drought and salt tolerances. Under normal conditions, transgenic poplar plants were shorter than the wild-type, but under drought and salt stresses, they maintained their normal rooting and stem growth rates, while those of the wild-type plants were suppressed. Under stress conditions, CarNAC3 expression
caused increases in proline and photosynthetic pigment levels and in antioxidant enzyme activities. Furthermore, the expression of CarNAC3 lowered malondialdehyde concentrations compared with the wild-type control. Overall, our results indicated that the CarNAC3 transgene enhanced drought and salt tolerance in transgenic poplar plants.
Plant Cell Tissue and Organ Culture 08/2014; · 2.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Poplar is a model system for the regeneration and genetic transformation of woody plants. To shorten the time required for studies of transgenic poplar, efforts have been made to optimize transformation methods that use Agrobacterium tumefaciens. In this study, an Agrobacterium infective suspension was treated at 4 °C for at least 10 h before infecting explants. By transforming the Populus hybrid clone "Nanlin895" (Populus deltoides × P. euramericana) with Agrobacterium harboring the PBI121:CarNAC6 binary vector, we showed that the transformation efficiency was improved significantly by multiple independent factors, including an Agrobacterium infective suspension with an OD600 of 0.7, an Agrobacterium infection for 120 min, an Agrobacterium infective suspension at a pH of 5.0, an acetosyringone concentration of 200 µM, a cocultivation at 28 °C, a cocultivation for 72 h and a sucrose concentration of 30 g/L in the cocultivation medium. We also showed that preculture of wounded leaf explants for two days increased the regeneration rate. The integration of the desired gene into transgenic poplars was detected using selective medium containing kanamycin, followed by southern blot analysis. The expression of the transgene in the transgenic lines was confirmed by northern blot analysis.
International Journal of Molecular Sciences 06/2014; 15(6):10780-10793. · 2.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein kinases are major signal transduction factors that have a central role in mediating acclimation to environmental changes in eukaryotic organisms. In this study, we cloned and identified three salt overly sensitive 2 (SOS2) genes in the woody plant Populus trichocarpa, designated as PtSOS2.1, PtSOS2.2, and PtSOS2.3, which were transformed into hybrid poplar clone T89 (Populus tremula× Populus tremuloides Michx clone T89) mediated by Agrobacterium tumefaciens. Southern and northern blot analyses verified that the three genes integrated into the plant genome, and were expressed at a stable transcription level. Meanwhile, overexpression of all three PtSOS2 genes did not retard the growth of plants under normal conditions. Instead, it promoted growth in both agar-medium and soil conditions in response to salinity stress. Under salt stress, overexpression of PtSOS2.1, PtSOS2.2, and PtSOS2.3 increased the concentrations of proline and photosynthetic pigments, and the relative water content (RWC), and the activity of antioxidant enzymes, and decreased the malondialdehyde (MDA) concentrations in transgenic lines compared to the control. These results suggest that overexpression of PtSOS2 plays a significant role in improving the salt tolerance of poplars, reducing the damage to membrane structures, and enhancing osmotic adjustment and antioxidative enzyme regulation under salt stress.
[Show abstract][Hide abstract] ABSTRACT: Galactinol synthase (GolS; EC 18.104.22.168) is a member of the glycosyltransferase eight family that catalyzes the first step in the biosynthesis pathway of the raffinose family of oligosaccharides (RFOs). The accumulation of RFOs in response to abiotic stress indicates a role for RFOs in stress adaptation. To obtain information on the roles of RFOs in abiotic stress adaptation in trees, we investigated the expression patterns of nine Populus trichocarpa GolS (PtrGolS) genes with special reference to stress responses. PtrGolS genes were differentially expressed in different organs, and the expressions of PtrGolS4 and PtrGolS6 were relatively high in all tested organs. The expression levels of all PtrGolS genes, except PtrGolS9, changed in response to abiotic stress in gene- and stress-type-specific manners. Moreover, short- and long-term stress treatments revealed that induction of PtrGolS by salt stress is obvious only in the early period of treatment (within 24 h), whereas water-deficit stress treatments continued to upregulate PtrGolS gene expression after two days of treatment, in addition to induction within 24 h of treatment. Consistent with these expression patterns, the galactinol content in leaves increased after four days of drought stress, but not under salt stress. Our findings suggest divergent roles for PtrGolS genes in abiotic stress responses in poplars.
