Phenotypic Characterization and QTL/Gene Identification for Internode Number and Length Related Traits in Maize

Internode number and length are the foundation to constitute plant height, ear height and the above-ground spatial structure of maize plant. In this study, segregating populations were constructed between EHel with extremely low ear height and B73. Through the SNP-based genotyping and phenotypic characterization, 13 QTL distributed on the chromosomes (Chrs) of Chr1, Chr2, Chr5-Chr8 were detected for four traits of internode no. above ear (INa), average internode length above ear (ILaa), internode no. below ear (INb), and average internode length below ear (ILab). Phenotypic variation explained (PVE) by a single QTL ranged from 6.82% (qILab2-2) to 12.99% (qILaa5). Zm00001d016823 within the physical region of qILaa5, the major QTL for ILaa with the largest PVE was determined as the candidate through the genomic annotation and sequence alignment between EHel and B73. Product of Zm00001d016823 was annotated as a WEB family protein homogenous to At1g75720. qRT-PCR assay showed that Zm00001d016823 highly expressed within the tissue of internode, exhibiting statistically higher expression levels among internodes of IN4 to IN7 in EHel than those in B73 (P Zm00001d016823 might provide novel insight into molecular mechanism beyond phytohormones controlling internode development in maize.

Genome-wide association with transcriptomics reveals a shade-tolerance gene network in soybean

Shade tolerance is essential for soybeans in inter/relay cropping systems.A genome-wide association study(GWAS)integrated with transcriptome sequencing was performed to identify genes and construct a genetic network governing the trait in a set of recombinant inbred lines derived from two soybean parents with contrasting shade tolerance.An improved GWAS procedure,restricted two-stage multi-locus genome-wide association study based on gene/allele sequence markers(GASM-RTM-GWAS),identified 140 genes and their alleles associated with shade-tolerance index(STI),146 with relative pith cell length(RCL),and nine with both.Annotation of these genes by biological categories allowed the construction of a protein–protein interaction network by 187 genes,of which half were differentially expressed under shading and non-shading conditions as well as at different growth stages.From the identified genes,three ones jointly identified for both traits by both GWAS and transcriptome and two genes with maximum links were chosen as beginners for entrance into the network.Altogether,both STI and RCL gene systems worked for shade-tolerance with genes interacted each other,this confirmed that shadetolerance is regulated by more than single group of interacted genes,involving multiple biological functions as a gene network.

A cluster of mutagenesis revealed an osmotic regulatory role of the OsPIP1 genes in enhancing rice salt tolerance

Aquaporins play important regulatory roles in improving plant abiotic stress tolerance.To better understand whether the Os PIP1 genes collectively dominate the osmotic regulation in rice under salt stress,a cluster editing of the Os PIP1;1,Os PIP1;2 and Os PIP1;3 genes in rice was performed by CRISPR/Cas9 system.Sequencing showed that two mutants with Cas9-free,line 14 and line 18 were successfully edited.Briefly,line 14 deleted a single C base in both the Os PIP1;1 and Os PIP1;3 genes,and inserted a single T base in the Os PIP1;2 gene,respectively.While line 18 demonstrated an insertion of a single A base in the Os PIP1;1gene and a single T base in both the Os PIP1;2 and Os PIP1;3 genes,respectively.Multiplex editing of the Os PIP1 genes significantly inhibited photosynthetic rate and accumulation of compatible metabolites,but increased MDA contents and osmotic potentials in the mutants,thus delaying rice growth under salt stress.Functional loss of the Os PIP1 genes obviously suppressed the expressions of the Os PIP1,Os SOS1,Os CIPK24 and Os CBL4 genes,and increased the influxes of Na+and effluxes of K^(+)/H^(+)in the roots,thus accumulating more Na+in rice mutants under salt stress.This study suggests that the Os PIP1 genes are essential modulators collectively contributing to the enhancement of rice salt stress tolerance,and multiplex editing of the Os PIP1 genes provides insight into the osmotic regulation of the PIP genes.

