A potassium-chloride co-transporter with altered genome architecture functions as a suppressor in glioma.

Gliomas, the most lethal tumours in brain, have a poor prognosis despite accepting standard treatment. Limited benefits from current therapies can be attributed to genetic, epigenetic and microenvironmental cues that affect cell programming and drive tumour heterogeneity. Through the analysis of Hi-C data, we identified a potassium-chloride co-transporter SLC12A5 associated with disrupted topologically associating domain which was downregulated in tumour tissues. Multiple independent glioma cohorts were included to analyse the characterization of SLC12A5 and found it was significantly associated with pathological features, prognostic value, genomic alterations, transcriptional landscape and drug response. We constructed two SLC12A5 overexpression cell lines to verify the function of SLC12A5 that suppressed tumour cell proliferation and migration in vitro. In addition, SLC12A5 was also positively associated with GABAA receptor activity and negatively associated with pro-tumour immune signatures and immunotherapy response. Collectively, our study provides a comprehensive characterization of SLC12A5 in glioma and supports SLC12A5 as a potential suppressor of disease progression.

Protein kinase MeSnRK2.3 positively regulates starch biosynthesis by interacting with the transcription factor MebHLH68 in cassava.

Starch biosynthesis involves numerous enzymes and is a crucial metabolic activity in plant storage organs. Sucrose non-fermenting related protein kinase 2 (SnRK2) is an abscisic acid (ABA)-dependent kinase and a significant regulatory enzyme in the ABA signaling pathway. However, whether SnRK2 kinases regulate starch biosynthesis is unclear. In this study, we identified that MeSnRK2.3, an ABA-dependent kinase, was highly expressed in the storage roots of cassava and was induced by ABA. Overexpression of MeSnRK2.3 in cassava significantly increased the starch content in the storage roots and promoted plant growth. MeSnRK2.3 was further found to interact with the cassava basic helix-loop-helix 68 (MebHLH68) transcription factor in vivo and in vitro. MebHLH68 directly bound to the promoters of sucrose synthase 1 (MeSUS1), granule-bound starch synthase I a (MeGBSSIa), and starch-branching enzyme 2.4 (MeSBE2.4), thereby upregulating their transcriptional activities. Additionally, MebHLH68 negatively regulated the transcriptional activity of sucrose phosphate synthase B (MeSPSB). Moreover, phosphorylated MebHLH68 by MeSnRK2.3 up-regulated the transcription activity of MeSBE2.4. These findings demonstrated that the MeSnRK2.3-MebHLH68 module connects the ABA signaling pathway and starch biosynthesis in cassava, thereby providing direct evidence of ABA-mediated participation in the sucrose metabolism and starch biosynthesis pathway.

Strike or ball? Batters know it better: an fMRI study of action anticipation in baseball players.

To assess whether the brain processes of action anticipation are modulated differently by perceptual and motor experiences, baseball batters, pitchers, and non-players were asked to predict the fate of pitching actions (strike or ball) while undergoing functional magnetic resonance imaging. Results showed both batters (perceptual experts of pitching action) and pitchers (motor experts) were more accurate than non-players. Furthermore, batters demonstrated higher perceptual sensitivity in discriminating strikes than non-players. All groups engaged the action observation network, putamen, and cerebellum during anticipation, while pitchers showed higher activity than non-players in the left premotor cortex, which has been implicated in the internal simulation of observed action. Only batters exhibited differences in strike versus ball pitches in their left ventral extrastriate cortex, which might be associated with the processing of relevant visual information conveyed by the observed pitcher's movement kinematics and pitch trajectory. Moreover, all groups showed higher activity selectively in the striatum, thalamus, sensorimotor cortices, and cerebellum during correct predictions than during incorrect ones, with most widespread activation in batters, reinforcing the greater involvement of the sensorimotor system in perceptual experience. Our findings demonstrate that perceptual experience might enhance action anticipation ability to a greater extent than motor experience, with overlapping but specific neural underpinnings.

Identification of two cassava receptor-like cytoplasmic kinase genes related to disease resistance via genome-wide and functional analysis.

