[Preliminary analysis of the role of GmSnRK1.1 and GmSnRK1.2 in the ABA and alkaline stress response of the soybean using the CRISPR/Cas9-based gene double-knockout system].

Sucrose non-fermenting related protein kinases (SnRKs) are a ubiquitous Ser/Thr protein kinase in the plant kingdom. These kinases play important roles in plant growth, development, metabolism and resistance to environmental stresses. The soybean (Glycine max L.) genome has four SnRK1 genes, of which GmSnRK1.1 and GmSnRK1.2 are predominant and participate in multiple stress response pathways. To dissect the mechanism of the role of GmSnRK1.1 and GmSnRK1.2 proteins in response to ABA and alkaline stresses, we constructed a dual-gRNA CRISPR vector to specifically knock out GmSnRK1.1 and GmSnRK1.2. The resultant constructs were transformed into soybean cotyledon nodes to induce hairy roots by agrobacteria (Agrobacterium rhizogenes). The soybean hairy roots obtained were genotyped, and the results showed that GmSnRK1.1 and GmSnRK1.2 were efficiently doubly knocked out in 48.6% hairy roots. We also generated control hairy roots that over-expressed GmSnRK1. The materials were treated with 25 µmol/L ABA for 15 days and the results showed that the growths of wild-type and GmSnRK1 over-expressed roots were significantly inhibited than GmSnRK1.1 GmSnRK1.2 double-knockout roots, as the controls displayed less root lengths and fresh weights. However, after treating with 50 mmol/L NaHCO3 for 15 days, we found that the growths of GmSnRK1.1 GmSnRK1.2 double-knockout roots were significantly inhibited than the wild-type and GmSnRK1 over-expressed control roots, as the knockout groups contained less root lengths and fresh weights. These results implied that the GmSnRK1.1 GmSnRK1.2 double knockout mitigated hairy root sensitivity to ABA and resistance to alkaline stress. Taken together, we established the CRISPR/Cas9 system to perform gene double knockout in the soybean and by using this technique, we determined the roles of GmSnRK1.1 and GmSnRK1.2 in response of abiotic stresses.

[Identification of calcium/calmodulin-binding receptor-like kinase GsCBRLK-interactive proteins using yeast two-hybrid system].

GsCBRLK (calcium/calmodulin-binding receptor-like kinase from Glycine soja) links ABA (abscisic acid)- and salt-induced calcium/calmodulin signal in plant cells. In order to study the molecular mechanisms of GsCBLRK, the salt-treated Glycine soja cDNA library was screened with pBT3-STE-CBRLK as bait plasmid using yeast two hybrid system. Two positive clones (SNARE and 14-3-3 protein) were identified by constructing cDNA library of wild soybean under salt treatment, membrane system yeast two hybrid screening, multiple screen, rotary validation, bioinformatic analysis and interaction identification in yeast.

[Study of coupling mechanism between hydrocarbon generation and structure evolution in low rank coal].

The structure evolution in the process of low rank coal hydrocarbon generation was studied using Fourier transform infrared spectroscopy and gas chromatography. Gaseous hydrocarbon yield and change law of functional groups were obtained. The results show that: the coal pyrolysis products are mainly gaseous hydrocarbon C1-5. Methane generation instantaneous yield curve contains four peak of hydrocarbon pyrolysis. Oxygen-containing functional group and alkyl side chain of low rank coal chemical structure reduced while aromatization degree increased along with coal rank. The characteristic absorption peak of coal structure of aliphatic hydrocarbons, aromatic hydrocarbon, methyl C=O base C=C of alkanes and aromatic structure of methyl and methylene were characterized by 2 950, 2 920, 2 860, 1 730, 1 705, 1 600 and 1 380-1 460 cm(-1) selected in FTIR spectra. Temperature 420 degrees C is the turning point, before the absorption peak intensity gradually decreases, and then increases slightly. Three major structural evolution stages of coalification mechanism were revealed. Finally, the low rank coal hydrocarbon structure evolution pattern was put forward.

