Promoter of Cassava MeAHL31 Responds to Diverse Abiotic Stresses and Hormone Signals in Transgenic Arabidopsis.

The AT-hook motif nuclear-localized (AHL) family is pivotal for the abiotic stress response in plants. However, the function of the cassava AHL genes has not been elucidated. Promoters, as important regulatory elements of gene expression, play a crucial role in stress resistance. In this study, the promoter of the cassava MeAHL31 gene was cloned. The MeAHL31 protein was localized to the cytoplasm and the nucleus. qRT-PCR analysis revealed that the MeAHL31 gene was expressed in almost all tissues tested, and the expression in tuber roots was 321.3 times higher than that in petioles. Promoter analysis showed that the MeAHL31 promoter contains drought, methyl jasmonate (MeJA), abscisic acid (ABA), and gibberellin (GA) cis-acting elements. Expression analysis indicated that the MeAHL31 gene is dramatically affected by treatments with salt, drought, MeJA, ABA, and GA3. Histochemical staining in the proMeAHL31-GUS transgenic Arabidopsis corroborated that the GUS staining was found in most tissues and organs, excluding seeds. Beta-glucuronidase (GUS) activity assays showed that the activities in the proMeAHL31-GUS transgenic Arabidopsis were enhanced by different concentrations of NaCl, mannitol (for simulating drought), and MeJA treatments. The integrated findings suggest that the MeAHL31 promoter responds to the abiotic stresses of salt and drought, and its activity is regulated by the MeJA hormone signal.

Functional Characterization of the MeSSIII-1 Gene and Its Promoter from Cassava.

Soluble starch synthases (SSs) play important roles in the synthesis of cassava starch. However, the expression characteristics of the cassava SSs genes have not been elucidated. In this study, the MeSSIII-1 gene and its promoter, from SC8 cassava cultivars, were respectively isolated by PCR amplification. MeSSIII-1 protein was localized to the chloroplasts. qRT-PCR analysis revealed that the MeSSIII-1 gene was expressed in almost all tissues tested, and the expression in mature leaves was 18.9 times more than that in tuber roots. MeSSIII-1 expression was induced by methyljasmonate (MeJA), abscisic acid (ABA), and ethylene (ET) hormones in cassava. MeSSIII-1 expression patterns were further confirmed in proMeSSIII-1 transgenic cassava. The promoter deletion analysis showed that the -264 bp to -1 bp MeSSIII-1 promoter has basal activity. The range from -1228 bp to -987 bp and -488 bp to -264 bp significantly enhance promoter activity. The regions from -987 bp to -747 bp and -747 bp to -488 bp have repressive activity. These findings will provide an important reference for research on the potential function and transcriptional regulation mechanisms of the MeSSIII-1 gene and for further in-depth exploration of the regulatory network of its internal functional elements.

A Transformation and Genome Editing System for Cassava Cultivar SC8.

Cassava starch is a widely used raw material for industrial production. South Chinese cassava cultivar 8 (Manihot esculenta Crantz cv. SC8) is one of the main locally planted cultivars. In this study, an efficient transformation system for cassava SC8 mediated with Agrobacterium strain LBA4404 was presented for the first time. Cassava friable embryogenic calli (FECs) were transformed through the binary vector pCAMBIA1304 harboring GUS- and GFP-fused genes driven by the CaMV35S promoter. The transformation efficiency was increased in the conditions of Agrobacterium strain cell infection density (OD600 = 0.65), 250 µM acetosyringone induction, and agro-cultivation with wet FECs for 3 days in dark. Based on the optimized transformation protocol, approximately 120-140 independent transgenic lines per mL settled cell volume (SCV) of FECs were created by gene transformation in approximately 5 months, and 45.83% homozygous mono-allelic mutations of the MePDS gene with a YAO promoter-driven CRISPR/Cas9 system were generated. This study will open a more functional avenue for the genetic improvement of cassava SC8.