Journal of Plant Research 11/2013; · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Thaumatin-like protein (TLP) genes constitute a large, highly complex gene family involved in host defense against abiotic stresses, as well as other physiological processes. However, their functions in the abiotic stress-induced defense response in poplar remain largely unknown. In this study, one full-length cDNA from a hybrid poplar (Populus
deltoides × P. euramericana cv. ‘Nanlin895’) that encodes a TLP was isolated by RACE and designated PeTLP. The deduced amino acid sequence of PeTLP encodes a protein of a predicted molecular mass 24.5 kDa and a pI of 8.19. Transcript of PeTLP accumulated in stems, but the gene showed low transcriptional levels in roots and leaves. PeTLP was upregulated in leaves after abiotic treatments, including 200 mM NaCl, 10 % PEG6000, 100 μM gibberellin, 1 mM salicylic acid and 100 μM abscisic acid. Subcellular localization analysis showed that GFP-tagged PeTLP predominantly localizes as discrete points in the cytoplasm and the periphery of the plasma membrane. Recombinant PeTLP obtained using Escherichia
coli expression produced antifungal activities to Rhizopus sp., Aspergillus
niger and Penicillium
citrinum. These results suggest that PeTLP is an abiotic stresses-induced protein and plays roles in physiological processes of poplar development, and is involved in the defense of poplar against abiotic stresses. Manipulation of PeTLP in poplar or other trees should improve their performance under various abiotic stress conditions.
Acta Physiologiae Plantarum 10/2013; · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We used large samples of expressed sequence tags to characterize the patterns of codon usage bias (CUB) in seven different Citrus species and to analyze their evolutionary effect on selection and base composition. We found that A- and T-ending codons are predominant in Citrus species. Next, we identified 21 codons for 18 different amino acids that were considered preferred codons in all seven species. We then performed correspondence analysis and constructed plots for the effective number of codons (ENCs) to analyze synonymous codon usage. Multiple regression analysis showed that gene expression in each species had a constant influence on the frequency of optional codons (FOP). Base composition differences between the proportions were large. Finally, positive selection was detected during the evolutionary process of the different Citrus species. Overall, our results suggest that codon usages were the result of positive selection. Codon usage variation among Citrus genes is influenced by translational selection, mutational bias, and gene length. CUB is strongly affected by selection pressure at the translational level, and gene length plays only a minor role. One possible explanation for this is that the selection-mediated codon bias is consistently strong in Citrus, which is one of the most widely cultivated fruit trees.
[Show abstract][Hide abstract] ABSTRACT: A tissue-specific promoter, Pt-RbcS, from Populus was isolated and cloned based on alignment of AtRBCS-2B cDNA with genomic Populus sequences. Sequence analysis of Pt-RbcS revealed cis-acting regulatory elements in the promoter region, including an ATCT-motif, BoxI, GAG-motif, I-box, G-box, BoxII, GATA-motif, and TCT-motif, which are involved in light responses. In transgenic tobacco lines carrying the β-glucuronidase (GUS) gene driven by the Pt-RbcS promoter, GUS expression was detected in leaves and stems, but not in roots. Transgenic poplar lines harboring constructs carrying the GUS gene driven by truncated Pt-RbcS promoters revealed distinctive expression patterns for five different promoter constructs. The Pt-RbcS promoter was expressed preferentially in photosynthetic tissues such as leaves and stems. Moreover, deletion analysis of the 1,547 bp Pt-RbcS promoter region revealed that a 927-bp DNA segment is critical for expression of Pt-RbcS in green tissues. Overall, our study suggests that the Pt-RbcS promoter from Populus could be applied to genetically improve the photosynthetic efficiency of woody plants.