[Isolation and functional analysis of GsTIFY11b relevant to salt and alkaline stress from Glycine soja].

Using homologous cloning and RT-PCR technology, we isolated a novel TIFY family gene, GsTIFY11b, from Glycine soja L. G07256, a species that is tolerant to saline and alkaline environments. Phylogenetic analysis indicated that GsTIFY11b was closely related to AtTIFY11a with 56% similarity in amino acid identity. Protein sequence analysis showed that GsTIFY11b protein also had conserved TIFY domain, N-terminal domain, and a C-terminal Jas motif. Quantitative realtime PCR analysis indicated that the expression of GsTIFY11b was induced by both saline and alkaline stresses. Two homozygous GsTIFY11b over-expressing transgenic Arabidopsis lines were obtained. Phenotypic analysis of the transgenic and wild-type Arabidopsis indicated that over-expressing GsTIFY11b in Arabidopsis did not enhance plant tolerance to saline and alkaline stresses, whereas it showed an increased sensitivity to saline stress during seed germination and seedling development. Expression analysis of saline stress response marker genes in transgenic and wild-type plants under stress condition indicated that GsTIFY11b regulated the expression of RD29B, KIN1, and DREB. The transient expression of a GsTIFY11b-GFP fusion protein in onion epidermal cells showed that GsTIFY11b was localized to the nucleus, suggesting a role as a transcriptional regulator in the saline stress response pathway.

Therapeutic effect of indirubin-loaded bovine serum albumin nanoparticules on ulcerative colitis.

Indirubin is considered to have promising potential in the treatment of ulcerative colitis (UC). However, poor aqueous solubility and low bioavailability limit its clinical application. We produced indirubin-loaded bovine serum albumin nanoparticles (INPs) and characterized their drug encapsulation efficiency, drug-loading capacity, capacity to release indirubin in vitro and short-term physical stability. We also investigated the pharmacokinetics of INPs in mice. We then compared the curative effects of INPs and indirubin against dextran sulfate sodium-induced colitis in mice and 3D cultured biopsies from patients with UC. In the mouse model, the outcomes of INP treatment, including the disease activity index and serous levels of interleukin (IL)-1ß and IL-10, were significantly different from those of indirubin treatment. Similarly, when we administered INPs and indirubin to the ex vivo colonic tissues of patients with UC, the effect of INPs was stronger than that of indirubin for most antioxidant and anti-inflammatory biomarkers. The results of both the animal trial and ex vivo experiment indicate that the therapeutic effect of indirubin was further enhanced by the carrier system, making it a highly promising medical candidate for UC.

Alkaline-stress response in Glycine soja leaf identifies specific transcription factors and ABA-mediated signaling factors.

Transcriptome of Glycine soja leaf tissue during a detailed time course formed a foundation for examining transcriptional processes during NaHCO(3) stress treatment. Of a total of 2,310 detected differentially expressed genes, 1,664 genes were upregulated and 1,704 genes were downregulated at various time points. The number of stress-regulated genes increased dramatically after a 6-h stress treatment. GO category gene enrichment analysis revealed that most of the differentially expressed genes were involved in cell structure, protein synthesis, energy, and secondary metabolism. Another enrichment test revealed that the response of G. soja to NaHCO(3) highlights specific transcription factors, such as the C2C2-CO-like, MYB-related, WRKY, GARP-G2-like, and ZIM families. Co-expressed genes were clustered into ten classes (P < 0.001). Intriguingly, one cluster of 188 genes displayed a unique expression pattern that increases at an early stage (0.5 and 3 h), followed by a decrease from 6 to 12 h. This group was enriched in regulation of transcription components, including AP2-EREBP, bHLH, MYB/MYB-related, C2C2-CO-like, C2C2-DOF, C2C2, C3H, and GARP-G2-like transcription factors. Analysis of the 1-kb upstream regions of transcripts displaying similar changes in abundance identified 19 conserved motifs, potential binding sites for transcription factors. The appearance of ABA-responsive elements in the upstream of co-expression genes reveals that ABA-mediated signaling participates in the signal transduction in alkaline response.

