Glucosinolates(GS), as one of important plant secondary metabolites containing nitrogen and sulfur, is one major flavor source of cruciferous vegetables. In this study we developed SSRs linked to GS content in Chinese cabbage(Brassica rapa ssp. pekinensis), aiming to exploit the variation in gene specific SSR loci or alleles crossed over Chinese cabbage accessions. According to the genomic sequence information of Arabidopsis thaliana and Chinese Cabbage, gene sequences related to GS biosynthesis were alignment and analysis, finding 102 candidate genes associated with GS content distributed in 10 chromosomes. Compared with Arabidopsis, the homologous genes for GS biosynthensis were detected in Chinese cabbage, with 8 zero copy, 13 single copies, 17 double copies, 14 more than 2 copies. By analyzing sequences of candidate genes and their up/down franking 5 kb nucleotides with software FASTPCR 6.0, 237 SSRs were developed in 102 genes, 16 of them had no SSRs, 72 of them had 1~4 SSRs, 5 of them had 5 SSRs, 4 of them had 6 SSRs, 4 of them had 7 SSRs, and 1 of them had 8 SSRs. Within all detected SSRs, four replicated types were found. Most of types were single nucleotide replication, with number of 122 and 51.4% of total SSRs; forty eight replication types of double nucleotide were found, with 20.3% of total SSRs; the most less types were three nucleotide replications, with only 7 SSRs and with 3.0%; the rest 60 replicated types had no clear replications. Seventy seven SSR specific primers linked to GS synthesis genes could be designed from 86 genes with SSR locus. Forty nine SSR primers could amplify available and clear bands in 75 inbred lines of Chinese cabbage, 16 of them (20.8%) were polymorphic, 18 of them (23.4%) had no polymorphism, 15 of them (19.5%) had more bands, 28 of them (36.4%) had no amplification products. Finally 11 pairs of primer were amplified in all accessions and produced 26 SSR alleles. The results can offer basic materials used for molecular marker assistant in breeding new varieties with high valuable GS compositions, and accelerate the process of breeding in nutritional quality in Chinese cabbage.

Myostatin gene(MSTN) is a negative skeleton muscle development control gene, and mutation of this gene will induce the over development of skeleton muscles, which is extremely valuable for the breeding of the meat animals. In this study, two pairs of zinc finger nucleases (ZFN) aimed at bovine (Bos taurus) MSTN second exon were used to introduce the mutation in bovine MSTN gene to achieve the purpose of knocking out this gene. Firstly, the transfection condition by two plasmids to bovine fibroblasts via lipofectamine was optimized using different combination of DNA and transfection reagent, and the highest transfection efficiency was 19.25%. Secondly, the bovine fibroblasts were transfected with two pair of ZFNs, respectively; single cells were picked to 96-well plate by mouth-pipetting. After PCR screening, eighteen MSTN mutated cell lines were obtained, which included one double allele mutated cell line. The mutation efficiencies of these two pairs of ZFNs were 6.05% and 3.84%, respectively. Finally, one of the mutated cell lines was used as the donor cells for somatic cell nucleus transfer (SCNT), and 24 reconstructed blastocysts were obtained. The trophoblast stem-like cells were derived after the zona pellucida removed and cultured in 2i medium. They had the same mutation at MSTN locus with the donor cells according to the PCR and sequencing analysis. In conclusion, ZFNs were very efficient to introduce the mutation to bovine MSTN gene in fibroblasts, and they had the ability to generate double allele mutated cells in one experiment, and the mutated cells were capable to generate the reconstructed embryos for transfer after SCNT. This study provides basic data for the breeding of the beef cattle by MSTN gene mutation.