[Show abstract][Hide abstract] ABSTRACT: The capacity to root from cuttings is a key factor for the mass deployment of superior genotypes in clonal forestry. We studied
the genetic basis of rooting capacity by mapping quantitative trait loci (QTLs) that control growth rate and form of root
traits in a full-sib family of 93 hybrids derived from an interspecific cross between two Populus species, P. deltoides and P. euramericana. The hybrid family was typed for different marker systems (including SSRs, AFLPs, RAPDs, ISSRs, and SNPs), leading to the
construction of two linkage maps based on the female P. deltoides (D map) and male P. euramericana (E map) with a pseudotestcross mapping strategy. The two maps were scanned by functional mapping to detect QTLs that control
early growth trajectories of two rooting traits, maximal single-root length and the total number of roots per cutting, measured
at five time points in water culture. Of the six QTLs detected for these two growth traits, only one is segregating in P. deltoides with poor rooting capacity, while the other five are segregating in P. euramericana showing good rooting capacity. Tests with functional mapping suggest different developmental patterns of the genetic effects
of these root QTLs in time course. Five QTLs were detected to change their effects on root growth trajectories with time,
whereas one detected to affect root growth consistently in time course. Knowledge about the genetic and developmental control
mechanisms of root QTLs will have important implications for the genetic improvement of vegetative propagation traits in Populus.
Tree Genetics & Genomes 07/2009; 5(3):539-552. · 2.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lycoris aurea exhibits parallel venation, the main vein with many lateral veins in a longitudinal parallel arrangement. There are secondary lateral veins (SLV) between each longitudinal veins. In general, SLVs are not remarkable. In this paper, the material was one kind of Lycoris aurea mutant called Raised Secondary Lateral Veins mutant (RSLV), because many Raised Secondary Lateral Veins are in abaxial surface of its leaves. Its growing potential is weaker than that of wild type and its blades are very thin. Moreover, the stamens of RSLV degenerate completely. Two cDNA libraries were constructed from RSLV mutant and wild type (WT) leaves. From the libraries, 3,122 ESTs, which are longer than 100 bp each after vector sequence removed, were acquired by single-pass sequencing from the 5'end. Following a multistep selection, 512 70-mer oligo-DNA probes were designed for attachment on the microarray slide based on the ESTs. The gene expression profile of RSLV mutant and WT leaves was compared through the microarray at transcriptional level. The microarray experiment results were further confirmed by Quantitative Real-Time PCR (QRT-PCR). We identified 5 genes whose expressions changed more than 2-fold between RSLV mutant and WT leaves. They encode phloem protein 2 (PP2), ferritin, pectin methyl esterase (PME), chlorophyll a/b binding protein (CAB protein) and pyruvate decarboxylase (PDC), respectively. Furthermore, the full-length cDNA sequences of the 5 genes were separately obtained from RSLV and WT by RACE. The relationship between differential expressions of the genes and the formation of the RSLV mutant phenotype were discussed.
[Show abstract][Hide abstract] ABSTRACT: In the present study, we cloned two lipoxygenase genes, PdLOX1 and PdLOX2 (GenBank accession no. DQ131178, DQ131179), from Populus deltoides cv. ‘Lux’ (I-69/55). A prokaryotic expression analysis of PdLOX1 and PdLOX2 revealed that the encoded exogenous proteins were identical to the predicted molecular weights and possessed the expected lipoxygenase activities. Chromatogram analysis indicated that the two lipoxygenase mainly possess 13-LOX activity. Phylogenetic analysis of the derived amino acid sequences of known lipoxygenases revealed that PdLOX1 and PdLOX2 were members of the type 2 13-LOX family of genes. This class of lipoxygenases is known to be involved in biotic and abiotic stress. Using real-time RT-PCR, we evaluated PdLOX1 and PdLOX2 expression following exposure to a Poplar fungal pathogen (Marssonina brunnea f. sp. Multigermtubi), mechanical injury, methyl jasmonate (MeJA), or salicylic acid (SA). We report that both PdLOX1 and PdLOX2 expression levels were increased following exposure to M. brunnea f. sp. Multigermtubi, with the pathogen exerting a relatively stronger influence on PdLOX1 expression. Furthermore, expression levels of the two genes were also up-regulated by mechanical damage and exposure to MeJA. In contrast, both PdLOX1 and PdLOX2 expression was down-regulated by SA treatment. We propose that the two novel lipoxygenases may play an important role in Poplar resistance to biotic and abiotic stress.