Characteristics and Controlling Factors of Nanopores of the Niutitang Formation Shale from Jiumen Outcrop, Guizhou Province.

This paper studies the characteristics and controlling factors of nanopores in organic-rich shale in the Niutitang Formation. Six samples were collected from the bottom of the formation at the Jiumen Outcrop, Guizhou Province. Experiments were conducted to investigate the pore structures of these high-maturity shale samples. The TOC contents vary between 4.81-17.51% with an average of 10.18%. The XRD data show that these samples are dominated by quartz (44%-71%), with a significant amount of clay minerals, such as illite, with a content of 8%-27.5%. Based on the low-pressure liquid N2 sorption measurements, the pore structures can be divided into two groups. Group A including samples of N-2, N-3 and N-4, mainly develop slit-shaped pores, mesopores and macropores. Group B shown from samples N-1, N-5 and N-6, are mainly composed of narrow slitlike pores, which may provide more space for shale gas than slit-shaped pores. The mesopores, macropores, porosity and specific surface areas of group B are more developed than those of group A. With the comparison of pore structures in shales with various organic matter and mineral contents, the dissolution of quartz and feldspar can be the important factor controlling pore development. The evolution of diagenesis is closely related to pore evolution. This diagenesis has various types and complex effects on the pores, mainly including compaction, dissolution and cementation.

Study on the Shale Gas Reservoir-Forming Characteristics of the Taiyuan Formation in the Eastern Qinshui Basin, China.

Shales are widely developed in the strata of the Carboniferous-Permian coal measures in the Qinshui Basin, and these shales have great potential for shale gas exploration. In this paper, the shales of the Taiyuan Formation in the eastern Qinshui Basin are studied. The shales of the Taiyuan Formation in the study area are investigated through field investigation, organic geochemical testing, X-ray diffraction, scanning electron microscopy, high pressure mercury injection, low temperature liquid nitrogen adsorption and PetroMod simulation and through other tests to study the reservoir characteristics, such as organic geochemistry, mineralogy, petrology, pore permeability, and gas burial history. The results show that the shales of the Taiyuan Formation are well developed over the whole area with a thickness of more than 60 m. The average organic matter content is 2.95%, and the kerogen type is type III. The shale maturity (average value is 2.45%) corresponds to the stage of high maturity evolution, indicating that a large amount of shale gas has been generated in this area. A high content of quartz and clay minerals indicates a high fracturability. The nanopores in the shale reservoir are well developed at pore sizes between 2˜10 nm and greater than 1000 nm; however, the pores at the other pore sizes are poorly developed, resulting in weak pore connectivity in the reservoir. According to the results of the PetroMod simulation, the shale of the Taiyuan Formation has undergone two subsidence and two uplift processes. The Yanshanian magmatic intrusion is the key factor for the rapid increase in gas production. In addition, the geological structure of the area is relatively simple, and the burial history and caprock thickness are also the main controlling factors of gas generation and preservation. The shale-sandstone-shale combination and shale-coal-shale combination are the main models of shale gas preservation. This comprehensive study suggests that the shale gas of the Taiyuan Formation in the eastern Qinshui Bain has good potential for exploration and development.

Analysis of Developmental Characteristics and Dominant Factors of Pore-Fracture Systems in Lower Cambrian Marine Shale Reservoirs: A Case Study of the Niutitang Formation, Fenggang Block, Southern China.

