Plant tonoplast Na+/H+ antiporter comparting sodium into vacuolar plays an important role in salinity tolerance. NHXFS1 is a novel tonoplast Na+/H+ antiporter gene which is obtained from AtNHX1 of Arabidopsis thaliana, OsNHX1 of Oryza sativa and DmNHX1 of Flos chrysanthemi by DNA family shuffling in vitro. To study the expression pattern and salt tolerance ability of NHXFS1 in plant, we transformed NHXFS1 into tobacco (Nicotiana benthamiana) by Agrobacterium-mediated leaf disc method, the transgenic plants were screened with hygromycin B and identified by PCR and Southern blot. 15 NHXFS1 positive plants were identified from 43 hygromycin-resistant tobacco plants that we had gotten. Southern blotting analysis with NHXFS1 probe indicated that the NHXFS1 gene was integrated into tobacco genome. According to segregate ratio, transgenic tobacco line with single copy was inbreeded to homozygote. The result of salinity tolerance test demonstrated that the growth vigor of transgenic plants under high salt stress was better than that of the control ones. The transgenic seeds could still germinate in MS plate with 400 mmol/L NaCl, but the controls could not. Also, the growth statuses of transgenic plants were better than that of controls in soil contained high NaCl concentration. When NaCl concentration reached 300 mmol/L, the wild-type plants were nearly wilting, while the transgenic ones were still vital. Results showed that the transgenic plants could accumulate more proline and Na+ ion. RT-PCR analysis showed that the NHXFS1 gene was expressed in tobacco. The result of Real-time PCR showed that the transcript level of NHXFS1 was significantly higher in different tissues after 200 mmol/L NaCl treatment, which increased 1.01, 0.60 and 1.79 fold in root, stem and leaf, respectively. The data suggest that this novel gene NHXFS1 can be applied in plant salt tolerance engineering and molecular breeding. And it also manifest that it's available to develop novel genes improving plant salt tolerance by DNA family shuffling method

The origin of Chinese sheep is unclear yet. There is a large controversy between the Dual Origins and the Three Origins. For the further study on the origin and genetic diversty of sheep, the complete sequences of mitochondrial DNA(mtDNA) D-loop of 133 individuals in 10 Chinese sheep (Ovis areis) breeds were determined with primer pairs which were designed by Primier Premier 5.0 based on the sheep mitochondrial genome sequence. And the results were analyzed by the Laser Gene, MEGA4, Clustalx1.83 and other software. The results showed that the length of whole mtDNA D-loop of Chinese sheep was 1 106~1 182 bp. There were 103 different haplotypes and 155 polymorphic sites. The results indicated that Chinese domestic sheeps had abundant genetic diversity. The NJ tree results indicated that there were two main branches in Chinese domestic sheeps, O. musimon formed one of the branchs, while O. vignei and O. ammon was clustered together, which indicated that O. musimon had more contribution to Chinese domestic sheep breeds. Ten Chinese domesticated sheep species measured in this study were divided into two branches A and B, which indicated that there were at least two maternal origins and the Asia-A was the main haplotype.From the species level, this study evaluated the genetic diversity of domesticated sheep in China. It is pointed out that the Chinese sheep is divided into two branches, and a theoretical basis is provided for the establishment of the domesticated sheep populations relations in China and the protection of germplasm resources