Detection of new candidate genes controlling seed weight by integrating gene coexpression analysis and QTL mapping in Brassica napus L.

Seed weight is a component of seed yield in rapeseed(Brassica napus L.).Although quantitative trait loci(QTL)for seed weight have been reported in rapeseed,only a few causal quantitative trait genes(QTGs)have been identified,resulting in a limitation in understanding of seed weight regulation.We constructed a gene coexpression network at the early seed developmental stage using transcripts of 20,408 genes in QTL intervals and 1017 rapeseed homologs of known genes from other species.Among the 10 modules in this gene coexpression network,modules 1 and 2 were core modules and contained genes involved in source–flow–sink processes such as synthesis and transportation of fatty acid and protein,and photosynthesis.A hub gene SERINE CARBOXYPEPTIDASE-LIKE 19(SCPL19)was identified by candidate gene association analysis in rapeseed and functionally investigated using Arabidopsis T-DNA mutant and overexpression lines.Our study demonstrates the power of gene coexpression analysis to prioritize candidate genes from large candidate QTG sets and enhances the understanding of molecular mechanism for seed weight at the early developmental stage in rapeseed.

Study of pathogenic genes in a pedigree with familial dilated cardiomyopathy

BACKGROUND Dilated cardiomyopathy(DCM)is a genetically heterogeneous cardiac disorder characterized by left ventricular dilation and contractile dysfunction.The substantial genetic heterogeneity evident in patients with DCM contributes to variable disease severity and complicates overall prognosis,which can be very poor.AIM To identify pathogenic genes in DCM through pedigree analysis.METHODS Our research team identified a patient with DCM in the clinic.Through invest-igation,we found that the family of this patient has a typical DCM pedigree.High-throughput sequencing technology,next-generation sequencing,was used to sequence the whole exomes of seven samples in the pedigree.RESULTS A novel and potentially pathogenic gene mutation-ANK2p.F3067L-was discovered.The mutation was completely consistent with the clinical information for this DCM pedigree.Sanger sequencing was used to further verify the locus of the mutation in pedigree samples.These results were consistent with those of high-throughput sequencing.CONCLUSIONS ANK2p.F3067L is considered a novel and potentially pathogenic gene mutation in DCM.

Molecular cloning and functional analysis of the pepper resistance gene Me3 to root-knot nematode

Root-knot nematodes(RKNs)cause severe diseases in peppers annually around the world.In pepper,the Me3 gene provides a heat-stable and broad-spectrum resistance to RKNs.In this study,several simple sequence repeat(SSR)markers and insertion/deletion(In Del)markers were developed to fine map the Me3 gene.Analysis of 2272 individuals(F2progenies)revealed that Me3 was located in a 45-kb DNA region between markers SSR784 and SSR339,in which there were three candidate genes.Among them,as a novel nucleotide binding site and leucine rich repeat(NBS-LRR)family gene,the DNA sequence of Capana09g000163 of pepper line‘HDA149’was 6348 bp in length,with a 2802-bp open reading frame encoding 933 amino acids,including NB-ARC and LRR domains.Tobacco transient expression assays demonstrated that expression of Capana09g000163 triggered a hypersensitive response(HR)in Nicotiana benthamiana leaves.Subcellular localization results showed that the Capana09g000163 protein was localized in the cell nucleus.Ectopic expression of Capana09g000163 in Arabidopsis significantly increased resistance against Meloidogyne incognita compared with the wild-type(WT)Arabidopsis.Furthermore,M.incognita was almost unable to develop in transgenic Arabidopsis expressing Capana09g000163.Taken together,we cloned the Me3 gene and verified that it induced resistance against M.incognita with the methods of map-based cloning and transgenic technology,which may be of great significance to pepper breeding for resistance against RKNs.