Due to breakthroughs in the Lower Silurian Longmaxi Formation in the Sichuan Basin and multiple strata around the basin, the northern part of Guizhou adjacent to the Sichuan Basin has become a key area for shale gas exploration. Compared with the Longmaxi Formation, the Niutitang Formation displays greater TOC (total organic carbon) content, depositional thickness and distribution area, but the details remain undetermined. In the study area, the Lower Cambrian Niutitang Formation typically has high TOC content, maturity and brittle mineral content. The study area has experienced multiple periods of tectonic movement, which have great influence on the fracture and pore characteristics. The fractures are mainly structural fractures and have obvious zoning. The primary types of pores are intraparticle pores, organic matter pores, and interparticle pores. Further, macropores and mesopores less than 50 nm contribute most of the pore volume, while pores less than 2 nm contribute most of the specific surface area. Many factors affect the pore-fracture system, such as tectonism, TOC content, mineral composition, and sedimentary environment. Tectonic movements produce fractures based on the changing stress field, but the degree of fracture development does not agree well with the degree of pore development. The TOC content has good positive correlations with the development of fractures and micropores, especially for nanoporosity, while clay minerals show a negative correlation with the development of fractures but a strong positive correlation with the growth of micropores. Quartz displays a positive correlation with the development of fractures but no good correlation with pore development. Finally, the lithofacies, lithologies and mineral compositions under the control of sedimentary environments are internal factors that can impact the development of pore-fracture systems.

Global transcriptome profiling of wild soybean (Glycine soja) roots under NaHCO3 treatment.

BACKGROUND: Plant roots are the primary site of perception and injury for saline-alkaline stress. The current knowledge of saline-alkaline stress transcriptome is mostly focused on saline (NaCl) stress and only limited information on alkaline (NaHCO3) stress is available. RESULTS: Using Affymetrix Soybean GeneChip, we conducted transcriptional profiling on Glycine soja roots subjected to 50 mmol/L NaHCO3 treatment. In a total of 7088 probe sets, 3307 were up-regulated and 5720 were down-regulated at various time points. The number of significantly stress regulated genes increased dramatically after 3 h stress treatment and peaked at 6 h. GO enrichment test revealed that most of the differentially expressed genes were involved in signal transduction, energy, transcription, secondary metabolism, transporter, disease and defence response. We also detected 11 microRNAs regulated by NaHCO3 stress. CONCLUSIONS: This is the first comprehensive wild soybean root transcriptome analysis under alkaline stress. These analyses have identified an inventory of genes with altered expression regulated by alkaline stress. The data extend the current understanding of wild soybean alkali stress response by providing a set of robustly selected, differentially expressed genes for further investigation.

Hydrocarbon Generation and Chemical Structure Evolution from Confined Pyrolysis of Bituminous Coal.

The molecular composition of organic matter formed during pyrolysis is complex. Fourier transform infrared spectroscopy (FTIR) is a good technique to investigate the coal chemical structural evolution. However, reports on the effects of chemical structure on the n-alkane yields and their relative functional groups are scarce in the literature. In our case, the chemical structural evolution process of bituminous coal obtained by pyrolysis at two different heating rates has been analyzed by pyrolysis-gas chromatography (Py-GC) and FTIR. Furthermore, some of the small molecular compounds (e.g., n-alkanes 24 can generate n-alkanes 20 or low-weight compounds) generated by gold-tube pyrolysis were identified using other GC techniques. Biomarkers were analyzed and compared to generated n-alkanes from the gold-tube pyrolysis experiments. We present the results of the relationship between the FTIR parameters and the molecular compositions that were analyzed. A good linear relationship can be seen between the FTIR parameters (C=O, C=C, and C-factor values), the carbon preference index (CPI), and the ratio of the pristane content and n-C17 alkane content (Pr/n-C17). Furthermore, the n-alkane fraction of the pyrolysates, in particular pristane, phytane, n-C17 alkane, and n-C18 alkane, changed upon maturation. Our conclusions indicate that FTIR is applicable as a structural and chemical change probe to explore the pyrolysis process.

[Cultivation of transgenic rice plants with OsCDPK7 gene and its salt tolerance].