MeNINV1: An Alkaline/Neutral Invertase Gene of Manihot esculenta, Enhanced Sucrose Catabolism and Promoted Plant Vegetative Growth in Transgenic Arabidopsis.

Alkaline/neutral invertase (A/N-INV) is an invertase that irreversibly decomposes sucrose into fructose as well as glucose and plays a role in plant growth and development, starch synthesis, abiotic stress, and other plant-life activities. Cassava is an economically important starch crop in tropical regions. During the development of cassava tuber roots, A/N-INV activity is relatively high, which indicates that it may participate in sucrose metabolism and starch synthesis. In this study, MeNINV1 was confirmed to function as invertase to catalyze sucrose decomposition in yeast. The optimal enzymatic properties of MeNINV1 were a pH of 6.5, a reaction temperature of 40 °C, and sucrose as its specific catalytic substrate. VB6, Zn2+, and Pb2+ at low concentrations as well as EDTA, DTT, Tris, Mg2+, and fructose inhibited A/N-INV enzymic activity. In cassava, the MeNINV1 gene was mainly expressed in the fibrous roots and the tuber root phloem, and its expression decreased as the tuber root grew. MeNINV1 was confirmed to localize in chloroplasts. In Arabidopsis, MeNINV1-overexpressing Arabidopsis had higher A/N-INV activity, and the increased glucose, fructose, and starch content in the leaves promoted plant growth and delayed flowering time but did not change its resistance to abiotic stress. Our results provide new insights into the biological function of MeNINV1.

Pathological Change of Chronic Hepatitis B Patients with Different Tongue Coatings by Circular Multi-Omics Integrated Analysis.

OBJECTIVE: To compare the circular pathological changes of chronic hepatitis B (CHB) patients according to the tongue diagnosis. METHODS: Totally 41 CHB patients with typical white tongue coating (WTC) or yellow tongue coating (YTC) were enrolled and 14 healthy volunteers with normal tongue manifestation served as controls. The mRNA expression of peripheral leukocytes was detected by GeneChips, and 9 genes were randomly selected for expression validation. Circular metabolites were detected by gas chromatographymass spectrometry. Biological information was analyzed based on ingenuity pathways analysis or metabolomics database and the integrated networks were constructed by ClueGO. RESULTS: A total of 945 and 716 differentially expressed genes were found in patients with WTC and YTC relative to healthy volunteers respectively. The biological information analysis indicated that CHB patients had obviously increased functions in cell death, apoptosis and necrosis (Z-score ⩾2, P<0.05) and decreased activation in T lymphocytes (Z-score ⩽-2, P<0.05), regardless of the tongue manifestation. Compared to patients with WTC, the YTC patients were predicted to be more active in functions related to virus replication (Z-score ⩾2, P<0.05), and the content of circular fatty acids, such as oleic acid (P=0.098) and lauric acid (P=0.035), and citric acid cycle-related metabolites were higher in the YTC patients (P<0.1). The integrated analysis based on differential genes and metabolites indicated that the most difference in the biological function network between the WTC and YTC patients was tumor necrosis factor receptor associated factor 6 mediated-nuclear factor kappa-B activation process. CONCLUSIONS: CHB patients with YTC had more severe inflammation and fatty acids metabolism aberrant than patients with WTC. The results facilitate the modern pathological annotation of Chinese medicine tongue diagnosis theory and provide a reference for the interpretation of pharmacological mechanisms of Chinese medicine treatment.

Low vitamin D levels are associated with high viral loads in patients with chronic hepatitis B: a systematic review and meta-analysis.