FAM134B plays a critical role in regulation of intramuscular fat(IMF) deposition. For a further understanding of the structure and function of FAM134B gene, as well as its impact on mutton (Capra hirus) quality, RT-PCR technique was adopted to amplify the FAM134B gene, during which Chuanzhong black goat was used as the experimental animal. The nucleotide and amino acid sequence was analyzed using bioinformatics tools. The expression levels of FAM134B in different tissues were detected by fluorogenic quantitative PCR. Additionally, correlation analysis between expression of FAM134B mRNA and IMF content in muscles was done. The results showed that the full length of goat FAM134B gene(GenBank accession No. KF684947.1) was 1 071 bp, and coding 356 amino acid sequence. Four N-glycosylation sites were predicted in the translated FAM134B protein, as well as more than 20 phosphorylation sites. A phylogenic tree analyse revealed that the nucleotide sequence of goat shared the highest identity with the cattle (Bos taurus) FAM134B. Fluorescence quantitative PCR analyse indicated that FAM134B expressed in heart, liver, spleen, lung, kidney, longissimus dorsi muscle and leg muscle, with the highest expression level in liver and the lowest expression level in spleen. Correlation analysis demonstrated that strong positive correlations were observed between expression of FAM134B mRNA and IMF content in longissimus dorsi and crureus. These results suggested that FAM134B gene might play an important role in regulation of IMF deposition in goat. This research provides a basic data for a further understanding of the function of FAM134B gene, and the role during the process of IMF metabolism as well as its mechanism of nutritional regulation in ruminant.

Scutellarin (Scu) has many beneficial effects clinically such as heat-clearing, detoxicating, expansion of blood vessels and improving microcirculation. The protective effects of scutellarin on improving the heat resistant of pig kidney proximal tubular (LLC-PK1) cells were studied by measuring expressions of heat shock protein 72 (HSP 72), B-cellymphoma/leukemia-2 (Bcl-2) and Bcl-2 associated X protein (Bax) of the cells treated with different concentrations of Scu. The LLC-PK1 cells cultured in vitro were randomly divided into five groups including 37 ℃ control group (Ⅰ), 42 ℃ heat stress group (Ⅱ), 1×10-5 mol/L Scu-treated group (Ⅲ), 1×10-6 mol/L Scu-treated group (Ⅳ) and 1×10-7 mol/L Scu-treated group (Ⅴ), respectively. The total RNA and protein in all groups of cells were extracted, then the mRNA and protein content of HSP72, Bax and Bcl- 2 in the cells were detected by qPCR and Western blot, respectively. Our results showed that the mRNA and protein expression of HSP72 and Bax all increased significantly (P＜0.05), Bcl-2/Bax ratio decreased significantly in groupⅡ compared with groupⅠ(P＜0.05), while expression of Bcl-2 had no significant difference (P＞0.05). Treatments with various concentrations of Scu significantly increased both expressions of Bcl-2 in group Ⅳ and HSP72 in groups Ⅳ andⅤcompared with groupⅡ(P＜0.05) at mRNA and protein level, while neither HSP72 nor Bcl-2 in group Ⅲ had any significant differences. The mRNA and protein expressions of Bax in Scu- treated groups were obviously decreased compared with groupⅡ. The protein ratios of Bcl-2/Bax in both group Ⅳ and Ⅴ were significantly increased compared with groupⅡ(P＜0.01). All above results indicated that Scu could induce HSP72 expression, increase Bcl-2/Bax ratio and enhance heat resistance of LLC- PK1 cells under heat stress condition. Our result enriches the theory of heat stress response in cells cultured in vitro, which provides theoretical basis for improving the cell resistance to heat shock in practice.

To explore candidate genes that affect chicken reproduction traits, 396 Jinghai yellow chicken(Gallus gallus) were genotyped for the 29 SNPs, which had been reported, using a kind of chicken 60 K single nucleotide polymorphism(SNP) chip, and the associations between the 29 SNPs and 11 reproduction traits were analyzed. The results showed that 12 of the 29 SNPs were associated with no less than 1 reproduction trait in Jinghai yellow chicken (P＜0.05). Moreover, 7 of 12 SNPs were identified to be associated with 2 traits (P＜0.05). In total, there was 1 SNP that was identified to have effects on age at first egg (P＜0.05), 2 SNPs had effects on egg number at the age of 462 days, 6 SNPs had effects on egg weight, 6 SNPs had effects on body weight at first egg, 2 SNPs had effects on multiple selection index and 1 SNP had effect on healthy offspring percentage. Unfortunately, no significant SNP (P＜0.05) was found to be associated with egg number at the age of 300 days, egg number between the ages of 300 and 462 days and offspring hatchability. The genetic parameters indicated that continued selection of Jinghai yellow chicken was possible and necessary. Moreover, most of the homozygote had better reproduction performance. The functions of a few function genes nearest to 12 SNPs: Armadillo repeat gene deleted in velocardiofacial syndrome(ARVCF), colony stimulating factor 3 receptor(CSF3R) and odz, odd Oz/ten-m homolog(2ODZ2) were discussed and found to affect chicken reproduction traits by different ways. All these results furtherly supply basic materials for researches of molecular markers and candidate genes of chicken reproduction traits.