Receptor-like cytoplasmic kinases (RLCKs) play important roles in various developmental processes and stress responses in plants. Whereas, the detailed information of this family in cassava has not clear yet. In this study, A total of 322 MeRLCK genes were identified in the cassava genome, and they could be divided into twelve clades (Clades I-XII) according to their phylogenetic relationships. Most RLCK members in the same clade have similar characteristics and motif compositions. Over half of the RLCKs possess cis-elements in their promoters that respond to ABA, MeJA, defense reactions, and stress. Under Xpm11 infection, the expression levels of four genes show significant changes, suggesting their involvement in Xpm11 resistance. Two RLCK (MeRLCK11 and MeRLCK84) genes potentially involved in resistance to cassava bacterial blight were identified through VIGS experiments. This work laid the foundation for studying the function of the cassava RLCK genes, especially the genes related to pathogen resistance.

Influence of Drought Stress on the Rhizosphere Bacterial Community Structure of Cassava (Manihot esculenta Crantz).

Drought presents a significant abiotic stress that threatens crop productivity worldwide. Rhizosphere bacteria play pivotal roles in modulating plant growth and resilience to environmental stresses. Despite this, the extent to which rhizosphere bacteria are instrumental in plant responses to drought, and whether distinct cassava (Manihot esculenta Crantz) varieties harbor specific rhizosphere bacterial assemblages, remains unclear. In this study, we measured the growth and physiological characteristics, as well as the physical and chemical properties of the rhizosphere soil of drought-tolerant (SC124) and drought-sensitive (SC8) cassava varieties under conditions of both well-watered and drought stress. Employing 16S rDNA high-throughput sequencing, we analyzed the composition and dynamics of the rhizosphere bacterial community. Under drought stress, biomass, plant height, stem diameter, quantum efficiency of photosystem II (Fv/Fm), and soluble sugar of cassava decreased for both SC8 and SC124. The two varieties' rhizosphere bacterial communities' overall taxonomic structure was highly similar, but there were slight differences in relative abundance. SC124 mainly relied on Gamma-proteobacteria and Acidobacteriae in response to drought stress, and the abundance of this class was positively correlated with soil acid phosphatase. SC8 mainly relied on Actinobacteria in response to drought stress, and the abundance of this class was positively correlated with soil urease and soil saccharase. Overall, this study confirmed the key role of drought-induced rhizosphere bacteria in improving the adaptation of cassava to drought stress and clarified that this process is significantly related to variety.

Comparative genome analyses uncovered the cadmium resistance mechanism of enterobacter cloacae.

Cadmium (Cd) can be transported into plants from polluted soils and may cause animal and human diseases through food chains, which requires the development of highly efficient methods for soil Cd remediation. Although we isolated an Enterobacter cloacae strain Cu6 with Cd resistance, this strain cannot be used for soil Cd remediation due to its lower resistance. Here, we domesticated Cu6 and obtained a highly Cd-resistant strain, LPY6, and found that this strain can attenuate the toxic effects of Cd on wheat seedling growth. We deciphered the high Cd-resistance mechanism of LPY6 by genome comparative and genetic analysis. Compared with Cu6, 75 genes were mutated in LPY6. Thirty-four of these genes were deleted, and 41 had single nucleotide polymorphisms (SNPs). Most of these mutated proteins are involved in basic metabolism, substrate transport, stress response and formate and hydrogen metabolism. RNA quantitative analysis and promoter activity assays showed that the transcription or mRNA levels of two operons (cadA and norVW) in these mutated genes were regulated by Cd, zinc (Zn) or lead (Pb) ions, suggesting that these two operons might be required for Cd, Zn or Pb resistance. Expression of cadA and norVW operons in LPY6 partially recovered Cd susceptibility, demonstrating that CadA and NorVW are involved in Cd resistance in E. cloacae. Our findings illustrate that E. cloacae acquires Cd resistance through different pathways and lay a foundation for developing highly efficient methods for soil Cd remediation.

Brain plasticity of structural connectivity networks and topological properties in baseball players with different levels of expertise.