BACKGROUND: Previous studies have investigated the vitamin D status in patients with chronic hepatitis B virus (HBV) infection and its relationship with HBV replication, the results however were inconsistent. The present meta-analysis was carried out to compare the vitamin D levels between patients with chronic hepatitis B (CHB) and healthy controls, and to determine whether vitamin D levels were correlated with HBV viral loads significantly. METHODS: A systematic search was conducted via PubMed, Web of Science, EMBASE and the Cochrane Library to identify eligible studies until September 28, 2017. We calculated pooled mean difference (MD) and 95% confidence intervals (CI) to quantitatively estimate the difference of vitamin D levels between CHB patients and controls. In addition, correlation between serum vitamin D levels and HBV viral loads was defined by summary correlation coefficient (r value) and the corresponding 95% CI. RESULTS: A total of 7 studies involving 814 CHB patients and 696 healthy controls were included. A significantly decreased vitamin D levels was found in CHB patients compared with healthy controls: pooled MD (95% CI) was - 2.03 ng/mL (- 2.60, - 1.46). Latitude-stratified subgroup analysis indicated this difference was more obvious in low latitude areas, with a bigger pooled MD (95% CI) of - 2.72 ng/mL (- 4.57, - 0.87). In addition, we observed an inverse correlation between serum vitamin D levels and HBV viral loads: pooled r (95% CI) was - 0.41(- 0.54, - 0.27). CONCLUSIONS: Our results showed that vitamin D levels were lower in CHB patients than that of healthy controls and inversely correlated with HBV viral loads, although future comprehensive studies are needed to clarify the underlying mechanisms.

Identification and expression analysis of MinD gene involved in plastid division in cassava.

Cassava is a tropical crop known for its starchy root and excellent properties. Considering that starch biosynthesis in the amyloplast is affected by its division, it appears conceivable that the regulation of plastid division plays an important role in starch accumulation. As a member of the Min system genes, MinD participated in the spatial regulation of the position of the plastid division site.In our studies, sequence analysis and phylogenetic analysis showed that MeMinD has been highly conserved during the evolutionary process. Subcellular localisation indicated that MeMinD carries a chloroplast transit peptide and was localised in the chloroplast. Overexpression of MeMinD resulted in division site misplacement and filamentous formation in E. coli, indicating that MeMinD protein was functional across species. MeMinD exhibited different spatial and temporal expression patterns which was highly expressed in the source compared to that in the sink organ.

Overexpression of MinE gene affects the plastid division in cassava.

The MinE protein plays an important role in plastid division. In this study, the MinE gene was isolated from the cassava (Manihot esculenta Crantz) genome. We isolated high quality and quantity protoplasts and succeed in performing the transient expression of the GFP-fused Manihot esculenta MinE (MeMinE) protein in cassava mesophyll protoplasts. The transient expression of MeMinE-GFP in cassava protoplasts showed that the MeMinE protein was located in the chloroplast. Due to the abnormal division of chloroplasts, overexpression of MeMinE proteins in cassava mesophyll protoplasts could result in fewer and smaller chloroplasts. Overexpression of MeMinE proteins also showed abnormal cell division characteristics and minicell occurrence in Escherichia coli caused by aberrant septation events in the cell poles.

Chinese Herbal Medicine Combined with Entecavir for HBeAg Positive Chronic Hepatitis B: Study Protocol for a Multi-Center, Double-Blind Randomized-Controlled Trial.