Legumes are often recognised as the pioneer plants in tailing areas because of their outstanding symbiotic nitrogen fixation. As the copper ions in the environment may lead to unstable expression of housekeeping genes, screening reference genes is important to identify the differentially expressed genes in black medic (Medicago lupulina L.) during the symbiotic nodulation with Sinorhizobium meliloti CCNWSX0020 under copper stress using qRT-PCR. In this study, the eight housekeeping genes(beta actin(ACT), histone H2A(H2A), ribosomal 18S(18S), tubulin beta(TUB), ubiquitin(UBI), elongation factor 1(EF1), glyceraldehyde-3-phosphate dehydrogenase(GAPDH) and cyclophilin(CYP)) from black medic were selected and used to screen the optimal reference genes. The genes were generated from the roots of black medic inoculated rhizobia at 30 d which were planted under the different levels of copper (50, 100, 150 and 200 mg/kg, respectively). The analyses of primers specificity and PCR amplification efficiency showed that all the primers were accordance with the requirements of the stability screening. The results of qRT-PCR indicated that the expression level and stability of candidate genes were various along with the different concentration of copper ion. With geNorm software and the online estimation of gene expression stabilities in all samples, the optimal combination of reference genes was found to be GAPDH and EF1. The expressions of candidate genes under different levels of copper were also online assessed separately and the results displayed that the best reference gene was GAPDH at low concentration of copper (≤50 mg/kg) and UBI at moderate or high copper level (≥100 mg/kg). The conclusions provide the appropriate reference genes for characterizing the genes related to symbiotic nodulation and copper stress of Medicago lupulina L., and basic data for the plant reference genes under other heavy metal stress.

Tobacco aroma sytles is affected by the ecological environment and cultivation conditions. In order to investigate the effect of ecological environment factors on tobacco(Nicotina tabacum L. cv.) aroma style formation, we characterized gene expression profiles in leaf of the tobacco cultivar K326, which were grown in three main growth areas in China: Pingdingshan County in Henan Province, where is famous for producing strong arama feature of tobacco leaf, Yuxi County in Yunnan Province, where displays mild aroma style of tobacco leaf, and Zunyi County in Guizhou Province, where is representative for producing aroma style between strong and mild of tobacco leaf. A total of 920 genes were found to express differently in three ecological zones. After pathway analysised by KEGG(kyoto encyclopedia of genes and genomes), our results showed that carbohydrate metabolism might have more contribution to the formation of different arama styles. While the biosynthesis of phenylpropanoids, biosynthesis of alkaloids and metabolism of terpenoids, which belong to the secondary metabolism, might have great influence on the formation of arama features. Besides, amino acid metabolism might also have a certain influence on aroma features' formation. Also, we found the gene expression of phosphoenolpyruvate carboxylase 1 (PPC1) (photosynthesis related), cell wall invertase 1 (CWINV1) (starch biosynthesis related), 3-hydroxy-3-methylglutaryl coA reductase (HMGR1) and terpene synthase 21 (TPS21) (terpenoids metabolism related), and some genes related to alkaloid biosynthesis were the highest in Henan Province and lowest in Yunnan Province. While the phytoene synthase (PSY) (key enzyme involved in carotenoid biosynthesis) expressed the highest level in Guizhou Province and the lowest in Henan Province. For the carotenoid degradation, the nine-cis-epoxycarotenoid dioxygenase (NCED4) showed the highest level in Yunnan and the lowest in Henan Province. As for the starch metabolism, the α-glucan phosphorylase (PHS1) expressed the highest level in Yunnan and the lowest in Guizhou Province. These results showed that the expression levels of these genes might play important roles in the formation of different aroma styles under the different ecological environments，which can provide some clues for the regulation of the formation of different aroma styles by proper agronomic practices in the future.