Brain plasticity in structural connectivity networks along the development of expertise has remained largely unknown. To address this, we recruited individuals with three different levels of baseball-playing experience: skilled batters (SB), intermediate batters (IB), and healthy controls (HC). We constructed their structural connectivity networks using diffusion tractography and compared their region-to-region structural connections and the topological characteristics of the constructed networks using graph-theoretical analysis. The group differences were detected in 35 connections predominantly involving sensorimotor and visual systems; the intergroup changes could be depicted either in a stepwise (HC < / = IB < / = SB) or a U-/inverted U-shaped manner as experience increased (IB < SB and/or HC, or opposite). All groups showed small-world topology in their constructed networks, but SB had increased global and local network efficiency than IB and/or HC. Furthermore, although the number and cortical regions identified as hubs of the networks in the three groups were highly similar, SB exhibited higher nodal global efficiency in both the dorsolateral and medial parts of the bilateral superior frontal gyri than IB. Our findings add new evidence of topological reorganization in brain networks associated with sensorimotor experience in sports. Interestingly, these changes do not necessarily increase as a function of experience as previously suggested in literature.

Effect of temperature and water potential on the germination of seeds from three different populations of Bidens pilosa as a potential Cd hyperaccumulator.

BACKGROUND: Bidens pilosa L., an annual herb, has recently been shown to be a potential Cd-hyperaccumulating plant. The germination characteristics of B. pilosa have been documented, while the difference among populations remains unclear. Understanding variability in seed germination among populations is crucial for determining which populations to use for soil remediation programs. RESULTS: Present study was conducted to compare the requirements of temperature and water potential for germination of B. pilosa cypselae (the central type, hereafter seeds) from three populations using the thermal time, hydrotime, and hydrothermal time models. Seeds of three populations were incubated at seven constant temperatures (8, 12, 15, 20, 25, 30, and 35 °C) and at each of four water potentials (0, -0.3, -0.6, and -0.9 MPa). The results showed that germination percentage and rate of B. pilosa seeds were significantly by population, temperature, water potential and their interaction except for the interaction of population and water potential. Seeds from Danzhou population displayed a higher base temperature (Tb) for germination than those from Guilin and Baoshan population, however the ceiling temperature (Tc) had no consistent level among the populations but varied according to the water potential. In addition, the median base water potential [ψb(50)] for germination of seeds from Danzhou population was higher than that for seeds from Baoshan and Guilin population at low temperatures (< 25 °C), which was opposite at high temperatures (≥ 25 °C). CONCLUSION: Seed germination requirements of B. pilosa on temperature and water differed significantly among populations. Differences in seed germination among populations may be complicated, which could not be simply explained by the temperature and rainfall conditions where the seeds were produced as previously reported. The results suggested that programme management should consider variation in seed germination traits when select which population could be applied to what kind of target remediation sites.

The lncRNA39896-miR166b-HDZs module affects tomato resistance to Phytophthora infestans.

The yield and quality of tomatoes (Solanum lycopersicum) is seriously affected by Phytophthora infestans. The long non-coding RNA (lncRNA) Sl-lncRNA39896 is induced after P. infestans infection and was previously predicted to act as an endogenous target mimic (eTM) for the microRNA Sl-miR166b, which function in stress responses. Here, we further examined the role of Sl-lncRNA39896 and Sl-miR166b in tomato resistance to P. infestans. Sl-miR166b levels were higher in Sl-lncRNA39896-knockout mutants than in wild-type plants, and the mutants displayed enhanced resistance to P. infestans. A six-point mutation in the region of Sl-lncRNA39896 that binds to Sl-miR166b disabled the interaction, suggesting that Sl-lncRNA39896 acts as an eTM for Sl-miR166b. Overexpressing Sl-miR166b yielded a similar phenotype to that produced by Sl-lncRNA39896-knockout, whereas silencing of Sl-miR166b impaired resistance. We verified that Sl-miR166b cleaved transcripts of its target class III homeodomain-leucine zipper genes SlHDZ34 and SlHDZ45. Silencing of SlHDZ34/45 decreased pathogen accumulation in plants infected with P. infestans. Additionally, jasmonic acid and ethylene contents were elevated following infection in the plants with enhanced resistance. Sl-lncRNA39896 is the first known lncRNA to negatively regulate resistance to P. infestans in tomato. We propose a novel mechanism in which the lncRNA39896-miR166b-HDZ module modulates resistance to P. infestans.