BACKGROUND: The domestic prevalence of chronic hepatitis B (CHB) in China is 7.18% in 2006, imposing great societal healthcare burdens. Nucleot(s)ide analogues (NUCs) anti-hepatitis B virus (HBV) therapies are widely applied despite the relatively low rate of seroconversion and high risk of drug-resistant mutation. More effective treatments for CHB deserve further explorations. Combined therapy of NUCs plus Chinese herbal medicine (CHM) is widely accepted in China, which is recognized as a prospective alternative approach. The study was primarily designed to confirm the hypothesis that Tiaogan-Yipi Granule (, TGYP) or Tiaogan-Jianpi-Jiedu Granule (, TGJPJD) plus entecavir tablet (ETV) was superior over ETV monotherapy in enhancing HBeAg loss rate. METHODS: The study was a nationwide, large-scale, multi-center, double-blind, randomized, placebo-controlled trial with a designed duration of 108 weeks. A total of 16 hospitals and 596 eligible Chinese HBeAg positive CHB patients were enrolled from November 2012 to September 2013 and randomly allocated into 2 groups in 1:1 ratio via central randomization system: experimental group (EG) and control group (CG). Subjects in EG received CM formulae (TGYP or TGJPJD, 50 g per dose, twice daily) plus ETV tablet (or ETV placebo) 0.5 mg per day in the first 24 weeks (stage 1), and CHM granule plus ETV tablet (0.5 mg per day) from week 25 to 108 (stage 2). Subjects in CG received CHM Granule placebo plus ETV tablet (0.5 mg per day) for 108 weeks throughout the trial. The assessments of primary outcomes (HBV serum markers and HBV-DNA) were conducted by a third-party College of American Pathologists (CAP) qualified laboratory. Adverse effects were observed in the hospitals of recruitment. DISCUSSION: The study was designed to compare the curative effect of CM plus ETV and ETV monotherapy in respect of HBeAg loss, which is recognized by the European Association for the Study of the Liver as "a valuable endpoint". We believe this trial could provide a reliable status for patients' "journey" towards durable responses after treatment discontinuation. The trial was registered before recruitment on Chinese Clinical trial registry (No. ChiCTR-TRC-12002784, Version 1.0, 2015/12/23).

A Time-Series Analysis of Severe Burned Injury of Skin Gene Expression Profiles.

BACKGROUND/AIMS: Major burn injury is one of the main severe forms of wound which lead to a mass of clinical debilitation, this study was to identify core biomarkers for the recovery of severe burned injury. METHODS: Gene expression profiles (GSE19743) from the Gene Expression Omnibus (GEO) was downloaded, followed by background correction, normalization of raw microarray dataset and identification of differential expression genes (DEGs) . Soft clustering of DEGs was used for the screening of gene clusters that with sustained increasing (SIG) and decreasing expression (SDG) profiles along with the recovery process of burned injury. The significantly enriched Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of SIGs and SDGs were obtained through the Database for Annotation, Visualization, and Integrated Discovery (DAVID), based on which the miRNA-gene regulation network for SIGs and SDGs were constructed from the miRWalk database. RESULTS: Ten clusters were obtained through soft clustering. The SIGs and SDGs were found to be closely associated with the biological processes of immune system. The miRNA-gene regulation network analysis suggested different roles between SIGs and SDGs in the recovery of severe burned injury. Furthermore, a bunch of important biomarkers were identified, which would be helpful in the treatment of burned patients. CONCLUSION: Our current findings suggest an interesting molecular link between transcriptional regulation potentially involved in immunosuppressive state after major burn injury, which warrants further exploration for their utilization in the treatment of major burn injury.

Efficacy and safety of thrombopoietin receptor agonists in children with chronic immune thrombocytopenia: a meta-analysis.

BACKGROUND AND AIM: Thrombopoietin receptor agonists (TPO-RAs) have been shown to be safe and effective for adults with chronic immune thrombocytopenia (ITP). The aim of this meta-analysis is to assess the efficacy and safety of thrombopoietin receptor agonists for children with chronic ITP. MATERIALS AND METHODS: Clinical randomized controlled trials (RCTs) evaluating the efficacy and safety of TPO-RAs in pediatric ITP patients published up to June 2017 were retrieved from PubMed, Cochrane Library, and Embase databases. Relevant data were extracted, and the Physiotherapy Evidence Database scale was used to assess the methodological quality. Stata/SE 12.0 was used to perform a meta-analysis. RESULTS: Seven RCTs were included, with 238 patients and 107 patients in the TPO-RA group and the control group, respectively. Assessing efficacy, better results were found in the TPO-RA group for the rate of overall platelet response, durable response, and rescue medication needed. Furthermore, the TPO-RA group yielded superior results in the incidence of clinically significant bleeding events but had a comparable result in the incidence of any bleeding events and severe bleeding events. No significant difference was found between the two groups in health-related quality of life and parental burden. Assessing safety, no significant difference was found between the two groups in the incidence of any adverse events and severe adverse events. CONCLUSIONS: TPO-RAs are effective and safe agents for the treatment of chronic ITP in pediatric patients. Eltrombopag appears to be better than romiplostim in terms of the rate of rescue medication needed and clinically significant bleeding events.

