Rab proteins have the conserved cystein residues at C terminus, which are the prenylational modification site after protein translation. In this study, we obtained Arabidopsis RabD2b mutant alleles with either lacking both two conserved cystein residues at 199 and 200 (AtRabD2bΔCC) or altering one conserved cystein residue at 199 to serine residue (AtRabD2bC199S) by site mutation. When the mutated genes were transiently transformed into tobacco(Nicotiana tabacum) leaves or stably transformed into Arabidopsis, it was found that they were both targeted to the cytoplasm, while wild-type AtRabD2b was mainly localized on Golgi stacks. The lethal phenotype at certain temperature of Yeast Ypt1, a temperature-sensitive mutant stain, could be recovered by the transformation of AtRabD2b. However, two mutant AtRabD2b proteins both lost the complementary ability. These results suggested that the mutation of single conserved cystein residue or lacking of the two conserved cystein residues at C terminus affected the localization and function of AtRabD2b. The results provide the reference data for further study on the mechanism of plant Rab prenylational modification.

The ecological risk of transgenic herbicide-resistant rice(Oryza sativa) has been seriously concerned by professional and public. Evaluating the size of ecological risk and the amount of benefit has important reference value in risk precaution, distinguishing right from wrong, weight the advantage and disadvantage, and scientific policy-making. The previous researches emphasised on individual risk analysis and mainly adopted the simulated environment, but there was less on comparison of risk and benefit and deficient on global and integrated analysis. This paper has comprehensively analysed the ecological risk and benefit of transgenic herbicide-resistant rice, and put forward technical approaches to control the ecological risk, that has vital pratical significance in promoting healthy and steady development of transgenic herbicide-resistant rice in China.

Human amniotic mesenchymal stem cells (hAMSCs) are adult stem cells, and have the characteristics of wide source, low immunogenicity and rapid proliferation. The isolation and culture condition of hAMSCs and its differentiation potency of three germ layers were investigated in this study. The amnion was mechanically separated from the placenta obtained by cesarean section after 38~40 week-pregnancy. The hAMSCs were isolated from human amnion digested twice with 0.05% trypsin-EDTA for 30 min and once with collaenase typeⅠfor 1 h. Therefore, the obtained cells were high purity human amniotic mesenchymal stem cells . The morphololgy of hAMSCs was observed under the microscope. The growth curve of hAMSCs was checked by cell counting. Chromosome analysis was checked, then the flow cytometry, immunofluorescence and reverse transcription-PCR techniques were adopted to identify the surface molecular markers and stem cell characteristics. Further more, hAMSCs were able to differentiate into multi-potential ability when cultured in the special inducing medium. The results showed that high-purified hAMSCs isolated from amnion in the most appropriate culture condition was established. The morphology of hAMSCs was identical with fibroblast and the cells showed active proliferative ability. Results of immunocytochemistry and immunofluorescence showed that they were positive for stage specific embryonic antigens (SSEA-4) and vimentin and had a strong proliferation and multipotential capacity. By FASC and RT-PCR analysis, the following cell surface antigens including CD29, CD49d and CD73 expressed in hAMSCs, but negatived for CD34, CD45 and HLA-DR. The stem cell special gene markers such as OCT4, SOX2 and NANOG expressed constantly in these cells, and these cells could differentiate into fat cells, osteoblasts cells, neuron-like cells and insulin secreting cells; special marker genes such as PPARγ2, Osteocalcin, MAP-2, PDX1 expressed in insulin secreting cells. hAMSCs could be successfully isolated, cultured and proliferation in vitro culture conditions. The results showed that hAMSCs can be separated and maintained a stable genetic characteristics in vitro. hAMSCs not only has the characteristics of mesenchymal stem cells, but also has the potential to differentiate the three germ layers. These results provide experimental basis for clinical applications of tissue engineering and regenerative medicine.