[Analysis of clinical phenotype and genetic variant in a Chinese pedigree affected with cleidocranial dysplasia].

OBJECTIVE: To analyze the clinical features and pathogenic variant in a Chinese pedigree affected with cleidocranial dysplasia (CCD). METHODS: Clinical data of 8 patients from the pedigree was collected, including physical examination and X-ray images of head, face, spine, limbs, and mouth. Peripheral blood samples were collected from 6 affected members for the extraction of genomic DNA. The proband and other 3 patients were subjected to trio-whole exome sequencing. Candidate variant was verified by Sanger sequencing of the other 2 affected members from the pedigree. RESULTS: This pedigree has included 22 members (8 affected) from four generations. Genetic testing revealed that the proband has harbored a novel pathogenic variant of the RUNX2 gene [NM_001024630: c.1268_1277del (p.P425Afs*56)], which was inherited from her mother and carried by all affected members in the pedigree. The same variant was not detected among the unaffected members, suggesting co-segregation with the phenotype. CONCLUSION: The c.1268_1277del (p.P425Afs*56) variant of the RUNX2 gene probably underlay the pathogenesis of CCD in this pedigree. Genetic testing has facilitated the definite diagnosis and enabled prenatal diagnosis.

Representational momentum in adolescent dancers.

An observer's memory for the final position of a previously viewed moving target is often displaced slightly forward in the direction of the target motion. This forward displacement, named representational momentum (RM), reflects the implied momentum of the target and is influenced by the level of familiarity that the observer has with the target observed. In this study, we investigated whether RM would be present in adolescents when they viewed actions from their domain of expertise, which would allow them to anticipate the sequence of familiar movements. We thus recruited adolescent ballet dancers and asked them to view a typical ballet jump (grand jeté) in photos as in a classical RM paradigm. The ascending, descending, and flying (between ascending and descending) phases of the jump were used to test the effects of the momentum of the jump combined with the effects of gravity, and adolescent dancers' performance was compared with age-matched non-dancers. Results revealed that all adolescents exhibited RM in the ascending and descending phases of the jump with a greater RM effect in the descending than in the ascending phases. Crucially, only dancers exhibited RM in the flying phase of the jump. Our findings provided evidence of the presence of RM phenomenon in adolescents along with the tendency of an amplified effect due to the level of expertise.

Procedural Pain in Hospitalized Children in a Chinese Children's Hospital.

BACKGROUND: Pain prevalence in pediatric hospitals has been investigated in many developed countries, but little is known about this topic in China. AIMS: This study sought to describe the frequency and pain intensity of procedures for medical care in hospitalized children in a Chinese children's hospital. DESIGN: A cross-sectional study was designed to include interviews with children, their parents and the nurses. SETTINGS: This survey was administered in a teaching hospital in southeast China. PARTICIPANTS/SUBJECTS: Infants and children up to 16 years old who were admitted to the study units for more than 6 days were eligible for inclusion. METHODS: Information regarding patient demographics, painful procedures and pain management strategies was obtained during the day shifts of the children's hospitalization. RESULTS: A total of 3886 procedures were performed on 342 children during the data collection period. The reuse of intravenous indwelling needles ( n = 577), removal of tape from the skin (n = 420) and venipuncture on the back of the hand ( n = 401) were the most frequently performed procedures on children. A total of 1941 procedures, accounting for 49.9% (1941/3886) of painful procedures caused moderate to severe pain (pain score ≥4.0). However, only 25.3% (984/3886) received a valid pain assessment, and only 14.4% (560/3886) received pain interventions. CONCLUSIONS: Most children, especially those who are younger (<4 years old), experienced moderate or severe pain during their hospitalization, but did not receive appropriate interventions.

Clinical Impacts and Outcomes With Possible Donor-Derived Infection in Infected Donor Liver Transplantation: A Single-Center Retrospective Study in China.