A 1,700 bp DNA fragment, OsCDPK7 gene, was cloned with RT-PCR from liaoyan241 leaf treated under a low temperature of 4. Compared to the OsCDPK7 gene reported before (GenBank accession No. AB042550), this fragment, lack of 26 amino acids, possesses the activity of Ca2+-dependent protein kinase because of a complete integration of the Ca2+ binding structure domain and Ser/Thr protein kinase activity center. Plant expression vector was constructed,, OsCDPK7 gene was regulated by E12 promoter. OsCDPK7 gene was transferred into rice via Agrobacterium-mediated method. After Km screening and Southern blot, 10 transgenic plants were obtained. The analysis on the salt tolerance showed that the expression of OsCDPK7 gene composition enhanced the salt tolerance of T2 transgenic plants, part of T2 transgenic seeds could germinate in 0.2 mol/L NaCl medium, and T2 transgenic young plants could rejuvenate after treatment with 0.4 mol/L NaCl for 10 days, while the controlled plants could not germinate and died in salt stress. This research finding proved that the regulation factor of the plant signal transduction could enhance the salt tolerance of transgenic plants, while OsCDPK7 expression was different in the different tolerence transgenic plants.

[Drought resistance and heredity of transgenic tobacco with OsMAPK4 gene].

OsMAPK4 gene was obtained by RT-PCR using gene special primer from Liaoyan241 leaf treated with low temperature. Plant expression vector pBME12 was constructed in which OsMAPK4 gene was regulated by E12 promoter. OsMAPK4 gene was transferred into tobacco via Agrobacterium-mediated method and 25 transgenic plants obtained. The results of drought resistance experiments showed that overexpression of OsMAPK4 gene conferred enhanced drought resistance of T1 transgenic plants. Kanamycin-resistance segregation in T1 transgenic plants showed that the most transgenic lines were in single gene inheritance.

Generation and analysis of expressed sequence tags from NaCl-treated Glycine soja.

BACKGROUND: Salinization causes negative effects on plant productivity and poses an increasingly serious threat to the sustainability of agriculture. Wild soybean (Glycine soja) can survive in highly saline conditions, therefore provides an ideal candidate plant system for salt tolerance gene mining. RESULTS: As a first step towards the characterization of genes that contribute to combating salinity stress, we constructed a full-length cDNA library of Glycine soja (50109) leaf treated with 150 mM NaCl, using the SMART technology. Random expressed sequence tag (EST) sequencing of 2,219 clones produced 2,003 cleaned ESTs for gene expression analysis. The average read length of cleaned ESTs was 454 bp, with an average GC content of 40%. These ESTs were assembled using the PHRAP program to generate 375 contigs and 696 singlets. The resulting unigenes were categorized according to the Gene Ontology (GO) hierarchy. The potential roles of gene products associated with stress related ESTs were discussed. We compared the EST sequences of Glycine soja to that of Glycine max by using the blastn algorithm. Most expressed sequences from wild soybean exhibited similarity with soybean. All our EST data are available on the Internet (GenBank_Accn: DT082443-DT084445). CONCLUSION: The Glycine soja ESTs will be used to mine salt tolerance gene, whose full-length cDNAs will be obtained easily from the full-length cDNA library. Comparison of Glycine soja ESTs with those of Glycine max revealed the potential to investigate the wild soybean's expression profile using the soybean's gene chip. This will provide opportunities to understand the genetic mechanisms underlying stress response of plants.

Molecular model and ReaxFF molecular dynamics simulation of coal vitrinite pyrolysis.

Vitrinite in coal, the mainly generating methane maceral, plays an important role in hydrocarbon generation of coal. This study aims at obtaining products formation mechanism of vitrinite pyrolysis, and hence determining the chemical bond, molecular liquefaction activity, and reactions mechanism of methane and C2-4 during pyrolysis. The ReaxFF molecular dynamics (MD) simulation was carried out at temperature of 1500 K in order to investigate the mechanism of vitrinite pyrolysis. Initially, a minimum energy conformational structure model was constrained by a combination of elemental and carbon-13 nuclear magnetic resonance ((13)C NMR) literature data. The model analysis shows the chemical and physical parameters of vitrinite pyrolysis are broadly consistent with the experimental data. Based on the molecular model, ReaxFF MD simulations further provide information of unimolecule such as bond length, and chemical shift, and hence the total population and energy of main products. Molecules bond and pyrolysis fragments, based on active bond analyzed, revealed pyrolysis products of single vitrinite molecule with aliphatic C-C bond, especially ring and chain aliphatic as liquefaction activity. The molecular cell whose density is 0.9 g/cm(3) with lowest energy accords with the experimental density 1.33 g/cm(3). The content of main products after pyrolysis, classifying as CH4, H2O, and H2, was changed along with the increasing temperature. The gas molecule, fragments and generation pathways of CO2, H2, CH4, and C2H6 were also elucidated. These results show agreement with experimental observations, implying that MD simulation can provide reasonable explanation for the reaction processes involved in coal vitrinite pyrolysis. Thus the mechanism of coal hydrocarbon generation was revealed at the molecular level.