In the present study, a cDNA-AFLP differential fragment was isolated from the stem of tuber mustard (Brassica juncea var. tumida) at different swelling stages, to explain the mechanism of swelling of tumor-like stems. The full-length cDNA (named orf451, GenBank accession: KM213220), was cloned by rapid amplification of cDNA ends (RACE). Both the nucleotide sequence and putative amino acid sequence were analyzed. The results showed that the length of orf451 was 1 512 bp, and it contained a 1 356 bp long open reading frame. The orf451 shared high similarity (90%) with polygalacturonases/glycoside hydrolase in nucleotide sequence, and its protein possessed of the functional domains of polygalacturonases (pectate lyase) and glycoside hydrolase. The expression analysis by Real-time quantitative PCR (qRT-PCR) revealed that orf451 highly expressed at the early stage during the swelling of tumor-like stems and was rich in epidermis and outer cortex. These results suggested that orf451 was highly involved in the hydrolysis of polysaccharides in epidermis and outer cortex during the swelling of tumor-like stems. These study provide basic data for exploring the molecular mechenism of tumor-like stems swelling.

Heat shock transcription factors (Hsfs) widely exist in plants and play a key role under extreme environmental conditions, especially under heat shock stress. Most researches focused on model plants such as Arabidopsis and tomato (Lycopersicon esculentum). There were few reports about maize Hsfs so far. In this study, a Hsf gene, named ZmHsf06 (GenBank accession: GRMZM2G115456_T01), was cloned from maize (Zea mays) young leaves treated by heat shock at 42 ℃ for 1 h using homologous cloning methods. The patterns of ZmHsf06 expression level in different organs and its subcellular location were analyzed. Sequence analysis showed that the coding sequences (CDS) of ZmHsf06 was 1 584 bp encoding a protein of 527 amino acids. ZmHsf06 contained not only the most conserved and typical DNA-binding domain (DBD) of Hsf family, but also other functional domains such as a nuclear localization signal (NLS) KKRR peptide, a nuclear export signal (NES) IGDLTEQM peptide and an aromatic, large hydrophobic and acidic amino residues (AHA) DSFWEQFL peptide. Real-time quantitative PCR (qRT-PCR) analysis showed that under normal growth conditions, ZmHsf06 expressed in roots, stems and leaves of maize seedlings with the highest expression level in roots, and also in functional leaves, ears, immature embryo and pollen at anther period with the highest expression level in pollen and the lowest in leaves. The expression level of ZmHsf06 was up-regulated by 42 ℃ heat shock, abscisic acid(ABA) and salt stress, respectively. In details, under heat shock of 42 ℃, the highest expression level of ZmHsf06 in roots was 16 folds of that in leaves, and it appeared later than in leaves. While treated with ABA, the highest expression level in leaves was 2 folds of that in roots, and it appeared later than in roots. Under salt stress, the expression levels of ZmHsf06 in leaves and roots were increased significantly and shared the similar patterns. Based on transient expression assay using onion (Allium cepa L.) epidermis, it was found that the ZmHsf06 was located in nucleus specifically both under normal condition and heat shock at 37 ℃ within 1 h. It suggested that ZmHsf06 probably participates in signal transduction process of pollen development and responses to abiotic stresses at transcription level, and functions in nucleus. The results of this work provide basic data for isolating and characterizing more Hsfs in maize, as well as exploring their biological functions in abiotic stresses.

5-enolpyruvyl-shikimate-3-phosphate synthase (epsps) is a new gene of high resistance to glyphosate and Bt cry1AcM as an insect-resistent gene can expresse at high level in monocotyledon. In order to breed transgenic maize with stacking traits of insect-resistance and glyphosate-tolerance, the plant expression vector pCAMBIA1302-P35S::epsps-Tnos-Pubi::cry1AcM-Tnos was constructed and then introduced into maize (Zea mays) inbred lines by Agrobacterium mediated shoot apical meristem transformation. After herbicide screening, survived plants were confirmed by PCR, and the expression of cry1AcM gene in plants was detected by RT-PCR and Western blot, respectively. The resistance of transgenic plants to Asian corn borer(Ostrinia furnacalis (Guenée)), was examined by indoor and field experiments. And the tolerance of transgenic plants to glyphosate was evaluated by the field trials. The analysis of transgenic maize resistances demonstrated that the plants were highly resistant to herbicide and the Asian corn borer. We obtained 6 transgenic maize lines with genetic stability, highly Asiatic corn borer-resistance and glyphosate-tolerance, which achieved the criterion of actual application. Among them lines Z1~Z3 came from elite inbred line Zheng58, and lines C1~C3 came from elite inbred line Chang7-2. In summary, the introduction of cry1AcM and epsps genes into maize elite inbred lines endued the Asiatic corn borer-resistance and glyphosate-tolerance of transgenic plants, which created new corn transgenic lines with excellent complex traits. It is very useful for large-scale planting of the insect-resistance and glyphosate-tolerance transgenic maize in China.