SUPERMAN family genes are important transcription factors in the plant growth and development. In order to understand physiological functions of SUPERMAN family genes in apple (Malus domestica Borkh.), the MdLIFa (GenBank accession No. HM370493) and its promoter were cloned using RT-PCR and anchored PCR from apple cultivar Fuji(M. domestica. cv. Fuji). The 11 genes of SUPERMAN family in apple were cloned by PCR amplification using gene-specific primers. All 12 genes were designated as MdLIFa/MdLIFb(GenBank accession No. HQ260429), MdSUP1a(HQ418477)/MdSUP1b(HQ418478), MdSUP4(HQ418475)/MdSUP12(HQ418476), MdSUP5a(HQ418469)/MdSUP13b(HQ418470), MdSUP9a(HQ418473)/MdSUP9b(HQ418474), and MdSUP5b (HQ418471)/MdSUP13a(HQ418472), located on chromosomes 11/3, 1/1, 4/12, 5/13, 9/unknown, and 5/13, respectively. The similarity of deduce amino acid sequences of each pair of genes was higher than 86%. The subcellular localization of MdLIFa was transient expressed in the cell nucleus by the particle bombardment into onion(Allium cepa) epidermal cells. Cis-acting elements, such as POLLEN1LELAT52 (AGAAA), GTGANTG10 (GTGA), ROOTMOTIFTAPOX1 (ATATT), DOFCOREZM (AAAG), and GT1CONSENSUS (GRWAAW), were found in the upstream of MdLIFa. Phylogenetic analysis showed that MdLIFa/b were clustered in one group, together with MdSUP9a/9b and MdSUP5a/13b while MdSUP1a/b, MdSUP5b/MdSUP13, and MdSUP4/12 in another group. These genes were putatively involved in plant morphogenesis and floral organogenesis. The SUPERMAN family genes in apple are highly homologous in pair with diverse biological functions, which provide useful information for further gene characterization and genetic transformation

The sex differentiation of melon is a complex system-regulation network, which is effected by different environment factors and level of hormone. In this study, a set of recombinant inbred lines of melon(Cucumis melo L.) was derived by crossing WI 998 with TopMark. The transcript profiles of monoecious lines and gynoecious lines in F2S6 group were acquired using the second-generation high throughput sequencing method, and genes associated with plant hormone biosynthesis pathways were analyzed by comparison of differentially expressed genes, Solexa sequencing and sequence-assembling, the results showed that 77 376 Unigenes were acquired from transcripts of monoecious lines with an average length of 435 bp, and 80 825 Unigenes were acquired from transcripts of gynoecious lines with an average length of 509 bp. Comparing the transcript profiles between monoecious and gynoecious lines, 8 966 genes were deemed as differentially expressed. In these genes, 4 296 genes were down-regulated while 4 670 genes were up-regulated. These genes were classified by gene ontology(GO)-ranking method, 2 352 Unigenes were related to biological process, 4 107 Unigenes were related to cellular process, and 2 507 Unigenes were related to molecular functions. The differential expressed genes participated in 121 pathways, gibberellin biosynthesis pathway, gibberellin3- oxidase(GA3-ox)(MU3674), GA7-ox(MU36987), GA2-ox(MU13098/MU13099), GA20-ox and GA2-ox (MU33020) were differentially expressed. Thirty eight genes expressed differentially involved in abscisic acid biosynthesis pathway, which 19 genes were up-regulated and 19 genes were down-regulated. In brassinolide biosynthesis, MU22012 (cytochrome P450) and MU26893 (cytochrome P450) were up-regulated, and MU56098 (cytochrome P450, CYP724B3) and MU76596 (CYP724A1) were down-regulated. In this study, the sex differentiation was also associated with the biosynthesis and signal transduction pathways of gibberellin, abscisic acid, brassinolide and zeatin. The results provide strong and important basis for the analysis of the possible mechanism of sex differentiation of melon, and for the further research on melon sex-modification genes cloning

Maize yield is quantitative trait with complex genetic basis. Used 191 F2 individuals derived from the cross Yandan14 of maize(Zea mays L.), Mo17×Huangzao4 as genotype, the linkage map was constructed with simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP ) markers 184 corresponding F2∶3 families were phenotyped for 7 main ear traits of maize in three environments. A mixed linear model approach and its software, QTLmapper/V2.0, were used to detect quantitative trait loci (QTLs) controling 7 ear traits. 76 QTLs in three environments, including 8 kernel weight per ear QTLs, 8 ear weight QTLs, 11 kernel product percent QTLs, 10 ear length QTLs, 9 bare tip length QTLs, 10 ear diameter QTLs and 20 axis diameter QTLs, were detected totally. Most QTLs were detected in only one environment. The phenotypic variation explained by single QTL was very small. There were only 5 QTLs with the phenotypic variation explained by additive effect of more than 10%, and most QTLs with the phenotypic variation explained by dominance effect of less than 1%. The percentage of 76 QTLs with the gene action model of additive , part dominance, dominance and over dominance were 29%，47%, 11% and 13% , respectively. The QTLs of ear traits in maize had the distribution characteristics of not uniform, clustered and adjacent in chromosome. The increased-effect allele and the decreased-effect allel on these QTLs were not well-uniform between two parents. Any part could provide the increased-effect or decreased-effect allel. The results of this study will be heuristic and instructive for molecular marker assisted selection (MAS) and parent selection etc in high yield molecular breeding of maize