In higher plant, dehydration responsive element-binding protein (DREB) family transcription factors play an important role under abiotic stress. Transcription factor binds specific DNA sequence to regulate related gene expression. DREB subfamily factors, one of the major subfamilies of the APETLA 2/ethylene responsive element binding protein (AP2/ERF) family transcription factor, were mainly divided into 6 groups, named A1 to A6 group. Carrot (Daucus carota L.), a biennial vegetable plant, is famous for its taproot and antioxidant compounds. In this study, the gene sequence, which encoded a DREB transcription factor of DcDREB-A6(GenBank accession No. KM386646), was isolated from carrot based on transcriptome and genome sequence data of carrot. The DcDREB-A6 gene was cloned by RT-PCR using cDNA as template from carrot cultivar Heitianwucun. Sequence analysis indicated that the length of DcDREB-A6 gene was 993 bp, which encoded 330 amino acids. Then, nucleic acid and deduced amino acid sequence, phylogenetic tree, and molecular modeling were further predicted and analyzed. It was predicted that the relative molecular mass of the DcDREB-A6 was 36.68 kD, and the pI was 6.00. The DcDREB-A6 was a hydrophilic protein. DcDREB-A6 from carrot was classified into A6 group of DREB subfamily transcription factor, which belonged to AP2/ERF family transcription factor. The sequence of DcDREB-A6 was highly conserved in the AP2 domain compared to the DREB transcription factors of other plants. The AP2 domain three-dimension structure of DcDREB-A6 of carrot was built with the template structure of 1gcc, which was the AP2 domain of the AtERF1 transcription factor. Protein three-dimensional structure prediction and analysis showed that the domain of DcDREB-A6 had typical structural features of AP2 domain, with one α-helix and three β-sheets. To elucidate the response of the DcDREB-A6 transcription factor gene of carrot to abiotic stresses (high temperature, low temperature, drought and high-salinity stresses), the gene was chosen for quantitative Real-time PCR experiment on Heitianwucun. Quantitative Real-time PCR analysis of the expression profiles showed that the DcDREB-A6 gene was induced by high temperature, low temperature, and high-salinity treatment, respectively. The DcDREB-A6 gene expression increased about 2 times by high temperature and low temperature after 4 and 2 h treatments in carrot, respectively. The DcDREB-A6 gene expression was induced about 4.6 times by salt stress treatments at 8 h treatment in carrot, and increased little by drought treatments in carrot. These results will be useful for elucidating the regulation roles of the DREB subfamily transcription factor under abiotic stress in carrot.

Using the Agrobacterium mediated transformation method, the stem tips of Xinhai13, Xinhai14 and Xinhai16 were used as transformation acceptors for studying the effects of different seedling age, bacteria concentration, infection time and co-culture time on the efficiency of genetic transformation of stem tips, and establishing an efficient genetic transformation system of sea island cotton (Gossypium barbadense L.) in this study. The optimum transformation system was constructed as follows: the seedlings at the age of 4 d were infected by Agrobacterium tumefaciens bacteria at a concentration of OD600=0.5 for 10 min, and co-cultured for 48~72 h. The stem tips were then transformed from co-culture medium to selective medium containing 150 mg/L kanamycin for screening resistant seedlings which was transformed to rooting medium for root regeneration 25 days later. A total of 29 resistant plants were obtained from shoot-tip by grafted or directly transplanted. The PCR and RT-PCR were used to detect the resistant seedlings, and preliminary indicated that the 5- enolpyruvylshikimate-3-phosphate synthase gene (EPSPS) had been inserted into the genome of cotton. The establishment of this transformation system provides the technical support for the transgenic breeding of sea island cotton.