The new germplasm WB13 is a large spike and grain type of wheat which is selected from the derived lines after crossing and backcrossing multi-generational between common wheat (Triticum aestivum L.) 7182 and two-rowed barley (Hordeum vulgare ssp. distichon Hsü.). In order to identify the genetic foundation of WB13, a research work was carried out using the method of morphology, cytology, molecular markers, and specific bands recovery and sequencing. The results showed that the genetic similarity coefficient(GS) was 97.0% between WB13 and wheat 7182 by analyzing their genetic background with several simple sequence repeat (SSR) markers of wheat, but WB13 had better agronomic traits than its parents and had larger spike and grain than wheat 7182. The chromosome number in root-tip cells of WB13 was 2n=42. No obvious hybridization signal was observed in genomic in situ hybridization (GISH) using two-rowed barley genomic DNA as probe. Using specific sequence-tagged site (STS) markers of barley, ABG054 and ABC305B, which were located in 4H and 7H chromosomes of Hordeum vulgare, respectively, two specific sequences (named WS1 and WS3) were amplified from WB13. Compared WS1 and WS3 with the sequences amplified from barley using the two same STS markers, 100% and 98% identities were observed, respectively. Furthermore, the sequences similar with both WS1 and WS3 were all from Hordeum vulgare by blasting with the published sequences in EMBL. Using 35 sets of SSR markers distributed among barley 4H and 7H chromosomes to detect WB13 and its parents, a specific band was amplified in WB13 with a marker MGB396 in barley 4H chromosome, which had been identified to link with a quantitative trait loci (QTL) related with thousand-grain weight by previous research. In summary, WB13 was an introgression line containing parts of the barley 4H and 7H chromosomes. What's more, the barley 4H segments in WB13 may contain some beneficial genes contributed to thousand-grain weight. Our results demonstrated that WB13 was a wheat-barley hybrid, which enriched the germplasm resources of wheat-barley hybrids and large spike and grain wheat. Furthermore, the research had revealed the possible causes of large spike and grain character of WB13 at the molecular level, and provides basic data for QTL mapping and utilizing this material.

Brassinosteroid (BR) is one of the most important hormones which regulate plant growth and development. To clarify the regulatory function of BR in rice (Oryza sativa) cell development, rice calli and suspension cells treated with different concentrations of exogenous BR and the mutant OsBZR1-OE in which the endogenous BR signal enhanced was used to analyze the effect of BR on the volume and cell distribution in rice callus, the shape and size of the single cell suspension, and we observed the cells in OsBZR1-OE transgenic rice seedlings, analyzed expression level of the key genes in regulating cell division and the accumulation of cytoskeletal protein (F-actin) which plays an important role in cell division. We found that low concentration (1×10-10 mol/L) BR could increase the calli volume but the high concentration (1×10-6 mol/L) BR blocked the growth of rice calli. Consistent with calli size, the rice calli treated with high concentrations of BR showed a high density nuclei distribution pattern (cell volume was small) whereas the calli treated with low concentrations of BR showed a low density nuclei distribution pattern (cell volume was large). In addition, BR treatments of single suspension cells showed similar results, low concentration of BR promoted the cell elongation but the high concentration blocked the cell elongation. Moreover, the length of hypocotyl cells in OsBZR1-OE transgenic rice plants (in which the BR signaling was enhanced) was shorter than that in wild type rice. Propidium iodide(PI) and fluorescein isothiocyanate isomerⅠ(FITC)-phalloidin staining showed that BR treatment could promote the division of suspension and cytoskeleton, and the promoted effect of the high concentration BR treatment was more obvious. Two dimensional electrophoresis results indicated that BR could promote the accumulation of actin cytoskeleton protein. RT-PCR results showed that BR could increase the expression of CDC48 (cell division cycle protein 48) gene which can promote mitosis and CYCD2 (cyclin D2) gene which can shorten the cell cycle. Taken together, these results suggested that low concentration of BR could promote the elongation of rice cells but high concentration inhibited the elongation of rice cells via affecting the expression of cytoskeleton protein F-actin and the cell cycle regulatory genes CDC48 which can promote mitosis and CYCD2 which can shorten the cell cycle. These results provide a theoretical basis for the application of BR in control of the rice height and increasing the lodging resistance.