Iron (Fe) is important and indispensable element for the normal physiological processes in the human body, and iron deficiency is a serious nutritional problem in the world, so it is economical and effective to breed and grow wheat cultivars with high grain iron content, especially for those population in poor-developed areas. In this paper, the soybean (Glycine max) ferritin gene was obtained by RT-PCR, and cloned into the pBAC47P vector. The soybean ferritin expression cassette without vector backbone sequence but including wheat endosperm-specific HMW-GS 1Dx5 promoter and NOS terminator was constructed by digestion and purification, and has been transformed through particle bombardment of PDS1000/He system into winter wheat(Triticum aestivum) cultivars, Jinghua No.1. After phosphinothricin (PPT) selection, differentiation and regeneration, 276 plants were regenerated from immature embryos callus. By PCR screening and PCR-Southern blot using specific primers for soybean ferritin gene 65 transgenic plants of T0 generation were selected, Among them, kernels of 29 plants of T1 generation were positive by RT-PCR on the RNA levels. The grain iron content of 8031 T1 transgenic lines have been determined. Compared to the control, there were 265 lines of them have increased iron content, ranging from 4.93 to 64.03 percent, and 22 lines of them increased significantly, which suggests that Fe content of wheat grain can be improved by the soybean ferritin expression cassette without vector backbone sequence

During the degrading of diet protein, protease is the first key enzyme which hydrolyses protein to peptide or amino acid. However there is few genetic information of protease from rumen microbiota due to the limitation of pure-cultured method. The objective of the experiment was to screen, sequence and bioinformatics analyze the protease clone from a Fosmid library of the dairy cow rumen microbiota. Using two different protease selective medium, including 1% skim milk or 1% isolated soybean protein, respectively, fourteen protease activity clones were obtained from rumen microbiota Fosmid library containing 30 000 clones. Different protease decomposition ability of the fourteen clones was measured by Folin-phenol reagent method. The results showed that each clone had its unique ability of protease decomposition. When casein was used as the substrate, the range of enzyme activity was from 0.59 to 2.74 U/mg. While the isolated soybean protein served as substrate, the range of enzyme activity was from 0.70 to 7.19 U/mg. Furthermore, the same clone had different sizes of enzyme activity for different substrates. After end sequences of ten positive cloning(GenBank Accession numbers: JY084410~JY084429), the sequences were blasted by Blastn and Blastx. The results showed that 45% of the genetic sequences could not match with the known genes encoding. The end sequence of pro10F could match to metal peptidase, with the similarity of 54%, which belonged to peptidase family M13.The optimal pH of the clone was 7.0.These results provide the basicdatum for the further study on the clone's enzymatic properties and gene characteristics

It focuses on the intestinal health that balances in microbial flora. The objective of this study was to examine the variation of gastrointestinal tract microflora in broilers raised under different immune status. Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) was conducted to evaluate this effect in broilers. Treatments consisted of a negative control, general vaccination, lipopolysaccharide(LPS) + general vaccination and cyclophosphamide(CYP )+ general vaccination. On the age of 21, 28, 35 and 42 d, digesta of duodenum, jejunum, ileum and cecum were collected to assay for gut microflora. The results showed that the DNA fingerprinting of ERIC-PCR had high stability and repetition. The molecular weights of gene fragment were almost from 200 to 2 000 bp after ERIC-PCR amplification. Different immune stress affected gut microflora to different degrees, and as age varied, the dominant microflora correspondingly changed. The diversity of cecal microflora was mostly obvious in LPS+ general vaccination treatment and CYP+ general vaccination treatment. In the present study, based on cluster analysis, the similarity of gut microflora in negative control was relatively stable, and among all treatments, the similarity of duodenal microflora was up to 75%, followed by cecum(40%), jejunum(38%) and ileum(39%). Under different immune status, between LPS group and no vaccination group, the microbial number and distribution in jejunum were different, but there were no significant difference in ileum and cecum between LPS group and CYP group