During culturing embryonic stem cells (ESCs), mouse embryonic fibroblasts (MEFs) are used as feeder layer, generally. However, MEFs in limited passages are available for feeder cells. Therefore, it takes much time for preparing MEFs, and expends lots of animals. It is important to find stable cell lines as substitution of MEFs for saving expenditure and reducing workload during ESCs culture. L-Wnt3a cells are immortal cell line that stably secrets activated Wnt-3A protein into culture medium. Recently, it is unclear if the cells can be used as feeder cells for culturing ESCs. In present study, L-Wnt3a cells were used to co-culture with mouse (Mus musculus) ESCs for feeder layer, and its conditional medium was examined to culture ESCs without feeder layer. The results showed that treatment L-Wnt3a cells with mitomycin C for 4 h (10 μg/mL) was effectively for inhibiting proliferation and keeping their viability. Transfer single C57BL/6 mouse blastocyst to wells of plate containing a feeder layer of mitotically inactivated L-Wnt3a cells, primary colonies were formed and an embryonic stem cell line was established. A conditional medium was prepared from L-Wnt3a. ESCs in the conditional medium could proliferate and keep pluripotency when removing feeder layer. Feeder-free cultured ESCs in L-Wnt3a conditioned medium were ALP positive, and immunostaining showed that the cells expressed pluripotent markers, Oct4, Sox2, Nanog, E-cadherin and SSEA-1. It formed embryoid body in vitro and teratoma in vivo, and differentiated into three germinal layers. When injecting feeder-free ESCs into blastocoels, the chimeric offsprings were achieved. In conclusion, the mouse ESCs were successfully isolated and cultured in L-Wnt3a feeder layer. The cells were available cell line for replacing MEFs as feeder cells. It refrained from limited passages and heavy works in MEFs culture. Feeder-free ESCs could be cultured in L-Wnt3a conditional medium, it properly avoides the negative effect of feeder layer during culturing ESCs.

Droplet digital PCR(ddPCR) is a new absolutely quantitative method based on Poisson distribution, which shows huge potential applicability in accurate quantification of nucleic acid. Herein, we developed the ddPCR method to evaluate the exogenous gene copy number in genetically modified organisms (GMOs) as the example from genetically modified rice(Oryza sativa) T1c-19 and transgenic human lactoferrin gene goat(Capra hircus) 134 examples, and the results was also compared with those from quantitative Real-time PCR (qRT-PCR) and Southern blot. The results from qRT-PCR and ddPCR for cry1C* in T1c-19 were comparatively unanimous (~2 copies), while 1 copy was reported in literatures by Southern blot. The copy number from ddPCR was higher than that of qRT-PCR for bar gene in T1c-19, 2.09 and 1.51 copies respectively. Same result (~1 copy) was obtained from qRT-PCR and ddPCR for HLF gene in goat 134. Therefore, the ddPCR was well developed as one novel method for estimating transgene copy number with high accuracy, and which may be widely used in the exogenous genes copy number analysis in GMOs.

The incidence and prevalence of corn disease are limiting factors, which can cause the decrease of corn yield. The maize(Zea mays L.) bacterial spot disease is currently one of potentially harmful diseases in China, has made some progress in the inheritance of resistance genes and chromosomal localization researches, but the progress in gene cloning and function identification researches is relatively slow. At present, using constructed fosmid library to clone the resistance candidate genes is one of efficient and effective methods. The construction of a genomic fosmid library of maize and screening of 2 receptor-like kinase genes (Psy1 and Psy2) were described in this study. The constructed maize fosmid library contained 3.7 × 105 clones, with an average inserts size of 32 kb, achieving the coverage probability of 4.73 fold maize genome, and screening probability of any gene or sequence reached 99.12%. Subsequently, using different primer combinations screening the library, 14 positive clones were obtained, of which 4 clones contained the complete Psy2 gene and Psy1 gene partial fragment, and 3 clones contained the complete Psy1 gene. Two positive clones 13-12F-23 and 32-4A-6 were sequenced, and the results showed that 2 exogenous fragments, one was 10.0 kb DNA fragment and the other was 13.5 kb DNA fragment, were inserted into the promoter and intron of Psy1 gene, respectively. The 13.5 kb DNA fragment was Huck LTR retrotransposon, and the 10.0 kb DNA fragment was a complicated retrotransposon containing helitron transposable element. Nevertheless, there was still a full-length cDNA of Psy1 gene was amplified, indicating that these 2 types of retrotransposon insertion did not make the Psy1 gene inactivation. This study provides a feasible research approach for gene cloning and functional analysis of maize.