Adiponectin can improve insulin sensitivity, regulate fatty acid metabolism and participate in the cell proliferation and differentiation. Based on the highly conservative region of adiponectin receptor 1 gene (AdipoR1) sequences from bovine (Bos taurus), human (Homo sapiens) and mouse (Mus musculus) in GenBank, the specific primers were designed. A full-length 2 032 bp cDNA sequence of AdipoR1 gene(GenBank accession GQ918145) was isolated and cloned in mammary gland of Xinong Saanen dairy goats (Capra hircus) using RT-PCR and RACE. AdipoR1 gene contained 185 bp 5' untranslated regions(UTR), 719 bp 3' UTR, and 1 128 bp coding sequences (CDS) which encode 375 amino acid (AA). AA sequence alignment showed that the AA of goat AdipoR1 had higher similarity (＞95%) with bovine, pig(Sus scrofa), mouse and human. The protein structure analysis showed that the predicted molecular weight was 42.44 kD, the isoelectric point was 7.19, and 7 transmembrane domains and no signal peptide were included in the entire sequence. Furthermore, expression analysis showed that AdipoR1 gene had the most abundant expression in lung, followed by the small intestine and the minimal expression was observed in heart. However, the expression of AdipoR1 in remaining eight tissues changed more gently. Expression analysis in the two different stages of mammary tissue revealed that there was significantly different (P＜0.05) between peak lactation and dry period. Goat mammary epithelial cells were treated with different concentrations of insulin and prolactin, and the result showed that the mRNA level of AdipoR1 in group treated with insulin decreased and there was the most obvious effect at insulin concentration of 50 nmol/L (P＜0.01); the mRNA level of AdipoR1 in group treated with prolactin increased and there was the most obvious effect at prolactin concentration of 100 mg/mL (P＜0.05). These results indicated that AdipoR1 had certain regulative effects in goat mammary epithelial cells. This work provides basic information to further reveal the function of AdipoR1 in goat mammary gland.

Adult half-smooth tongue-sole (Cynoglossus semilaevis) differs widely in size between male and female, and sex control is an effective measure to improve economic profit for half-smooth tongue-sole aquaculture and the key to achieving sex control is to reveal the genetic mechanism of sex determination. The pattern of half-smooth tongue-sole's genetic sex determination is ZW type. This kind of type may exist multi-factor dose effect, that is, sex determination is not only related with sex chromosomes, but also autosomes may involve in the regulation of sex. Therefore, genetic sex is the result of sex chromosomes and autosomes’ synergistic effect. In this study, we tried to investigate the mechanism of half-smooth tongue-sole’s sex determination from the perspective of quantitative trait loci (QTL) interaction. Sex was taken as a binary trait, all bi-marker interactions were scanned with logistic regression model by using of high density microsatellite genetic linkage map. Results showed that 5 bi-marker combinations were detected at significance level of P＜0.05, among them, SCAFFOLD149_7459 and SCAFFOLD7854_71905 was significantly at P＜0.01 level, 5 bi-marker combinations came from 9 markers among 7 autosomes. These detected markers were mainly located on chromosome 2, 10 and 11, especially 2 bi-marker combinations located on linkage groups 10-11 and both possessed of SCAFFOLD149_7459, which indicating that this marker might have a greater impact on the process of interaction. This study showed that genes close to these markers were involved into sex determination of half-smooth tongue-sole, which means that autosomes may participate in the process of sex determination. The results have important meaning for further study sex determination and sex control of half-smooth tongue-sole.