Gli-similar-1(Glis1) is high expressed in unfertilized oocytes and in embryos at the 1, 2-cell stage, and it plays an important role on mouse embryos devolpment. Glis1 gene was cloned from mouse(Mus musculus) kidney and constructed eukaryotic expression vector pEGFP-C1-Glis1. The expression and subcellular localization of pEGFP-C1-Glis1 was detected by Western blot and GFP signal. In addition, the expression of several transcription factors was examined by quantitative RT-PCR after overexpression of Glis1. These factors included Oct4, Sox2, c-Myc, N-Myc, Klf4, Nanog, Nrgn and Tspan18. The results showed that a new isoform of Glis1 transcript(Genbank accession no. JQ043365) was cloned. This transcript encoded a truncated protein that lacked four amino acids in the third NLS (nuclear localization signal), and lacked 124 amino acids at the C-terminus, including all amino acids in proline-rich region. This new Glis1 was localized in the nucleus and overexpression of Glis1 dramatically upregulates the mRNA level of Tspan18. The result indicated that subcellular localization of the new isoform of mouse Glis1 transcript was identical with other existing transcripts, it might play a different role on mouse embryos devolpment and in induced pluripotent stem cells (iPSCs)

Silver carp (Hypophthalmichthys molitrix) is a commercially important fish in China, and its natural resource declined seriously in recent years. Several molecular markers had been used to evaluate population genetics of silver carp, and more markers and samples are still necessary for long term conservation issue. In order to enhance genotypic efficiency of microsatellite DNA(SSR) in silver carp, 14 SSR primer sets with characters of high polymorphism and stable amplification were screened out. Those primers were grouped to three multiplex PCR with four or five primer sets. Parameters of multiplex PCR such as annealing temperature, contents of Taq DNA polymerase，dNTPs，Mg2+ and PCR Buffer were optimized. When the annealing temperature were optimized, the PCR content including 2 units of Taq DNA polymerase，0.4 mmol/L dNTPs，2.3 mmol/L Mg2+ and 3 μL 10×PCR Buffer in a total volume of 25 μL could work preferably for different multiplex PCR. The optimized multiplex PCR then were used to evaluate genetic diversity of silver carp from National Original Breeding Farms in Laojianghe of Jianli and Laohe of Shishou. Two hundred specimens were collected from the two farms and used in this study. Number of alleles ranged from 7 to 24 and from 8 to 20 with mean 13.36 and 12.71 per locus for Jianli and Shishou Farms, respectively. The observed heterozygosity ranged from 0.83 to 1 and from 0.66 to 0.99, with means 0.8479 and 0.9443, and the expected heterozygosity from 0.6462 to 0.9327 and from 0.6075 to 0.9063, with means 0.7967 and 0.8134, respectively. There were 1 and 4 loci being deviations from Hardy-Weinberg equilibrium for Jianli and Shishou Farms, respectively. Analysis of molecular variance (AMOVA) among populations revealed FST=0.0864. It suggested that the genetic diversity level of H.molitrix in both Shishou and Jianli farms were high and no genetic divergence have taken place between H.molitrix populations in the two farms. The results show that the use of those three multiplex PCR can be more rapid and more economic in genetics analysis of H.molitrix

