Cry2Aa gene from prokaryotic organism Bacillus thuringiensis consists of low GC content and some elements that affect gene expression in eukaryotic organisms, and the introduction of the unmodified gene into rice resulted in low levels of protein expression and insect resistance. In this paper, the proportion of rice (Oryza sativa L.) preferred codons and GC content in optimized Cry2Aa gene were increased, the potential Poly(A) addition signal sequences and the intron-exon boundary sequences were eliminated by synonymous codon substitution; then the optimized Cry2Aa gene was added with PR1a signal peptide sequence at 5' end and KDEL sequence at 3' end, and the secondary structure of its mRNA was optimized by using Mfold software. As a result, the GC content of the optimized gene Cry2Aa# was 57.29%，25.87% of the total nucleotides and 67.51% of the total codons were replaced in the optimized Cry2Aa# gene; the sequences which had been shown to destabilize mRNA in eukaryotic organisms were eliminated, codon usage frequency in the optimized Cry2Aa# gene kept consistent with the rice genome; In addition, nine stable mRNA stem-loop structures were eliminated, and the free energy of mRNA of the optimized Cry2Aa# gene decreased by 23.9 kcal/mol comparing with the free energy before optimization. The plant expression vector containing the optimized Cry2Aa# gene was constructed, and the optimized Cry2Aa# gene was transformed into rice via Agrobacterium-mediated method; the results of ELISA showed that the Cry2Aa# protein concentration ranged from 8.62 to 18.88 μg/g fresh weight (FW) of leaf at tillering stage in different transgenic lines; the results of bioassay showed that the transgenic lines were well resistant to rice leaf roller (Cnaphalocrocis medinalis); that indicated the approaches for optimization of Cry2Aa# gene were viable. This study not only validats the methods for gene optimization, but also provides an excellent insect-resistant genetic resource for rice breeding.

Cajal body (CB) is a significant component of nucleus, playing roles in growth and deveplopment of animal and plants, and the progress of virus infection, coilin is the protein marker of CB. In order to understand the function and subcellular localization of Arabidopsis thaliana coilin (Atcoilin), the cDNA sequence of Atcoilin was amplified by RT-PCR from Arabidopsis thaliana, then cloned and identified by sequencing. The subcellular localization and functional domains of Atcoilin were predicted using bioinformatics softwares. Atcoilin was ligated into expression vector pEarley101 bearing yellow fluorescent protein (YFP). Recombinant protein Atcolin-YFP were transiently expressed on the leaf epidermis cells of Nicotiana benthamiana by using Agrobacterium-infiltration, the subcellular localization of protein was observed under the confocal laser scanning microscope (CLSM). The bioinformatics analysis showed that Atcoilin localized to the nuclear, encoded 608 amino acids and contained three functional domains (N-terminal ordered domain, central internal disordered domain and C-terminal domain). Atcoilin had higher homologous in amino acid with human coilin, and its homology was 50%. The expression of Atcoilin-YFP was also verified by Western bolting analysis, under the CLSM, Atcoilin-YFP localized in the nuclear. Function prediction and subcelluar localization analysis can provide the foundation for the further research on coilin.

Optimizing an efficient system is the basis of related research by using fluorescent in situ hybridization. In this study, the key factors which effect on fluorescence in situ hybridization results, such as chromosomal pretreatment, denaturing conditions and denaturing ways, were screened in order to optimize a suitable system for fluorescent in situ hybridization in sugarcane(Saccharum). Sugarcane root tips of YC96-66 which is F1 between Erianthus arundinaceus and Saccharum officinarum were pretreated with three following ways, p-dichlorobenzene under the condition of in vitro, α-bromidenaphthalene under the condition of in vitro and 4℃ low temperature water under the condition of non in vitro to screen chromosome morphology. The chromosome was denatured in two different ways with the optimal temperature on simultaneous denaturation at 70, 80, 90 and 98℃ and separate denaturation at 60, 70, 80 and 90℃ to compare the effects of hybridization signal. Results showed that chromosome morphology was stubby when using p-dichlorobenzene under the condition of in vitro which was suitable for sugarcane genomic in situ hybridization study on the count of chromosomal inheritance patterns research. Using the methods of α-bromonaphthalene under the condition of in vitro and 4℃ low temperature water under the condition of non in vitro, chromosome morphology was long and its centromere constriction was more obvious, so they both were suitable for chromosome mapping, karyotype analysis or other research. The effects of hybridization signal were better in the treatment of simultaneous denaturation at 80℃ and separate denaturation at temperature ranged from 70 to 80℃. Base on the screening results, separate denaturation at 80℃ was adopted to optimize an fluorescent in situ hybridization system. The effect of the system was detected using different pretreatment according the different aims. Pretreated with α-bromidenaphthalene, got the effect of longer, scattering chromosome, strong signal of 45S rDNA and high-resolution. Pretreated with p-dichlorobenzene, got the effect of shorter, scattering chromosome and uniform fluorescence signal. This study creates an important platform to following fluorescent in situ hybridization research, provides the technical support for utilization of closely relatives wild germplasm and the basic data for further cytogenetics research in the distant hybridization progeny materials.