BACKGROUND: Information on possible donor-derived transmission events in China is limited. We evaluated the impacts of liver transplantation from infected deceased-donors, analyzed possible donor-derived bacterial or fungal infection events in recipients, and evaluated the etiologic agents' characteristics and cases outcomes. METHODS: A single-center observational study was performed from January 2015 to March 2017 to retrospectively collect data from deceased-donors diagnosed with infection. Clinical data were recorded for each culture-positive donor and the matched liver recipient. The microorganisms were isolated and identified, and antibiotic sensitivity testing was performed. The pathogens distribution and incidence of possible donor-derived infection (P-DDI) events were analyzed and evaluated. RESULTS: Information from 211 donors was collected. Of these, 82 donors were infected and classified as the donation after brain death category. Overall, 149 and 138 pathogens were isolated from 82 infected donors and 82 matched liver recipients, respectively. Gram-positive bacteria, Gram-negative bacteria, and fungi accounted for 42.3% (63 of 149), 46.3% (69 of 149), and 11.4% (17 of 149) of pathogens in infected donors. The incidence of multidrug-resistant bacteria was high and Acinetobacter baumannii was the most concerning species. Infections occurred within the first 2 weeks after liver transplantation with an organ from an infected donor. Compared with the noninfection recipient group, the infection recipient group experienced a longer mechanical ventilation time (P = .004) and intensive care unit stay (P = .003), a higher incidence of renal dysfunction (P = .026) and renal replacement therapy (P = .001), and higher hospital mortality (P = .015). Possible donor-derived infection was observed in 14.6% of cases. Recipients with acute-on-chronic liver failure were more prone to have P-DDI than recipients with other diseases (P = .007; odds ratio = 0.114; 95% confidence interval, .025-.529). CONCLUSIONS: When a liver recipient receives a graft from an infected deceased-donor, the postoperative incidence of infection is high and the infection interval is short. In addition, when a possible donor-derived, drug-resistant bacterial infection occurs, recipients may have serious complications and poor outcomes.

Nonlinear engagement of action observation network underlying action anticipation in players with different levels of expertise.

The goal of this study was to reconcile inconsistency of neural engagement underlying action anticipation between experts and nonexperts, as well as between correct and incorrect anticipations. Therefore, we asked novice, intermediate, and skilled baseball batters (N, IB, and SB) to anticipate their swing decisions in response to pitching videos of a strike or ball, using functional magnetic resonance imaging. Behavioral results confirmed the effect of expertise that is generally shown in a linear fashion. Imaging results instead revealed a nonlinear relationship between expertise level and the evoked response amplitude of nodes within the action observation network. The relationship was best captured by an inverted U-shaped quadratic response profile across the three groups such that IB exhibited higher activation than did both SB and N. These empirical findings extend the framework of predictive coding as well as of neural efficiency in anticipating the action of others, and they might be associated with the underlying process to interpret the goal of the observed action and prepare one's own response. Furthermore, the right anterior cerebellum showed different levels of activation for correct and incorrect anticipations in all groups, adding novel evidence of its subtle involvement in anticipation processes irrespective of expertise status.

Identification of QTL TGW12 responsible for grain weight in rice based on recombinant inbred line population crossed by wild rice (Oryza minuta) introgression line K1561 and indica rice G1025.

BACKGROUND: Limited genetic resource in the cultivated rice may hinder further yield improvement. Some valuable genes that contribute to rice yield may be lost or lacked in the cultivated rice. Identification of the quantitative trait locus (QTL) for yield-related traits such as thousand-grain weight (TGW) from wild rice speices is desired for rice yield improvement. RESULTS: In this study, sixteen TGW QTL were identified from a recombinant inbred line (RIL) population derived from the cross between the introgression line K1561 of Oryza minuta and the rice cultivar G1025. TGW12, One of most effective QTL was mapped to the region of 204.12 kb between the marker 2,768,345 and marker 2,853,491 of the specific locus amplified fragment (SLAF). The origin of TGW12 was tested using three markers nearby or within the TGW12 region, but not clarified yet. Our data indicated thirty-two open reading fragments (ORFs) were present in the region. RT-PCR analysis and sequence alignment showed that the coding domain sequences of ORF12, one MADS-box gene, in G1025 and K1561 were different due to alternative slicing, which caused premature transcription termination. The MADS-box gene was considered as a candidate of TGW12. CONCLUSION: The effective QTL, TGW12, was mapped to a segment of 204.12 kb using RILs population and a MADS-box gene was identified among several candidate genes in the segment. The region of TGW12 should be further narrowed and creation of transgenic lines will reveal the gene function. TGW12 could be applied for improvement of TGW in breeding program.