Flammulina velutipes is an important commercial edible fungus. Exploring the genomic regions controlling important agronomic traits is helpful to the genetic improvement of F. velutipes. A total of 90 F. velutipes strains were used as tested materials, and 5 agronomic traits including primordium period, stipe diameter, stipe length, pileus diameter and yield per bottle were detected. Total 95 polymorphism simple sequence repeat(SSR) markers that developed from the whole genome sequence of F. velutipes were used to scan the genome of materials in present study. On the basis of analyzing population structure and linkage disequilibrium (LD) of the experimental materials. The association analysis between 5 agronomic traits and SSR loci were performed by using GLM (general linear model) program in TASSEL software. The results showed that the differences between the corresponding agronomic traits of the materials were highly significant(P＜0.01). A total of 582 alleles were found among 95 pairs of SSR markers with 6.13 alleles per locus. The polymorphism information content (PIC) value ranged from 0.204 to 0.818, with an average of 0.542. Through the mathematical model-based cluster in structure software, all the accessions were divided into 8 subgroups and the information on the subgroup was used for the next association analysis. A high degree of LD was detected among syntenic markers, and the loci pairs with D'＞0.5 accounting for 33.01% of the total. Twenty-eight markers were found to associate with the 5 traits, of which 7 marker associations were significantly different at P＜0.01. Thirty-one marker-trait associations were detected herein; two for primordium period with an explanation of phenotypic variation (R2) range of 13.87%~17.41%; twelve for stipe diameter with a R2 range of 7.23%~23.02%; three for stipe length with a R2 range of 13.48%~23.55%; nine for pileus diameter with a R2 range of 5.11%~17.04%; five for yield per bottle with a R2 range of 9.47%~18.94%. The present study suggested that the genetic diversity and linkage disequilibrium of the tested F. velutipes germplasm were suitable for association analysis. The marker-trait association SSR primers developed in present study provide reference for the construction of germplasm for F. velutipes germplasm and molecular marker assisted breeding.

Rhizopus oryzae is a major postharvest pathogen of tropical fruits and vegetables, and quite harmful to storage and preservation wihch result in huge economic losses. It can cause Rhizopus soft rot in fruit, destroy fruit quality and flavor, and also produce the ergot alkaloid agroclavine which is toxic to humans and animals. In the present study, we isolated the main pathogen from naturally infected strawberries (Fragaria ananassa L. cv. Zhangji), and identified it by morphological observation and internal transcribed spacer region of ribosomal DNA (rDNA-ITS) analysis. After that, five exogenous antifungal substances including sodium bicarbonate (SB), boric acid (BA), cinnamon oil (Co), sodium nitroprusside (SNP) and phosphite (Phi) were selected for known antifungal activities and commercially availability. And then, inhibitory effects of five substances on pathogen development and pathogenicity were evaluated via assessment of physiological properties, biochemical indexes and inoculation experiments on tomato (Solanum lycopersicum cv. Ailsa Cragi) fruits. Results indicated that the fungus isolated from strawberry was demonstrated as the main pathogen through an artificial infection test and it was highly pathogenic. The infected position of fruit appeared obvious browning, softening and hydration, and the symptoms were similar to those of soft rot caused by Rhizopus stolonifer. Meanwhile, after amplification using ITS universal primers, an approximately 600 bp fragment of ITS rRNA gene sequence was obtained and perfectly matched with that of the R. oryzae strains. In addition, antifungal activities of five substances were positive and dose-dependent. When the concentration of SB, BA, SNP, Co and Phi reached 0.2%, 0.2%, 0.5%, 0.02% and 5 mmol/L, respectively, spore germination rates of R. oryzae were all reduced to below 10% after 6 h of incubation, versus the germination rate was over 90% in the control at the same time. Therefore, these concentrations were considered to be the minimum inhibitory concentrations (MICs) of five substances. Under the MICs, despite the difference of antifungal efficacies, all of five exogenous antifungal could significantly delay germ tube elongation and mycelium extension of R. oryzae, lower fungal biomass accumulation, disturb carbohydrate absorption of pathogen, and effectively control soft rot of tomato caused by R. oryzae. Among five substances, BA, Co and Phi showed more inhibitory efficacies than those of SB and SNP in in vitro experiments; SB, BA and Phi worked better than Co and SNP in in vivo experiments. Our results explored the possibility of employing those antifungal substances as part of general program for management of postharvest diseases caused by fungal pathogens, and will provide some theoretical basis for integrated control of R.oryzae.