MAR (matrix attachment regions) are specific DNA sequences that can combine with nuclear matrix. They are very conservative on the evolution, and they have a very important role in chromosome fold of the eukaryotic genomes. When the MAR sequences are constructed to both sides of the transgene, the expression of transgene will be increased and the differences among individuals can be reduced. Therefore, MAR have become one of the highlights research in the field of animals and plants gene engineering. This review was mainly introduced the following aspects of MAR：the characteristics, the distribution and function, the influence of transgenic animals and the possible mechanisms involved.

OsDSR4 is a gene of unkown function in DUF966 gene family, and the function of DUF966 family genes have not been reported until now. In this study, the bioinformatic analysis showed that the cDNA of OsDSR4 had 2 167 bp containing an open reading frame (ORF) of 1 149 bp, and it encoded a putative protein of 372 amino acids with a highly conserved DUF966 domain. The gene expression profile analysis indicated that OsDSR4 was expressed mainly in internode and leaf blade of rice(Oryza sativa L.), and it was repressed markedly by drought, salt and cold stresses, and induced significantly by abscisic acid(ABA). OsDSR4 was cloned using overlap extension PCR, and the fusion construct containing OsDSR4 was introduced into rice(Oryza sativa L. ssp. japonica) by Agrobacterium-mediated transformation method. Thirty-two OsDSR4-overexpressing transgenic plants were obtained and identified by PCR and qRT-PCR, which was demonstated that OsDSR4 had been integrated into rice genome and was overexpressed in some positive transgenic plants. These results establish the foundation for further study of the precise function of OsDSR4.

Molecular marker is DNA sequence which can directly reflect the nucleotide sequence variation among individuals and the genetic polymorphism. The identification of hybrid rice by molecular marker will provide strong evidence for the registration and protection of new cultivars, test of seed quality and evaluation of genetic resources. In this study, in order to develop new molecular markers and establish the corresponding molecular marker system, PCR was carried out with molecular markers ISSR (inter-simple sequence repeat) and SCAR (sequence-characterized amplified region) in some new blast resistant varieties such as rice (Oryza sativa) Junyou 522 (JY522) and other common rice accessions. Results demonstrated that the amplification of 17 out of 40 ISSR primers were good polymorphic with all 41 accessions. There were 65 out of 119 polymorphic bands ranged from 250~2 000 bp. The frequency of polymorphism was 54.6%. The combination of 4 SCAR markers (822-CGA-1, 40N23R, NBS2PI9 and Pibdom) deriving from the specific PCR products in ISSR primers could identify the authenticity and purity of JY522 effectively. The genetic similarity coefficient ranged from 0.53 to 1.00 among all accessions. The identification of hybrid rice by the combination of molecular markers will provide strong evidence for the registration and protection of new cultivars, JY522, test of seed quality and evaluation of genetic resources.

Natural-resistance-associated marophage protein 1 gene (Nramp1) plays an important role in controling body's immune regulation. To explore genetic variation and its relevant molecular markers in piglet diarrhea of the swine (Sus scrofa) Nramp1 gene, this research detected the polymorphisms of Nramp1 gene in five pig populations by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) technique and analyzed their associations with piglet diarrhea by adopting the generalized least squares method. Two alleles (A and T) and three genotypes (AA, AT and TT) were detected in the Nramp1 gene exon 2, two alleles (C and T) and three genotypes (CC, CT and TT) were detected in its intron 6. The distributions of genotypes were significantly different among Hezuo pig and other four pig breeds (Landrace, Large White, Duroc and Gansu Black pigs) (P＜0.01); the genotypic distributions of Hezuo pigs were significantly deviated from Hardy-Weinberg equilibrium in both exon 1 and intron 6 (P＜0.05), whereas in other pig populations were all consistent with Hardy-Weinberg equilibrium (P＞0.05). The least square linear contrasts suggested that diarrhea score of genotype AA was significantly higher than that of TT (P＜0.05), and extremely significantly higher than that of AT (P＜0.01) in exon 2; CC genotype had significantly higher diarrhea score than that of CT (P＜0.05). The results indicate that there are important effect on piglet diarrhea of different Nramp1 gene genotypes, which can provide basic data for the genetic makers of diarrhea anti-disease breeding.