Isolation and Characterization of Ftsz Genes in Cassava.

The filamenting temperature-sensitive Z proteins (FtsZs) play an important role in plastid division. In this study, three FtsZ genes were isolated from the cassava genome, and named MeFtsZ1, MeFtsZ2-1, and MeFtsZ2-2, respectively. Based on phylogeny, the MeFtsZs were classified into two groups (FtsZ1 and FtsZ2). MeFtsZ1 with a putative signal peptide at N-terminal, has six exons, and is classed to FtsZ1 clade. MeFtsZ2-1 and MeFtsZ2-2 without a putative signal peptide, have seven exons, and are classed to FtsZ2 clade. Subcellular localization found that all the three MeFtsZs could locate in chloroplasts and form a ring in chloroplastids. Structure analysis found that all MeFtsZ proteins contain a conserved guanosine triphosphatase (GTPase) domain in favor of generate contractile force for cassava plastid division. The expression profiles of MeFtsZ genes by quantitative reverse transcription-PCR (qRT-PCR) analysis in photosynthetic and non-photosynthetic tissues found that all of the MeFtsZ genes had higher expression levels in photosynthetic tissues, especially in younger leaves, and lower expression levels in the non-photosynthetic tissues. During cassava storage root development, the expressions of MeFtsZ2-1 and MeFtsZ2-2 were comparatively higher than MeFtsZ1. The transformed Arabidopsis of MeFtsZ2-1 and MeFtsZ2-2 contained abnormally shape, fewer number, and larger volume chloroplasts. Phytohormones were involved in regulating the expressions of MeFtsZ genes. Therefore, we deduced that all of the MeFtsZs play an important role in chloroplast division, and that MeFtsZ2 (2-1, 2-2) might be involved in amyloplast division and regulated by phytohormones during cassava storage root development.

Identification, Expression, and Functional Analysis of the Fructokinase Gene Family in Cassava.

Fructokinase (FRK) proteins play important roles in catalyzing fructose phosphorylation and participate in the carbohydrate metabolism of storage organs in plants. To investigate the roles of FRKs in cassava tuber root development, seven FRK genes (MeFRK1-7) were identified, and MeFRK1-6 were isolated. Phylogenetic analysis revealed that the MeFRK family genes can be divided into α (MeFRK1, 2, 6, 7) and ß (MeFRK3, 4, 5) groups. All the MeFRK proteins have typical conserved regions and substrate binding residues similar to those of the FRKs. The overall predicted three-dimensional structures of MeFRK1-6 were similar, folding into a catalytic domain and a ß-sheet ''lid" region, forming a substrate binding cleft, which contains many residues involved in the binding to fructose. The gene and the predicted three-dimensional structures of MeFRK3 and MeFRK4 were the most similar. MeFRK1-6 displayed different expression patterns across different tissues, including leaves, stems, tuber roots, flowers, and fruits. In tuber roots, the expressions of MeFRK3 and MeFRK4 were much higher compared to those of the other genes. Notably, the expression of MeFRK3 and MeFRK4 as well as the enzymatic activity of FRK were higher at the initial and early expanding tuber stages and were lower at the later expanding and mature tuber stages. The FRK activity of MeFRK3 and MeFRK4 was identified by the functional complementation of triple mutant yeast cells that were unable to phosphorylate either glucose or fructose. The gene expression and enzymatic activity of MeFRK3 and MeFRK4 suggest that they might be the main enzymes in fructose phosphorylation for regulating the formation of tuber roots and starch accumulation at the tuber root initial and expanding stages.