In using Real-time quantitative PCR (qRT-PCR) detection of genetically modified (GM) ingredients, the standardization of data analysis is of great significance for the accuracy of the experimental data and the comparability of inter-laboratory data. This research was designed to promote the standardization of data analysis in GM ingredients quantitative detection. Taking the GM maize (Zea mays) NK603 as an example in qRT-PCR analysis, maize endogenous reference gene alcohol dehydrogenase 1 (Adh1) and NK603 event-specific sequence were set as amplified targets. Two certified reference materials (GM content of 0.98% and 4.91%, respectively) were used as blind samples and the GM content were measured. In qRT-PCR assays, standard curves were generated based on the least-square method, and GM content of blind samples was measured through the approach of absolute quantification. The construction of standard curves and analysis of blind samples were repeated 3 times, and 3 parallel reactions were included in one plate for each time. Calibration curves were produced using the mean Ct values of three parallels or using the individual Ct values of three parallels. Corresponding, the GM content of blind samples should be calculated firstly based on mean of Ct values and then transformed to copies, or calculated firstly based on individual copies which was transformed from individual Ct values and then taking the geometric mean or arithmetic mean of copies. It was considered that different treatments on data lead to varied accuracy of the results. In order to enhance the comparability of inter-laboratory data, our results suggested that standard curves should be produced using the original Ct values rather than the mean of the Ct values, and the GM content of blind samples should be based on arithmetic mean of Ct values or geometric mean of copies. The accuracy of the test should be judged through comparing the measurement results with the certified value (UΔ＞Δm, which indicating there was no significant difference between the measurement result and the certified value). This work provides useful information for the standardization of qRT-PCR data analysis in GMO absolute quantification.

The Qinchuan cattle is mainly breeding in Guanzhong area of Shaanxi province, and has good reputation for its purple hair, capacity for drafting as well as superior performance in growth and productivity. The governments and scientific researchers have put significant emphasis on improvement and industrialization of Qinchuan cattle. The process of breeding and improving has been accelerated into the direction of producing beef instead of laboring. With maintaining the inherent advantages of Qinchuan cattle as the basic breed，new strain with significant advances in shape and meat performance has been bred by Northwest Agriculture and Forestry University through more than 30 years. Meanwhile, by introducing the foreign breeds, crossbred breeds were well developed to improve the meat performance. Qinchuan beef industry has fundamentally taken shaped and brought significantly benefits to both economy and society. In this article, the process of selection and improvement, features and popularization situation of the new strain of Qinchuan cattle were introduced. Critical issues related to key techniques have been discussed in yellow cattle selection and improvement in China. This paper will be very important to guide the improvement and industrialization of Chinese indigenous cattle, and promote the development of China beef cattle breeding industry.

Chemosensory proteins(CSPs) widely express in insect organs. Since its first being identified in Drosophila melanogaster, it has been identified from almost all insects. CSPs had been suggested to evolve from the same ancestor because gene clusters of CSP had been found in many species of animals. This review summarized the physical and chemical characters, expression features, molecule structures and evolution modes of CSPs, focused on their functions. Finally, a new functional view of CSPs has been proposed on the basis of previous study, including our lab's study, which is as protector and transfer of small molecules that are involved in metabolism.