The new variety of large yellow croaker (Pseudosciaena crocea) named Donghai No.1 was selected from wild P. crocea which were captured from Daiquyang in Zhejiang Province in 2000. The group selection technology was performed by taking the traits of growth rate and low temperature tolerance as the selection index. The breeding line has got new aquatic variety certificate after the continuous five-generation mass selection in more than 10 years. Under the same cultured conditions, the body length and body weight of Donghai No.1 were 6.06% higher and 15.57% longer than those of commercial larvae at 19-month old. The survival rate of Donghai No.1 (10 month old) was 22.5% higher than that of commercial larvae at 6 ℃. During 2010 to 2012, the pilot scale of Donghai No.1 reached 70 000 m3 seawater, and the demonstration culture gained better social and economic benefits. The breeding process, germplasm characteristics, fine traits and pilot test of Donghai No.1 were described in this paper. This information will be helpful for the extension of Donghai No.1 and the breeding of other aquatic varieties.

Induced pluripotent stem cells (iPSCs) can be generated from somatic cells by ectopic-expression of specific inducing factors, which are capable of maintaining self-renewal and an undifferentiated state just like embryonic stem cells (ESCs). Moreover, the application of iPSCs can avoid the immunological rejection and ethical challenges faced by ESCs research and clinical application. iPSCs were originally derived from Mus musculus somatic cells by ectopic-expression of 4 transcription factors, octamer-binding transcriptionfactor 4(Oct4), sex determing region Y-box 2(Sox2), proto-oncogene protein(Myc) and Krüppel-like factor 4(Klf4), with retrovirus vectors in 2006. Over past years, many scientists made great efforts to improve reprogramming technique and elucidate the mechanism of it. Inducing methods of iPSCs are therefore diversified, since it has been made available to transfer reprogramming factors using DNA virus vectors, plasmid vectors, and minicircle DNA vectors. Although DNA transfection-based method ologies are ostensibly safe, they nonetheless entail some risk of genomic recombination or insertional mutagenesis. RNA molecules and purified reprogramming functional proteins fused with a cell penetrating peptide were used to reprogram differentiated cells to a pluripotent state, respectively, with considerable conversion efficiencies and kinetics, where there is absolutely no genomic recombination or insertional mutagenesis. At the same time, some researchers used a number of small-molecule compounds aiming at replacing some transcription factors and finally succeeding in obtaining iPSCs completely induced by a group of chemicals without any genetic manipulation at a frequency up to 0.2%, which is even more efficient than viral protocols, also contributing to the generation of chimera. iPSCs research of big domestic animals can't be parallel with that of mouse(Mus musculus) or human(Homo sapiens), which have been much focused on, and ESCs of many big domestic animals haven't been derived yet. However, iPSCs derived from the somatic cells of pig(Sus scrofa), bovine(Bos taurus) and sheep(Ovis aries) were obtained by the transduction of serial transcription factors, while only iPSCs of pigs and sheep could generate chimeras after injected into the early embryo. The big domestic animals, such as pigs, are similar to human beings in anatomy and physiology, which can be a good model of organ transplantation and regenerative medicine, and they provide the main sources of meat and milk for human beings. Thus the iPSCs of the big domestic animals play significant roles in manufacture and clinical application. This review presents a general progress on the inducing methods, efficiency and mechanism of the iPSCs as well as the research status of iPSCs of the big domestic animals.

In order to decrease the blindness and to increase the efficiency in lily(Lilium) breeding, mainly based on the author's research results and experience, this paper reviewed the relationship of lily cultivars and wild species, the difference between intra-sectional and inter-sectional hybridizations, the breeding ways of modern lily cultivars and aneuploid lilies, and the mechanism of special phenomena and a new hypothesis (i.e., five same genomes of endosperm are essential for its development of triploid×diploid/tetraploid crosses of Lilium) of lily breeding. Hopefully, it would provide references for lily breeders.