Vibrio vulnificus induces mTOR activation and inflammatory responses in macrophages.

Vibrio vulnificus (V. vulnificus), a Gram-negative marine bacterium, can cause life-threatening primary septicemia, especially in patients with liver diseases. How V. vulnificus affects the liver and how it acts on macrophages are not well understood. In this report, we demonstrated that V. vulnificus infection causes a strong inflammatory response, marked expansion of liver-resident macrophages, and liver damage in mice. We demonstrated further that V. vulnificus activates mTOR in macrophages and inhibition of mTOR differentially regulates V. vulnificus induced inflammatory responses, suggesting the possibility of targeting mTOR as a strategy to modulate V. vulnificus induced inflammatory responses.

Structure, Expression, and Functional Analysis of the Hexokinase Gene Family in Cassava.

Hexokinase (HXK) proteins play important roles in catalyzing hexose phosphorylation and sugar sensing and signaling. To investigate the roles of HXKs in cassava tuber root development, seven HXK genes (MeHXK1-7) were isolated and analyzed. A phylogenetic analysis revealed that the MeHXK family can be divided into five subfamilies of plant HXKs. MeHXKs were clearly divided into type A (MeHXK1) and type B (MeHXK2-7) based on their N-terminal sequences. MeHXK1-5 all had typical conserved regions and similar protein structures to the HXKs of other plants; while MeHXK6-7 lacked some of the conserved regions. An expression analysis of the MeHXK genes in cassava organs or tissues demonstrated that MeHXK2 is the dominant HXK in all the examined tissues (leaves, stems, fruits, tuber phloems, and tuber xylems). Notably, the expression of MeHXK2 and the enzymatic activity of HXK were higher at the initial and expanding tuber stages, and lower at the mature tuber stage. Furthermore, the HXK activity of MeHXK2 was identified by functional complementation of the HXK-deficient yeast strain YSH7.4-3C (hxk1, hxk2, glk1). The gene expression and enzymatic activity of MeHXK2 suggest that it might be the main enzyme for hexose phosphorylation during cassava tuber root development, which is involved in sucrose metabolism to regulate the accumulation of starch.

[Correlation between Constitution of Yin Deficiency Syndrome and Polymorphism of HLA-DQA1/Treatment Response of Peg-IFNalpha Therapy in HBeAg Positive Chronic Hepatitis B Patients].

OBJECTIVE: To observe the correlation between constitution of yin deficiency syndrome (YDS) and polymorphism of HLA-DQA1/treatment response of Peg-lFNalpha therapy in HBeAg positive chronic hepatitis B (CHB) patients, and to explore constitution of Chinese medicine (CM) in response of interferon therapy. METHODS: Totally 120 HBeAg positive CHB patients who were treated with Peg-IFNalpha were enrolled, and assigned to YDS group (59 cases) and non-YDS group (61 cases) according to classification of CM constitutions. All patients were subcutaneously injected with Peg-IFNalpha-2b (1.0 microg/kg body weight) or Peg-IFNalpha-2a (180 microg), once per week. Effective efficacy was primarily judged when complete response (CR) or partial response (PR) was obtained at month 6. Those with CR or PR completed 1 year therapeutic course. HLA-DQA1 gene types were detected by polymerase chain reaction sequence specific primers (PCR-SSP). The distribution difference of CM constitutions in patients with CR or PR and their inter-group HLA-DQA1 allele frequency were compared. RESULTS: Different treatment responses of Peg-IFNalpha were observed in CHB patients of two different CM constitutions. The ratio of CR + PR was 61.0% (36/59) in YDS group, obviously lower than that in NYDS group [78.7% (48/61), P < 0. 05]. Patients with CR had a lower allele frequency of HLA-DQA1 * 0501 than those with no-response [14.8% (8/54) vs. 30.6% (22/72)] with statistical difference (P < 0.05). Patients with CR had a higher allele frequency of HLA-DQA1 * 0601 than those with no-response [18.5% (10/54) vs. 5.6% (4/72)] with statistical difference (P < 0.05). The allele frequency of HLA-DQA1 * 0301 was lower in YDS group than in non-YDS group [2. 5% (3/118) vs. 9.8% (12/122)] with statistical difference (P < 0.05). The allele frequency of HLA-DQA1 * 0501 was higher in YDS group than in non-YDS group [33.9% (40/118) vs. 18.9% (23/122)] with statistical difference (P < 0.05). Yet statistical significance was lost after adjustment (Pc > 0.05 for both). CONCLUSIONS: Both constitutions of CM and HLA-DQA1 gene polymorphism af- fect HBeAg positive CHB patients' response to Peg-INFalpha. Constitutions of YDS and HLA-DQA1 * 0501 was not favorable to response, their association needed to be further studied.