Genome-wide association study reveals candidate genes for gummy stem blight resistance in cucumber

Gummy stem blight(GSB),caused by Didymella bryoniae,is a serious fungal disease that leads to decline in cucumber yield and quality.The molecular mechanism of GSB resistance in cucumber remains unclear.Here,we investigated the GSB resistance of cucumber core germplasms from four geographic groups at the seedling and adult stages.A total of 9 SNPs related to GSB resistance at the seedling stage and 26 SNPs at the adult stage were identified,of which some are co-localized to previously mapped Quantitative trait loci(QTLs)for GSB resistance(gsb3.2/gsb3.3,gsb5.1,and gsb-s6.2).Based on haplotype analysis and expression levels after inoculation,four candidate genes were identified within the region identified by both Genome-wide association study(GWAS)and previous identified QTL mapping,including Csa3G129470 for gsb3.2/gsb3.3,Csa5G606820 and Csa5G606850 for gsb5.1,and Csa6G079730 for gsb-s6.2.The novel GSB resistant accessions,significant SNPs,and candidate genes facilitate the breeding of GSB resistant cucumber cultivars and provide a novel idea for understanding GSB resistance mechanism in cucumber.

Identification and expression analysis of chlorogenic acid biosynthesis key gene PpHCT in peach

Shikimic acid/quinic acid hydroxy cinnamyl transferase(HCT)is one of the key enzymes in the phenylpropanoid pathway.However,the role of the HCT gene in chlorogenic acid(CGA)biosynthesis in peach fruit remains unclear.For this,we identified the accumulation pattern of CGA in four peach cultivars,cloned and characterized 11 PpHCT gene members,and further analyzed the expression patterns of these PpHCT genes during fruit development.The contents of CGAs in the four peach cultivars all exhibited a trend of increasing and then decreasing during the fruit growth and development.Moreover,the contents of CGAs in the peel and flesh were tissue-specific.Gene structure analysis indicated that the PpHCT genes were highly conserved,containing two exons and one intron.The protein structure analysis demonstrated that the PpHCT proteins contained two conserved motifs(HXXXD,DFGWG)and a transferase domain(PF02458),which belonged to the BAHD acyltransferase family.The cis-acting element analysis suggested that the promoters of PpHCT genes contained many light-related,hormone-related,stress-related,tissue-specific,and circadian-related elements,and they could participate in a variety of biological processes.Phylogenetic analysis showed that the HCT proteins of peach were closely related to the HCT proteins of plum and had a close evolutionary relationship.The qRT-PCR analysis indicated that the expression levels of PpHCT1 and PpHCT2 showed an opposite trend to the accumulation of CGA,whereas the expression levels of PpHCT4,PpHCT5,PpHCT7,PpHCT8,and PpHCT11 demonstrated the same trend as CGA accumulation.It was worth noting that only PpHCT4 and PpHCT5 were highly expressed in the two high-CGA cultivars but showed low levels of expression in the two low-CGA cultivars.Therefore,it was hypothesized that these two genes might be key genes to the synthesis of CGA in peach fruit.Those findings provide a theoretical basis for further study on the biological functions of the HCT gene and help to reveal the molecular mechanism of CGA.

Transplantation of Nogo-66 receptor gene-silenced cells in a poly(D,L-lactic-co-glycolic acid) scaffold for the treatment of spinal cord injury

Inhibition of neurite growth, which is in large part mediated by the Nogo-66 receptor, affects neural regeneration following bone marrow mesenchymal stem cell transplantation. The tissue engineering scaffold poly（D,L-lactide-co-glycolic acid） has good histocompatibility and can promote the growth of regenerating nerve fibers. The present study used small interfering RNA to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells and Schwann cells, which were subsequently transplanted with poly（D,L-lactide-co-glycolic acid） into the spinal cord lesion regions in rats. Simultaneously, rats treated with scaffold only were taken as the control group. Hematoxylin-eosin staining and immunohistochemistry revealed that at 4 weeks after transplantation, rats had good motor function of the hind limb after treatment with Nogo-66 receptor gene-silenced ceils prus the poly（O,L-lactide-co-glycolic acid） scaffold compared with rats treated with scaffold only, and the number of bone marrow mesenchymal stem cells and neuron-like cells was also increased. At 8 weeks after transplantation, horseradish peroxidase tracing and transmission electron microscopy showed a large number of unmyelinated and myelinated nerve fibers, as well as intact regenerating axonal myelin sheath following spinal cord hemisection injury. These experimental findings indicate that transplantation of Nogo-66 receptor gene-silenced bone marrow mesenchymal stem cells and Schwann cells plus a poly（D,L-lactide-co-glycolic acid） scaffold can significantly enhance axonal regeneration of spinal cord neurons and improve motor function of the extremities in rats following spinal cord injury.