M-phase promoting factor (MPF) is an important protease in regulating cell cycle progression, explore the expression of MPF in different stages of spermatogenesis in mice will provide insight into the molecular mechanisms of spermatogenesis. The object of this experiment was to expounded the chang of MPF activity in different ages of the mice(Mus musculus) and the relationship between MPF activity and sperm production, which by measuring MPF activity of testicular cells in different ages(8, 14, 18, 20, 28 and 56 d), using flow cytometry to analysis the DNA ploidy of germ cell and analysing the expressing of two subunits of MPF-CDK1 and Cyclin B1 by immunohistochemistry. The results showed that there was no tetraploid cells in the testis of mice before 18-day old, and then suddenly increased to the peak, and then gradually decreased with the age increased. Haploid germ cells appeared from 18-day old and the ratio of this type of cell enlarged rapidly at 20-day old, followed by a further increasing with the age increasesd. The trend of the MPF activity in different ages was similar to tetraploid cells, that was, the MPF activity reached the highest level at 18 d and then decreased, but it incresed once again at 56 d. From the results of immunohistochemistry, CDK1 and Cyclin B1 which was the subunits of MPF expressed in all testis of different ages, its expression increased with age and reached the maximum at 18 d and 20 d, followed by gradually decreased. These results indicate that, tetraploid cells begin to appeare in the testis of mice of 18-day old which indicating that the starting of spermatogenesis and the germ cells would go into primary spermatocytes stage of the first meiosis,while the MPF activity and the expression of its subunits-CDK1 and Cyclin B1 in the testicular tissue are highest at 18 d, suggesting that MPF may play an important role in regulating the meiosis process of mouse spermatogenic cells.

Hepatocyte nuclear factor 4α (HNF4α) is a member of the nuclear receptor superfamily, it plays an crucial role in liver organogenesis and hepatocyte differentiation. In order to investigate the role of HNF4α in the formation of goose fatty liver, we amplified the goose(Anser anser) HNF4α gene full-length cDNA sequence, and detected the expression levels in 8 tissues(heart, liver, spleen, lung, kidney, pectorale, crureus and abdominal fat) as well as the effect of overfeeding on HNF4α gene in liver by qRT-PCR. The results showed that the cDNA sequence of HNF4α gene was 1 371 bp and encoded 456 amino acids, and the amino acids had high homology with chicken(99.12%). In addition, HNF4α gene was mainly expressed in liver and kidney of goose, but it had lower expression level in other tissues. After overfeeding, the expression of HNF4α gene in liver was significantly decreased compared with the control group(P<0.05). The results indicated that the HNF4α gene might play an important role in lipid metabolism. It provides the basic dates for the further study that analysis the function of HNF4α gene during formation of geese fatty liver.

Robinia pseudoacacia L. is a common leguminous plant and the pioneer species for soil and water conservation, which plays a significant role in the maintenance of regional ecological environment. To better understand the diversity and phylogeny of rhizobia collected from nodules of Robinia pseudoacacia in different regions of Shaanxi Shangluo, We estimated genetic diversity using 16S rRNA PCR-RFLP and complete sequence analysis of 16S rRNA gene sequencing. Restriction fragment length polymorphism(RFLP) results showed that twelve genotypes were tested from 68 strains of six collection pointsand and differences existed in different strains . The Jaccard similarity coefficient between the strains isolated from Shanyang and those isolated from other regions were low while the strains isolated from Shangzhou and Luonan had rich diversity, with Simpson indices of 0.887 and 0.880, respectively, and Shannon-Wiener indices of 1.555 and 1.798, respectively. 16S rRNA sequencing revealed that these rhizobial strains belonged to Mesorhizobium. Through comprehensive analysis we could draw the following conclusions: Rhizobia isolated from Robinia pseudoacacia in Shangluo region showed some differences in different geographic sampling sites, and in the evolution the regional leguminous plants of Robinia pseudoacacia majorly selected mesorhizobia in symbiotic nodulation. This study provides basic information for the phylogeny as well as development and utilization of rhizobia in Shangluo.

Mineral nutrient assimilation and utilization play a critical role in rice yield. Reference genes suitable for nutrient deficiency must be selected, prior to qRT-PCR analysis of rice functional gene. In this study, a total of 12 rice housekeeping genes were selected as candidate reference genes for qRT-PCR of rice under nutrient deficiency. Total sample pool consisted of rice(Oryza sativa L. ssp. japonica) plants grown under 1-week and 2-week nutrient deficiency of 6 macro elements, including nitrogen, phosphorus, potassium, calcium, magnesium, sulphur. Stability of candidate reference genes under nutrient deficiency was analysed and reference gene eIF4a was identified as the most suitable gene using geNorm, and two genes with most stable expression were found to be optimal for reliable normalisation, based on the result of actual normalisation effect under different nutrient deficiency using various reference gene groups, reference gene selection was been proved to be of importance on qRT-PCR results under nutrient deficiency treatment of rice, and effectiveness of gene stability analysis using geNorm was further confirmed. Finally, single gene normalisation of eIF4a was validated as an improvement and complement for geNorm software. Taken together, all the results provide good reference for further research in qRT-PCR under nutrient deficiency.