[Show abstract][Hide abstract] ABSTRACT: By using assembled expressed sequence tags (ESTs) from 14 different cDNA libraries that contain 84 132 sequences reads, 556 Populus candidate single nucleotide polymorphisms (SNPs) were identified. Because traces were not available from dbEST (http://www.ncbi.nlm.nih.gov/dbEST/index.html), stringent filters were used to identify reliable candidate SNPs. Sequences analysis indicated that the main types of substitutions among candidate SNPs were A/G and T/C transitions, which accounted for 22.0% and 30.8%, respectively. One hundred and ten candidate SNPs were tested. As a result, 38 candidate SNPs were confirmed by directed sequencing of PCR products amplified from six different individuals. Thirteen new SNPs in intron regions were found and multiple SNPs were found to be located in both intron and exon regions of four contigs. Heterozygosis was found in all 47 candidate sites and five SNP sites were heterozygous in all six samples. This is the first report of SNP identification in a tree species which reveals that assembled ESTs from multiple libraries of the public database may provide a rich source of comparative sequences for an SNP search in the poplar genome.(Managing editor: Li-Hui ZHAO)
[Show abstract][Hide abstract] ABSTRACT: The sequences of nrDNA regions of 17 species and Phyllostachys edulis (outgroup) sampled, which are of represent and type species for different taxa in the genus Arundinaria,were analyzed by PCR amplification and direct DNA sequencing. The phylogenetic trees generated from maximum parsimony analysis showed that the sampled bamboos were naturally monophletic, appearing that these species of the bamboos belong to the genus Arundinaria. The internal transcribed spacers (ITS) data indicated that the species were divided into two branches, one including A. oleosa, A. hsienchuensis, A. chino, A. amara, A. yixingensis, A. amabilis, A. fortunei, and A. pygmaea, the other including A. graminea, A. fargesii, A. faberi, A. hupehensis, Pseudosasa japonica cv. Tsutsumiana, P. japonica, Brachystachyum densiflorum, A. oedogonata, and A. sulcata. The result also showed that there was close relationship between A. graminea and A. fargesii, Pseudosasa japonica cv. Tsutsumiana and P. japonica, A. sulcata, Brachystachyum densiflorum and A. oedogonata, (99%, 100% and 82% boot-strap support respectively). Moreover, there was very close relationship between A. amabilis and A. hsienchuensis, indicating that A. amabilis belongs to the genus Arundinaria. It was shown in the phylogenetic tree that A. pygmaea and A. fortunei had close relationship, and were a sister branch to the bamboos of Pleioblastus.
[Show abstract][Hide abstract] ABSTRACT: To obtain a primary overview of gene diversity and expression pattern in Lycoris longituba, 4,992 ESTs (Expressed Sequence Tags) from L. longituba bud were sequenced and 4,687 cleaned ESTs were used for gene expression analysis. Clustered by the PHRAP program, 967 contigs and 1,343 singlets were obtained. Blast search showed that 179 contigs and 227 singlets (totally 1,066 ESTs) had homologues in GenBank and 3,621 ESTs were novel.
[Show abstract][Hide abstract] ABSTRACT: The fingerprints of 13 species in genus Lycoris were generated by use of RAPD method. Forty-one primers were screened from 520 random primers, and a total of 350 DNA fragments were amplified ranging from 0.3-3.0 kb, 253 (72.3%) of which were polymorphic. The average number of DNA band produced by each primer was 6.2. Nei's similarity coefficients and genetic distances were calculated by use of the software of TFPGA version 1.3 and dendrogram of Lycoris was constructed using UPGMA. It is indicated that the 13 species of the genus Lycoris were divided into two groups, and five species of the genus including L. rosea, L. haywardii, L. straminea, L. sprengeri and L. radiata with monotype karyotypes (I-shaped) were clustered together respectively. The basic chromosome number was x = 11. The others which have two-types karyotypes (I-shaped and V-shaped) were clustered together respectively. They were L. houdyshelii, L. albiflora, L. chinensis, L. longituba, L. anhuiensis, L. squmigera, L. caldwellii and L. aurea. The closest relationship was between L. rosea and L. haywardii. L. radiata is highly divergent from L. aurea. The results were in consistence with that of the analysis of chromosome karyotype. The present paper discussed the problems whether L. rosea, L. haywardii and L. stramina originated as natural hybrids and taxonomy position of L. albiflora, L. straminea and L. houdyshelii based on the RAPD analysis.