Transcriptional Regulation of 10 Mitochondrial Genes in Different Tissues of NCa CMS System in Brassica napus L. and Their Relationship with Sterility

Northern blot analysis was conducted with mitochondrial RNA from seedling leaves, floral buds, and developing seeds of NCa CMS, maintainer line and fertile F1 using ten mitochondrial genes as probes. The results revealed that 9 out of the 10 mitochondrial genes, except for atp6, showed no difference in different tissues of the corresponding materials of NCα CMS system and that they might be constitutively expressed genes. Eight genes, such as orf139, orf222, atpl, cox1, cox2, cob, rm5S, and rm26S, showed no difference among the three tissues of all the materials detected. So the expression of these eight genes was not regulated by nuclear genes and was not tissue-specific. The transcripts of atp9 were identical among different tissues, but diverse among different materials, indicating that transcription of atp9 was neither controlled by nuclear gene nor tissue-specific. Gene atp6 displayed similar transcripts with the same size among different tissues of all the materials but differed in abundance among tissues of corresponding materials and its expression might be tissue-specific under regulation of nuclear gene. Moreover, three transcripts of orf222 were detected in the floral buds of NCa cms and fertile F1, but no transcript was detected in floral buds of the maintainer line.The transcription of orf139 was similar to that of orf222 but only two transcripts of 0.8 kb and 0.6 kb were produced. The atp9 probe detected a single transcript of 0.6 kb in NCa cms and in maintainer line and an additional transcript of 1.2 kb in fertile F1. The relationship of expression of orf222, orf139, and atp9 with NCa sterility was discussed.

Assessment of Genetic Diversity of Argania spinosa L. Growing in Arid and Semi-arid Areas of Morocco as Revealed by Inter-Simple Sequence Repeats

Argania spinosa L. （Sapotaceae family）, endemic to Morocco, is a multipurpose tree with an important ecological and socio-economical interest. Nevertheless, the sustainability of the agro-forestry system is now threatened by overgrazing and over-exploitation leading to an alarming decline in the number of trees. In the present study, inter-simple sequence repeats （ISSR） markers were used to evaluate the genetic variation and to assess the genetic diversity distribution within and among 17 argan populations growing naturally under semi-arid and arid climate. Thus, a total of seven primers generated 260 well-defined bands, with an average of 37.14 bands per primer. Studied populations showed a relatively high level of genetic diversity at species level and low level of genetic diversity at population level. The percentage of polymorphic bands, Nei＇s gene diversity and Shannon＇s information index at population and species level were 30.15%, 0.164, 0.217 and 98.8%, 0.172, 0.293, respectively. A relative low level of genetic differentiation （Gst = 0.39） was in agreement with the results obtained from the analysis of molecular variance （AMOVA）. Estimated gene flow was Nm = 0.781 among populations. Mantel test revealed a non-significant correlation between genetic and geographic distance （r = 0.065, P 〈 0.05）, suggesting that the geographic distribution is not the major factor that shaped the present population genetic structure. The results can help preserve A. spinosa L. tree as an endangered species, however, refining these finding using co-dominant markers is recommended in order to establish conservation strategies.