The R. solanacearum pathogenicity variation strain Po82 has the characteristics of both strains pathogenic to banana and strains not pathogenic to banana(NPB). In order to elucidate the molecular mechanism of pathogenicity variation, the method of comparative genomics was used. Genomic DNA of the Po82 strain was compared with that of the NPB strain RUN292 by suppressive subtractive hybridization (SSH). The total genomic DNA was digested by RasⅠ, AluⅠand HaeⅢ , respectively. The results of electrophoresis showed that fragment which digested by HaeⅢ , ranged in size from 50 bp to 500 bp and were too short to use for SSH. In contrast, the fragments which digested by RasⅠ and AluⅠ, ranged in size from 500 bp to 2 000 bp, so they were appropriate to use. Detection and analysis of the library construction showed that the technique was efficient in the adaptors ligation and the subtraction. The average size of insert fragment was 300 bp. Plasmids were extracted from positive clones randomly selected from the library and then sequenced and forty-four strain-specific genes of Po82 were identified. The results of bioinformatics analysis indicated that they related to many biological processes including metabolism, transposase, membrane structure, secretion protein, virulence factor, transcription regulation and some unknown function. The mutant of c00283 gene was constructed by using homologous recombination and named Po82Δc00283. Based on the mutant strain, the complement strain was also constructed, named Po82Δc00283-pML123-c00283. The pathogenicity and the biological function of wild type, mutant strain and the complement strain were tested. Pathogenicity test showed that disease index of the mutant Po82Δc00283 was decreased compared with the wild type Po82 and the complement strain. The pathogenicity of the complement strain Po82Δc00283-pML123-c00283 was restored to the wild-type level. Growth curve analysis indicated that Po82Δc00283 mutant grew as fast as the Po82 strain in both rich medium and Boucher's minimal medium. There were no significant differences among the wild-type, mutant and the complement strain in the ability of the mobility and the biofilm formation. In this study, the SSH libraries were successfully constructed and forty-four Po82 strain specific genes were obtained. The function of one pathogenicity related gene was analyzed by using homologous recombination and other biological tests. In conclusion, the results provide basic information further elucidate the molecular mechanism of pathogenicity variation

Class-Ⅰ KNOX(KNOX1) gene is highly conserved homeobox transcription factor in eukaryote, and play a vital role in the process of plant embryogenesis and organ development. Homologue gene of KNOX1 gene was clonded from protocorm-like body of Rosa canina L., through comparison amino acid sequences with other KNOXI gene and phylogenetic analysis, it was named RcKN1 gene. The full length cDNA of RcKN1 was 1 509 bp, with open reading frame(ORF) of 1 185 bp, encoding a protein of 395 amino acids, 5'UTRs of 32 bp and 3'UTRs of 291 bp (GenBank accession No. JN998201). RT-PCR results showed that the gene was only expressed in young organ including leaf bud, floral bud, protocorm-like body, and so on. Constitutive plant expression vector of RcKN1 gene was constructed, its function was analysed by introducing it into tobacco(Nicotiana tabacum L.). A series of novel phenotype were observed on transgenic tobacco, including lobbed leaf, disorder leaf vein, deficit leaves, even leaf like compound leaf, and so on. The results suggest RcKN1 gene performed important function during plant development, which is valuable target gene can be used to study mechanism of leaf development and breed new flower varieties with novel leaf type

Pig is used as an important model animal, and induced pluripotent stem cells (iPS cells) have been established in pig now. However, the efficiency of cryopreserved survival and passage is very low. Rho-associated protein kinase(ROCK) inhibitor Y-27632 enhanced the survival of cryopreserved of human embryonic stem (ES) cells, and improved the ES colony formation. In this study, we used Y-27632 for porcine(Sus scrofa) induced pluripotent stem cells. 5 and 10 μmol/L Y-27632 was used for cryopreservation and passage of porcine iPS cells, and it suggested that Y-27632 could greatly suppress cryopreserved-induced apoptosis and increase thaw-survival rates of porcine iPS cells. Meanwhile, it was able to help the adhesion of porcine iPS cell in passage when 5 and 10 μmol/L Y-27632 was mixed into culture medium, and it also contributed to porcine iPS colonies formation in 24 h. Furthermore, 10 μmol/L Y-27632 would change the morphology of porcine iPS cells, and made the colonies become flat and loose, but it did not affect alkaline phosphatase (AP) activity and the expression level of pluripotenct genes, octamer-binding transcription factor-4 (Oct4), SRY-related high-mobility-group (HMG)-box protein-2 (Sox2) and homeobox transcription factor (Nanog) in porcine iPS cells treated with Y-27632. Besides, PB[Act-RFP] DS, the transposon reporter construct, was introduced into porcine iPS cells through electric transfected, and RFP positive cells were sorted by flow-cytometry. After treated with 10 μmol/L Y-27632 in culture medium, these sorted cells were easier to grow. After these sorting porcine iPS cells injected into porcine early embryos through micromanipulation, the cells treated with Y-27632 were easier to integrate into porcine parthenogenetic embryos than that without treated cells. Above all Y-27632 were able to improve the cryopreserved survival and passage of porcine iPS cells, and suppress the apoptosis induced by single cell dissociated and fluorescence-activated cell sorting, and enhance the integration of RFP carrying porcine iPS cells in porcine embryos. This study contributes to cryopreservation and associated research for porcine and other species pluripotenct stem cells