Differential expression of the Hsf family in cassava under biotic and abiotic stresses.

Heat shock transcription factors (Hsfs) are important regulators of biotic and abiotic stress responses in plants. Currently, the Hsf gene family is not well understood in cassava, an important tropical crop. In the present study, 32 MeHsf genes were identified from the cassava genome database, which were divided into three types based on functional domain and motif distribution analyses. Analysis of the differential expression of the genes belonging to the Hsf family in cassava was carried out based on published cassava transcriptome data from tissues/organs (leaf blade, leaf midvein, lateral buds, organized embryogenic structures, friable embryogenic callus, fibrous roots, storage roots, stem, petiole, shoot apical meristem, and root apical meristem) under abiotic stress (cold, drought) or biotic stress (mealybugs. cassava brown streak disease, cassava bacterial blight). The results show the expression diversity of cassava Hsfs genes in various tissues/organs. The transcript levels of MeHsfB3a, MeHsfA6a, MeHsfA2a, and MeHsfA9b were upregulated by abiotic and biotic stresses, such as cold, drought, cassava bacterial blight, cassava brown streak disease, and mealybugs, indicating their potential roles in mediating the response of cassava plants to environment stresses. Further interaction network and co-expression analyses suggests that Hsf genes may interact with Hsp70 family members to resist environmental stresses in cassava. These results provide valuable information for future studies of the functional characterization of the MeHsf gene family.

Genome-Wide Analysis of Dynamin Gene Family in cassava (Manihot esculenta Crantz) and Transcriptional Regulation of Family Members ARC5 in Hormonal Treatments.

The Dynamin gene family play a significance role in many physiological processes, especially ARC5 (Accumulation and replication of chloroplasts 5) in the process of plastid division. We performed a genome-wide analysis of the cassava Dynamin family based on the published cassava genome sequence and identified ARC5. 23 cassava Dynamins (MeDynamins) were identified and renamed. 23 MeDynamins were further divided into five major groups based on their structural and phylogenetic characteristics. The segmental duplication events have a significant impact on the expansion of MeDynamins. ARC5 expression analysis showed that there were differences between leaves and roots of cassava at different developmental stages. The tissue-specific expression analysis of the MeDynamins showed that most of MeDynamins were expressed in stem apical meristem and embryogenesis, whereas ARC5 was mainly expressed in leaves. The processing of IAA (Indole-3-acetic Acid) and MeJA (Methyl Jasmonate) verified the prediction results of cis-elements, and ACR5 was closely related to plant growth and positively correlated. It also indicated that high concentrations of MeJA treatment caused the cassava defense mechanism to function in advance. In conclusion, these findings provide basic insights for functional validation of the ARC5 genes in exogenous hormonal treatments.

Nonlinear neuroplasticity corresponding to sports experience: A voxel-based morphometry and resting-state functional connectivity study.

We aimed to investigate the structural neuroplasticity associated with different levels of sports experience and its effect on the corresponding resting-state functional circuitry. We recruited 18 skilled baseball batters (SB), 19 intermediate baseball batters (IB), and 17 healthy controls (HC), and used magnetic resonance imaging methods to compare their regional gray-matter volume (GMV) and seed-based resting-state functional connectivity (rsFC). Our results revealed that a quadratic function could better depict intergroup differences in regional GMV than a linear function. In particular, the IB showed lower or higher regional GMV than the other two groups. The difference in GMV in the supplementary motor area and areas belonging to the ventral stream, including the middle temporal gyrus and middle temporal pole, might be possibly related to baseball-specific motor and perceptual experience, such as inhibitory action control and pitch identification. On the other hand, the stronger rsFC seeded from the right middle temporal pole to the default mode network, particularly in the precuneus, in the SB and IB relative to that in the HC might be possibly associated with the theory of mind, such as deciding whether to swing or not against the pitcher by detecting the spatial information of pitches. In conclusion, our three-group design enabled the capture of the unique and transient changes that occur during the intermediate phase of expertise development. Our findings indicated that structural and functional brain changes do not necessarily linearly increase as a function of experience as previously suggested by the literature.

pXST, a novel vector for TA cloning and blunt-end cloning.