Construction of Recombinant Adenovirus Vector Containing CEVB2L Gene

[Objective] Sheep contagious ecthyma virus B2L gene recombinant adenovirus was built by adenovirus vector system.[Method] Genome DNA extracted from sheep contagious ecthyma virus strain JLSY04 as a template,Gene fragments obtained from B2L by PCR amplification;B2L gene cloning was cloned into PDNR-CMD vector,screening positive clones and plasmid CTC572-6 was obtained;CTC572-6 plasmid for homologous was recombined with the adenoviral vector.Screening positive clones and bacilli PCR,digestion and sequencing and so on were identified.[Result] After identified by enzyme digestion and gene sequencing,recombinant adenovirus vector CTC572Ade-30 of carrying sheep contagious ecthyma virus B2L gene was constructed successfully.[Conclusion] Which laid the foundation for sheep contagious ecthyma genetically engineered vaccine.

Construction and analysis of a plant transformation binary vector pBDGG harboring a bi-directional promoter fusing dual visible reporter genes

The constitutive promoter of cauliflower mosaic virus 35S （CaMV 35S） is a polar unidirectional promoter and is widely used in plant genetic engineering. In the present study, the unidirectional CaMV 35S promoter has been modified to a bi-directional promoter by fusing its minimal promoter element to the 5＇ end of CaMV 35S promoter in the opposite orientation. To qualitatively and quantitatively estimate its bi-directional transcriptional function and activity, two visible reporter genes, gusA （13-glucuronidase, GUS） and gfp （green fluorescent protein, GFP）, were fused to the two ends of the promoter in bi-orientations ending with NOS terminator sequences, respectively. Stable expression of gusA and gfp genes in transgenic tobacco （Nicotiana tabacum L.） was visulized by histochemically staining for GUS and fluorescence microscopic observation under UV for GFP in transgenic plants. The expression of two reporter genes showed that the constructed bi-directional promoter did have the bi-directional transcriptional function in both expected orientations. The quantitative estimation of GUS and GFP were determined on a HITACHI F1000 Fluorescence Spectrophotometer with various wavelengths of excitation and emission. The GUS activity varied from g to 250 pmol 4-MU/min/mg protein and the GFP content varied from 0.9 to 1.8 μg/ mg protein in various lines of transgenic tobacco plants. Higher GUS activity generally coupled with lower GFP content, and vice versa.

Identification and Gene Mapping of Completely Dominant Earliness in Rice

The completely dominant earliness was identified in a genie male-sterile and early maturing indica line 6442S-7. F1 progenies from 6442S-7 crossed with thirteen various types of medium- or late-maturing varieties, shared the same heading date as 6442S-7. The segregation of heading date in the F2 and B1F1 populations showed that the earliness of 6442S-7 is mainly controlled by two dominant major genes. The local linkage map of one dominant earliness gene harbored in 6442S-7 was constructed with F2 population and four kinds of molecular marker techniques. The results showed that the gene was located between a RFLP marker C515 and a RAPD marker OPI 11.557 on the terminal region of short arm of rice chromosome 3, 10.9cM and 1.5cM from C515 and OPI11.557, respectively. The genetic distances from the target gene to two SSR markers, RM22 and RM231, and one AFLP marker, PT671, were 3.0, 6.7 and 12.4 cM, respectively. This gene, being identified and mapped first, is designated tentatively as Ef-cd(t). As a new genetic resource of completely dominant earliness, 6442S-7 has splendid future in rice improvement.

Genetic transformation of loblolly pine using mature zygotic embryo explants by Agrobacterium tumefaciens

Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression frequency of (-glucuronidase reporter gene (GUS) varied among genotypes after mature zygotic embryos were infected with Agrobacterium tumefaciens cultures. The highest frequency (27.8%) of GUS expressing embryos was obtained from genotype E-Mc with mean number of 21.9 blue GUS spots per embryo. Expression of (-glucuronidase reporter gene was observed on cotyledons, hypocotyls, and radicles of transformed mature zygotic embryos, as well as on organogenic callus and regenerated shoots derived from co-cultivated mature zygotic embryos. Nineteen regenerated transgenic plants were obtained from GUS expression and kanamycin resistant calli. The presence and integration of the GUS gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. These results suggested that an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for the future studies on transferring economically important genes to loblolly pine.

Agrobacterium-mediated transformation and assessment of factors influ-encing transgene expression in loblolly pine (Pinus taeda L.)