[Characterizing Fracture Characteristics of Heterogeneous Oil and Gas Reservoir by Using Microscopic Infrared Spectroscopy Technique ­­Taking Carbonate Reservoir of the Wumishan Formation of Jixian System in Renqiu Burried Hill as an Example].

Oil and gas reservoir fracture is not only the important reservoir space, but also the main passage of the oil and gas migration. Therefore, the characterization of fracture is of great importance. However, reservoir rocks have strong heterogeneity, how to characterize heterogeneous reservoir fracture accurately is an urgent problem to be solved. Microscopic infrared spectroscopy imaging technique can be used to analyze spectral curve of the mineral molecules, different peak characteristics were got, and rock medium composition, the size of the rock fracture, and fracture filler properties and other characteristics were obtained accurately. In this work, carbonate heterogeneous reservoir of Renqiu buried hill was taken as an example, based on the microscopic infrared spectroscopy imaging technique, by the analysis of core sample infrared imaging spectrogram and spectral curves of different characteristic regions, the physicochemical characteristics of the target sample and spatial distribution of the rock medium were obtained, the regions where rock fracture maybe occurred were predicted and the effectiveness of the fractures was analyzed. The results show that the main medium of rock sample is dolomite. The rock fractures contain hydrocarbon organic and salt-water inclusion, which mainly exist in dolomite medium. The proportion of salt-water inclusion is 51.7%, and that of alkane organic matter is 26.0% in the fracture filler. The fracutres extend from the upper left region of core sample to the lower right region, which shows that the extended region of the core sample may be the passage of fluid migration. The salt-water inclusions can impede the migration of oil and gas, resulting in the decrease of fractures permeability. The experimental results show that the width of fractures is 1～1.5 mm, which belongs to large fracture, oil and gas can pass the fractures smoothly, therefore, the effectiveness of the fracture is good. The results showed that it is feasible to characterize the fracture of heterogeneous reservoir by means of microscopic infrared spectroscopy imaging technique, and it provides a new method for the accurate characterization of the fracture of heterogeneous reservoir.

[Theoretical investigation on the structure and vibration spectrum of D-luciferin].

In the present study, the geometry of D-Luciferin was fully optimized by the density functional theory at the B3LYP/6-311++G** and B3PW91/6-311++G** level, and the Cartesian coordinate force constant was calculated at the same level. The scaled quantum mechanism force field (SQM) method was performed to analyze the vibration spectrum. The local internal symmetry coordinates were defined using the method given by Pulay. The theoretical force field matrix, which was obtained through molecular vibration calculation programs, was transformed from Cartesian coordinates into the local internal coordinates. A normal coordinate analysis was carried out using GF matrix method developed by Wilson to give the scaled vibration frequencies and the potential energy distributions (PEDs). In order to make the vibration frequencies in good agreement with the experimental values, we empirically scale the theoretical force fields. According to PEDs, all vibration modes were assigned reliably to certain vibration frequencies. The calculated results show that the D-Luciferin molecule belongs to the point group C1 and involves 66 free degrees of vibration. All vibration modes are infrared and Raman activity. In the Infrared spectrum, the vibration frequency of the strongest absorption peak is 1,780 cm(-1), and the absorption intensity is 507 KM · mol(-1), which is mainly contributed by the stretching vibration mode of the C21==O22, double bond with the PEDs of 93%. In the Raman spectrum, the vibration frequency in the range of 1,200-1.700 cm(-1) presented strong Raman activity, the frequency of the strongest absorption peak is 1,573 cm(-1), and the absorpiton intensity is 297 KM · mol, which is mainly contributed by the stretching vibration made of the C21==N22 double bond in the five-membered ring. The results are helpful to further studying the structure and the luminescence activity of Luciferin derivatives in experiment and theory.