BACKGROUND: With the rapid development of sequencing technologies, increasing amount of genomic information has been accumulated. To clone genes for further functional studies in large scale, a cheap, fast and efficient cloning vector is desired. RESULTS: A bifunctional vector pXST has been constructed. The pXST vector harbors a XcmI-ccdB-XcmI cassette and restriction site SmaI. Digestion the vector with XcmI generates a single thymidine (T) overhang at 3' end which facilitates TA cloning, and SmaI gives blunt end that enables the blunt-end ligation. Multiple products with various sizes were amplified from cassava genome by PCR and each PCR fragment was separately cloned into a pXST using TA cloning and blunt-end ligation methods. In general, the TA cloning gave higher transformation efficiency than blunt-end ligation for inserts with all different sizes, and the transformation efficiency significantly decreased with increasing size of inserts. The highest transformation efficiency (8.6 × 106 transformants/µg) was achieved when cloning 517 bp DNA fragment using TA cloning. No significant difference observed in the positive cloning efficiency between two ligation methods and the positive cloning efficiency could reach as high as 100% especially for small inserts (e.g. 517 and 957 base pairs). CONCLUSIONS: We describe a simple and general method to construct a novel pXST vector. We confirm the feasibility of using pXST vector to clone PCR products amplified from cassava genome with both TA cloning and blunt-end ligation methods. The pXST plasmid has several advantages over many currently available vectors in that (1) it possesses XcmI-ccdB-XcmI cassette and restriction site SmaI, enabling both TA cloning and blunt-end ligation. (2) it allows direct selection of positive recombinant plasmids in Escherichia coli through disruption of the ccdB gene. (3) it improves positive cloning efficiency by introducing the ccdB gene, reducing the possibility of self-ligation from insufficient digested plasmids. (4) it could be used by high performance and cost-effective cloning methods. Therefore, this dual function vector would offer flexible alternatives for gene cloning experiments to researchers.

Genome-Wide Identification, Expression, and Functional Analysis of the Sugar Transporter Gene Family in Cassava (Manihot esculenta).

The sugar transporter (STP) gene family encodes monosaccharide transporters that contain 12 transmembrane domains and belong to the major facilitator superfamily. STP genes play critical roles in monosaccharide distribution and participate in diverse plant metabolic processes. To investigate the potential roles of STPs in cassava (Manihot esculenta) tuber root growth, genome-wide identification and expression and functional analyses of the STP gene family were performed in this study. A total of 20 MeSTP genes (MeSTP1-20) containing the Sugar_tr conserved motifs were identified from the cassava genome, which could be further classified into four distinct groups in the phylogenetic tree. The expression profiles of the MeSTP genes explored using RNA-seq data showed that most of the MeSTP genes exhibited tissue-specific expression, and 15 out of 20 MeSTP genes were mainly expressed in the early storage root of cassava. qRT-PCR analysis further confirmed that most of the MeSTPs displayed higher expression in roots after 30 and 40 days of growth, suggesting that these genes may be involved in the early growth of tuber roots. Although all the MeSTP proteins exhibited plasma membrane localization, variations in monosaccharide transport activity were found through a complementation analysis in a yeast (Saccharomyces cerevisiae) mutant, defective in monosaccharide uptake. Among them, MeSTP2, MeSTP15, and MeSTP19 were able to efficiently complement the uptake of five monosaccharides in the yeast mutant, while MeSTP3 and MeSTP16 only grew on medium containing galactose, suggesting that these two MeSTP proteins are transporters specific for galactose. This study provides significant insights into the potential functions of MeSTPs in early tuber root growth, which possibly involves the regulation of monosaccharide distribution.