Omega 3 fatty acid desaturase (FAD) enables algae to produce a series species of ω3 fatty acids of high-valued quality. Based on the sequence of a ω3FAD gene cloned from Myrmecia incisa Reisigl., its open reading frame(ORF) was amplified by RT-PCR and sub-cloned into the shuttle vector pYES2 to generate the recombinant vector pY-ω3FAD. This recombinant plasmid was transformed into a defective mutant INVSc1 strain of Saccharomyces cerevisiae for expression by electroporation. The target gene integrated in the yeast genome was confirmed by screening and sequencing. The transgenic yeast was cultured at 5℃ for 72 h with linoleic acid as a substrate and galactose as an inducer. Gas chromatography(GC) of fatty acid methyl esters and GC-mass spectrometry analysis showed that the transgenic yeast with the recombinant vector pY-ω3FAD could catalyze linoleic acid into α-linolenic acid at the Δ15 position of carbon chain. This result suggested that the ω3FAD cloned from M. incisa at least functioned as a Δ15 FAD for the conversion of linoleic acid to α-linolenic acid. GC analysis demonstrated that the new product of α-linolenic acid could not be detected from the transgenic yeast if it was cultured at 30℃. However, the exogenous precursor linoleic acid could be converted into α-linolenic acid by the transgenic yeast with target gene while it was cultured at 25℃and lower than this temperature. As the temperature decreased, the conversion of linoleic acid by the transgenic yeast would be enhanced and it reached 29.73% at 5℃. GC analysis also indicated that the conversion of linoleic acid by the transgenic yeast would be increased when the incubation time was delayed at 5℃. The conversion reached 38.86% while the transgenic yeast was cultured for 4 d. The results suggest that the protein encoded by the ω3 FAD gene from M. incisa can be a low-temperature inducible enzyme. The successful expression of the ω3 FAD gene from M. incisa in yeast induced by low temperature may be the reason that yeast has possessed a complete adaptation system to low temperature in fatty acid desaturation

Magnesium is the most abundant divalent cation in cells and acts as a critical role in organism growth and development. It has the largest hydrated radius, the smallest ionic radius, and the highest charge density. It is very important for organism to transport magnesium within or among cells because of its specially chemical and physical characteristics. Previous research focused on structural features, physiological function and pathology of magnesium deficiency, and have made great progress in photosynthesis, activating enzymes, stabilizing genomic, inhibiting aging , alleviating Al toxicity and adjusting N metabolism. Our understanding of magnesium crystal structure, signaling of magnesium stress and cellular homeostasis is still in its infancy. Some magnesium transporters have been cloned from organism, such as prokaryote, yeast, mammal and plant, also analyzed the structure, function and subcelluar localization of this magnesium transporters. According to the different structure or function of this magnesium transports, they are divided into six disparate family: Cobalt resistance A (Cor A), Arabidopsis thaliana magnesium-proton exchanger(AtMHX), cation channal, P-type ATPase, magnesium transport E(MgtE) family and other magnesium transports. The structure or function of different members which belong to the same family appears to be closely related. The CorA family is the most extensively studied magnesium transport system, and appears to be the primary magnesium transport system, which exists widely in fungi, bacteria, animals and plants with effectively mediating both influx and efflux of magnesium. This paper reviews the research progress of those magnesium transporter systems, and aims to provide some useful references for the later research