Genome-wide identification, 3D modeling, expression and enzymatic activity analysis of cell wall invertase gene family from cassava (Manihot esculenta Crantz).

The cell wall invertases play a crucial role on the sucrose metabolism in plant source and sink organs. In this research, six cell wall invertase genes (MeCWINV1-6) were cloned from cassava. All the MeCWINVs contain a putative signal peptide with a predicted extracellular location. The overall predicted structures of the MeCWINV1-6 are similar to AtcwINV1. Their N-terminus domain forms a ß-propeller module and three conserved sequence domains (NDPNG, RDP and WECP(V)D), in which the catalytic residues are situated in these domains; while the C-terminus domain consists of a ß-sandwich module. The predicted structure of Pro residue from the WECPD (MeCWINV1, 2, 5, and 6), and Val residue from the WECVD (MeCWINV3 and 4) are different. The activity of MeCWINV1 and 3 were higher than other MeCWINVs in leaves and tubers, which suggested that sucrose was mainly catalyzed by the MeCWINV1 and 3 in the apoplastic space of cassava source and sink organs. The transcriptional levels of all the MeCWINVs and their enzymatic activity were lower in tubers than in leaves at all the stages during the cassava tuber development. It suggested that the major role of the MeCWINVs was on the regulation of carbon exportation from source leaves, and the ratio of sucrose to hexose in the apoplasts; the role of these enzymes on the sucrose unloading to tuber was weaker.

Cloning, 3D modeling and expression analysis of three vacuolar invertase genes from cassava (Manihot Esculenta Crantz).

Vacuolar invertase is one of the key enzymes in sucrose metabolism that irreversibly catalyzes the hydrolysis of sucrose to glucose and fructose in plants. In this research, three vacuolar invertase genes, named MeVINV1-3, and with 653, 660 and 639 amino acids, respectively, were cloned from cassava. The motifs of NDPNG (ß-fructosidase motif), RDP and WECVD, which are conserved and essential for catalytic activity in the vacuolar invertase family, were found in MeVINV1 and MeVINV2. Meanwhile, in MeVINV3, instead of NDPNG we found the motif NGPDG, in which the three amino acids GPD are different from those in other vacuolar invertases (DPN) that might result in MeVINV3 being an inactivated protein. The N-terminal leader sequence of MeVINVs contains a signal anchor, which is associated with the sorting of vacuolar invertase to vacuole. The overall predicted 3D structure of the MeVINVs consists of a five bladed ß-propeller module at N-terminus domain, and forms a ß-sandwich module at the C-terminus domain. The active site of the protein is situated in the ß-propeller module. MeVINVs are classified in two subfamilies, α and ß groups, in which α group members of MeVINV1 and 2 are highly expressed in reproductive organs and tuber roots (considered as sink organs), while ß group members of MeVINV3 are highly expressed in leaves (source organs). All MeVINVs are highly expressed in leaves, while only MeVINV1 and 2 are highly expressed in tubers at cassava tuber maturity stage. Thus, MeVINV1 and 2 play an important role in sucrose unloading and starch accumulation, as well in buffering the pools of sucrose, hexoses and sugar phosphates in leaves, specifically at later stages of plant development.