[Show abstract][Hide abstract] ABSTRACT: DNA markers linked to resistance locus of Marssonina leaf spot in poplars were found by bulked segregant analysis(BSA). The bulks consisted of individual with a extreme phenotype taken from a population of 91 F1 clones,which is a progeny of Populus deltoides Bartr.cv."Lux"(I-69/55)(Resistance) and P.euramericana cv.I-45(Susceptible). Out of 114 RAPD primers, four markers showed polymorphisms between the resistance-bulk and the susceptible-bulk.By using selective genotype linkage analysis,OPAI17-1550 and OPAI13-900 were found linked to the resistance locus. The genetic distances between the two markers and the resistance locus were 29.9cM and 37.4cM,respectively.
[Show abstract][Hide abstract] ABSTRACT: We report molecular genetic linkage maps for an interspecific hybrid population of Populus, a model system in forest-tree biology. The hybrids were produced by crosses between P. deltoides (mother) and P. euramericana (father), which is a natural hybrid of P. deltoides (grandmother) and P. nigra (grandfather). Linkage analysis from 93 of the 450 backcross progeny grown in the field for 15 years was performed using random amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphisms (AFLPs), and inter-simple sequence repeats (ISSRs). Of a total of 839 polymorphic markers identified, 560 (67%) were testcross markers heterozygous in one parent but null in the other (segregating 1:1), 206 (25%) were intercross dominant markers heterozygous in both parents (segregating 3:1), and the remaining 73 (9%) were 19 non-parental RAPD markers (segregating 1:1) and 54 codominant AFLP markers (segregating 1:1:1:1). A mixed set of the testcross markers, non-parental RAPD markers, and codominant AFLP markers was used to construct two linkage maps, one based on the P. deltoides (D) genome and the other based on P. euramericana (E). The two maps showed nearly complete coverage of the genome, spanning 3801 and 3452 cM, respectively. The availability of non-parental RAPD and codominant AFLP markers as orthologous genes allowed for a direct comparison of the rate of meiotic recombination between the two different parental species. Generally, the rate of meiotic recombination was greater for males than females in our interspecific poplar hybrids. The confounded effect of sexes and species causes the mean recombination distance of orthologous markers to be 11% longer for the father (P. euramericana; interspecific hybrid) than for the mother (P. deltoides; pure species). The linkage maps constructed and the interspecific poplar hybrid population in which clonal replicates for individual genotypes are available present a comprehensive foundation for future genomic studies and quantitative trait locus (QTL) identification.
[Show abstract][Hide abstract] ABSTRACT: Xinjiang is a center of distribution and differentiation of genus Dianthus in China, and has a great deal of species resources. The sequences of ITS region (including ITS-1, 5.8S rDNA and ITS-2) of nuclear ribosomal DNA from 8 species of genus Dianthus wildly distributed in Xinjiang were determined by direct sequencing of PCR products. The result showed that the size of the ITS of Dianthus is from 617 to 621 bp, and the length variation is only 4 bp. There are very high homogeneous (97.6%-99.8%) sequences between species, and about 80% homogeneous sequences between genus Dianthus and outgroup. The sequences of ITS in genus Dianthus are relatively conservative. In general, there are more conversion than transition in the variation sites among genus Dianthus. The conversion rates are relatively high, and the ratios of conversion/transition are 1.0-3.0. On the basis of phylogenetic analysis of nucleotide sequences the species of Dianthus in China would be divided into three sections. There is a distant relationship between sect. Barbulatum Williams and sect. Dianthus and between sect. Barbulatum Williams and sect. Fimbriatum Williams, and there is a close relationship between sect. Dianthus and sect. Fimbriatum Williams. From the phylogenetic tree of ITS it was found that the origin of sect. Dianthusis is earlier than that of sect. Fimbriatum Williams and sect. Barbulatum Williams.