This investigation reports a protocol for transfer and expression of foreign chimeric genes in loblolly pine (Pinus taeda L.). Transformation was achieved by co-cultivation of mature zygotic embryos with Agrobacterium tumefaciens strain LBA4404 which harbored a binary vector (pBI121) including genes for β-glucuronidase (GUS) and neomycin phosphotransferase (NPTII). Factors influencing transgene expression including seed sources of loblolly pine, concentration of bacteria, and the wounding procedures of target explants were investigated. The expression of foreign gene was confirmed by the ability of mature zygotic embryos to produce calli in the presence of kanamycin, by histochemical assays of GUS activity, by PCR analysis, and by Southern blot. The successful expression of the GUS gene in different families of loblolly pine suggests that this transformation system is probably useful for the production of the genetically modified conifers.

Genetic Diversity of Recurrent Selection Populations with Ms2 Gene Assessed by Gliadins in Common Wheat (Triticum aestivum L.)

The male-sterile lines with Ms2 gene were highly evaluated in recurrent selection in wheat （Triticum aestivum L.）. Three populations C6 （population after six cycles of selection）, C7 （population after seven cycles of selection）, and C8 （population after eight cycles of selection） were constructed through recurrent selection with 12 parental materials （P）. Acid polyacrymide gel electrophoresis （A-PAGE） analysis was used to identify gliadin patterns and evaluate the genetic diversity in 12 parents and three populations. A total of 63 bands were identified, of which 17 polymorphic bands and 7 unique bands were present in populations and seven polymorphic bands and four unique bands were present in parents. The number of polymorphic and unique bands decreased gradually from C6 to C8, especially for to- and y-gliadins. The genetic distances in C6, C7, and C8 were calculated. The distributions of genetic distance were different in three recurrent selection populations. From C6 to C8, the genetic distance was 0.2687, 0.2652 and 0.1987, respectively. Statistically significant differences were detected between C7 and C8 with the T value of 37.9718. The result of cluster analysis based on genetic similarity matrix of three populations fitted well to those of principle coordinates analysis （PCoA）. Compared with 12 parents, almost all individuals of three populations are new genotypes. Most of the individuals from C6 and C7 could be divided into two groups, while most individuals of C8 were in one cluster. In conclusion, the results indicated that the genetic diversity was decreased severely according to the information revealed by A-PAGE, although some variations could be created in the recurrent selection. It was necessary to introduce diverse germplasm based on the genetic database of recurrent population to maintain and improve the breeding efficiency in the further program.

Isolation of a Plasmalemma Aquaporin Encoding Gene StPIP1 from Solanum tuberosum L. and Its Expression in Transgenic Tobacco

Aquaporin （AQP） belongs to a highly conserved group of membrane proteins considered as major intrinsic proteins, which facilitate water transport across biological membranes. The discovery of AQPs in plants has resulted in a paradigm shift in the understanding of plant-water relations, however, the potential relationship between the role of aquaporins in regulating plant water balance and drought tolerance still remains elusive. In this study, the gene encoding potato AQP cDNA, StPIP1 （GenBank accession no. DQ999080）, was cloned from the leaf of potato cultivar Gannongshu 2 by reverse transcription-PCR （RT-PCR）. Sequence alignment was made by BLASTn in GenBank, the phylogenetic analysis was conducted using PHYLIPWY, the 3D structure was predicted in Swiss-Model server. Subcellular localization of StPIP1 was performed by constructing CaMV35S-StPIP1-GFP and rd29A-StPIP1-GFP fusion proteins and transient expression in onion epidermis. To understand StPIP1 physiological functions in potato under various stress conditions, the StPIP1 gene in a reverse orientation was transformed into tobacco driven by the Cauliflower mosaic virus （CMV） 35S promoter. The expression levels of transgenic and wild-type plants were assessed under various abiotic stress conditions using semi-quantitative RT-PCR, and the morphological and physiological responses of transgenic plants to different stress conditions were investigated. The expression of StPIP1 mRNA decreased in transgenic plants under non-stress and stress conditions, however, the reduction was more severer under drought stress. In both non-stress and stress conditions, StPIP1 was expressed predominantly in root. The morphological and physiological investigation showed no significant differences in growth rate, germination rate, and root fresh weight （FW） between transgenic and wild-type plants when grown under favorable conditions. In contrast, under drought stress, the reduction in StPIPI expression leads to a delay in seed germination and seedling growth, accelerated seedling wilt, and leaf morphological abnormity. Under ＂enough＂ water conditions （i.e., water culture）, the aerial parts of anti-sense plants showed no differences. However, for the aerial parts to accumulate the same amount of biomass, transgenic plants needed about 3 times more abundant root system to transport water for plant growth than wild-type plants. Morphological investigation showed that the reduction in StPIP1 expression increased the root system in transgenic plants under drought stress. As a result, the increase of root mass might compensate the reduced cellular water permeability in order to ensure a sufficient water supply for the plant. Results demonstrated that StPIP1 plays an important role for water transportation in potato, especially under drought stress conditions. The reduced expression of StPIP1 decreases the cellular water transport and influences the expression of endogenous AQPs genes and thereby, has impacts on seed germination, seedling growth, and stress responses of potato to drought conditions.