[Analysis of codon usage in potato and its application in the modification of t-PA gene].

Bioperl-1.0 was used under Hongqi LINUX system to program the codon analysis software. According to the analysis of 98 codon DNA sequences with this software, the codon usage in potato was calculated and 4 codons have been inferred to the optimal codons. The codons of tissue plasminogen activator (t-PA) gene sequence have been reconstructed according to the results. The t-PA gene sequence containing the optimal codons of potato will be used for t-PA production by potato bioreactor.

[Using of selective marker gene in transgenic plants and its removal].

Selective marker gene is usually used to select transformed cells or tissue during gene transfer. However,the use of selective marker gene,especially antibiotic-resistant gene, is harmful to environment, plant development and affects multi-transformation. A new strategy that offers a approach for the elimination of those disadvantages caused by the selectable marker gene is developed. We summarized correlative marker genes used in transgenic plants and some methods of its removal.

MicroRNA319 positively regulates cold tolerance by targeting OsPCF6 and OsTCP21 in rice (Oryza sativa L.).

The microRNA319 (miR319) family is conserved among diverse plant species. In rice (Oryza sativa L.), the miR319 gene family is comprised of two members, Osa-miR319a and Osa-miR319b. We found that overexpressing Osa-miR319b in rice resulted in wider leaf blades and delayed development. Here, we focused on the biological function and potential molecular mechanism of the Osa-miR319b gene in response to cold stress in rice. The expression of Osa-miR319b was down-regulated by cold stress, and the overexpression of Osa-miR319b led to an enhanced tolerance to cold stress, as evidenced by higher survival rates and proline content. Also, the expression of a handful of cold stress responsive genes, such as DREB1A/B/C, DREB2A, TPP1/2, was increased in Osa-miR319b transgenic lines. Furthermore, we demonstrated the nuclear localization of the transcription factors, OsPCF6 and OsTCP21, which may be Osa-miR319b-targeted genes. We also showed that OsPCF6 and OsTCP21 expression was largely induced by cold stress, and the degree of induction was obviously repressed in plants overexpressing Osa-miR319b. As expected, the down-regulation of OsPCF6 and OsTCP21 resulted in enhanced tolerance to cold stress, partially by modifying active oxygen scavenging. Taken together, our findings suggest that Osa-miR319b plays an important role in plant response to cold stress, maybe by targeting OsPCF6 and OsTCP21.

GsSRK, a G-type lectin S-receptor-like serine/threonine protein kinase, is a positive regulator of plant tolerance to salt stress.

Receptor-like protein kinases (RLKs) play vital roles in sensing outside signals, yet little is known about RLKs functions and roles in stress signal perception and transduction in plants, especially in wild soybean. Through the microarray analysis, GsSRK was identified as an alkaline (NaHCO3)-responsive gene, and was subsequently isolated from Glycine soja by homologous cloning. GsSRK encodes a 93.22kDa protein with a highly conserved serine/threonine protein kinase catalytic domain, a G-type lectin region, and an S-locus region. Real-time PCR results showed that the expression levels of GsSRK were largely induced by ABA, salt, and drought stresses. Over expression of GsSRK in Arabidopsis promoted seed germination, as well as primary root and rosette leaf growth during the early stages of salt stress. Compared to the wild type Arabidopsis, GsSRK overexpressors exhibited enhanced salt tolerance and higher yields under salt stress, with higher chlorophyll content, lower ion leakage, higher plant height, and more siliques at the adult developmental stage. Our studies suggest that GsSRK plays a crucial role in plant response to salt stress.