Identification and Characterization of Tonoplast Sugar Transporter (TST) Gene Family in Cucumber

Tonoplast sugar transporters(TSTs)play essential roles in regulating plant growth,development,and response to various biotic and abiotic stresses.In this study,a total of three TST genes were identified by a genome-wide analysis in cucumber.Phylogenetic analysis showed that TST proteins from cucumber and other plant species can be classified into five groups,and nearly all TST members in the same groups displayed similarmotif distributions,transmembrane(TM)domains,and gene structures.All of the three CsTST genes possess a number of development-,stress-,and hormone-related cis-elements in the promoter sequences.Meanwhile,qRT-PCR assays revealed that the CsTST1 was expressed in fruits,flowers,leaves,and other tissues,and its expression varied significantly under various abiotic stresses such as cold,salt,drought(PEG),and abscisic acid(ABA).Finally,functional analysis of CsTST1 in yeast revealed that it was able to complement the deficiency in galactose,mannose and sucrose transport.These results revealed that CsTST1 can act as a functional sugar transporter to play important roles in cucumber growth and response to abiotic stress probably through affecting carbohydrate distribution.

Suppressive Effects of Genomic Imprinted Gene PEG10 on Hydrogen Peroxide-induced Apoptosis in L0_2 Cells

The effects of PEG10 on hydrogen peroxide （H2O2）-induced apoptosis in human normal liver cell line L02 were investigated. The PEG10 gene was transfected into L02 cells by lipofectamine, the positive clone was screened by G418 and defined as L02/PEG10, while the cell transfected with empty expression vector （pEGFP-N1） was defined as L02/vector. L02/vector and parental L02 cells served as control. RT-PCR and Western blotting were employed to detect the expression of target genes. H2O2 （50–400 mmol/L） was administered to induce the apoptosis of L02 cells. Cells viability was measured by MTT and the morphological changes of apoptotic cells were determined by fluorescence microscopy using hoechst33342 nuclei staining. DNA fragmentation was observed by agarose gel electrophoresis. PEG10 mRNA and protein levels in L02/PEG10 cells were significantly increased as compared with those in the control cells. After treatment with 400 mmol/L H2O2 for 24 h, the cellular growth inhibition rate of L02/PEG10 cells was significantly lower （58.2%） than that of L02 （92.5%） and L02/vector （88%）. Distinct morphological changes characteristic of cell apoptosis such as karyopyknosis and conglomeration were not observed in L02/PEG10. Ladder-like DNA fragmentation in a dose-dependent manner was observed in both L02 and L02/vector cell lines, but not in L02/PEG10. PEG10 over-expression significantly inhibited cytotoxicity induced by H2O2 on human normal liver cell line L02 by antagonizing H2O2-induced apoptosis.