Flavonoids are polyphenolic secondary metabolites, existing in maize(Zea mays L.). They possess natural antioxidant. It is important to comprehensively study the QTL mapping for fine mapping and positional cloning of pigmentation-related genes. In this paper, two connected F2:3 populations derived from crosses of Mu6×SDM and Mo17×SDM were used to identify QTLs for flavonoid content in maize kernels.The population was grown at Chongqing and Yunnan. A total of 8 QTLs were detected on two related populations. Eight QTLs were located on chromosome 1, 4, 7, 9 and 10, respectively explaining 5.27%~13.76% of phenotypic variation. One QTL located on chromosome 7 was linked with the same marker umc2332 at bin7.04 in two environments, and the QTL explained above 10% of phenotypic variation. These results suggest that the flavonoid content are controlled with a pattern of quantitative trait inheritance, and molecular markers closely linked with these QTLs can be applied in marker-assisted selection of pigment components in maize.

In order to confirm the popularity and mechanism of female fertility of triploid lilies(Lilium), Longiflorum-Asiatic lily Brindis(3x) and Pavia(3x) were used as maternal parents to cross with Asiatic lily Monte Negro(2x) and Tresor(4x) as male with normal pollination in the present paper. 21 fruits were obtained from 3 combinations, which were mostly developed well, while, the number of developmental embryos and endosperm from different fruits ranged from 6~42. Finally, 97 seedlings were obtained after embryo rescue within these fruits. The maternal parents Brindis and Pavia and their seven progenies were analyzed by using genomic in situ hybridization(GISH). The results showed that the seedlings were true progenies from the hybridizations. All of them were aneuploid whose chromosome numbers ranged from 36 to 63. The progenies contained different recombinant chromosomes from their two maternal parents. It further supports that, regardless of male sterility, triploid lilies can be used as maternal parents to cross with appropriate males to produce aneuploid progenies. Due to aneuploids may cause great variation in morphological and physiological traits, it is expected that allotriploid lilies can be used to create more variable new lily forms with foreign genes.

KANADI(KAN) genes are involved in controlling the dorsal-ventral polarity of plant lateral organs in Arabidopsis. For further research on the molecular mechanism of plant leaf polarity establishment, all members of KANADI family in Nicotiana benthamiana were cloned by RT-PCR method and their organ expression profile was examined. The Results showed that there were 8 KANADI gene family members in N. benthamiana genome, their encoding proteins shared around 70%~93% similarity to the homologs in Solanaceae and bear the specific conserved region and GARP domain that were found in plant KANADI protein family. Bioinformatic analysis indicated that NbKAN were non-secreted hydrophilic proteins without identifiable transmembrane structures. The results of Real-time qRT-PCR showed that NbKAN genes were present in all plant organs examined and they displayed different expression patterns.Therefore, the experiment provides the important basis for further study the relationships of KANADI gene function and other gene family.

Exploring the quantitative trait loci (QTL) of yield-related traits in melon (Cucumis melo L.) is helpful to the genetic improvement of melon yield. Total 221 melon landraces collected from various regions in China were used as experimental population in present study. The melon population was genotyped with 66 SSR markers that evenly distribute through the 12 melon chromosomes. On the basis of identification of population structure, genome-wide association study (GWAS) was applied to analyze the association between the SSR markers and the six yield-related traits, including fruit diameter, fruit length, thickness of fruit flesh, fresh weight of single fruit, 1000-seed weigh, and yield per plot. The results showed that the melon landraces were rich in variation at phenotype and molecular level, with phenotypic Shannon's index of 1.69 and SSR polymorphic information content of 0.576, and this population with diverse accessions was suitable for GWAS. All the accessions were divided into three subgroups by the mathematical model-based cluster in Structure software; the information on the subgroup was used for the next GWAS. The GLM (general linear mode) program in TASSEL software was run to make regression analysis of the phenotypic data and molecular marker data. Twenty four markers were found to associate with the six traits, of which 8 marker associations were significantly different at P<0.01. These markers distributed throughout the 11 linkage groups (LGs) besides LG 12, implying a complex genetic composition of the yield-related traits. Fourty seven marker-trait associations were detected herein, five for fruit diameter with an explanation of phenotypic variation (R2) range of 5.50%~14.08%, six for fruit length with a R2 range of 6.07%~14.22%, 7 for thickness of fruit flesh with a R2 range of 4.85%~11.22%, 11 for fresh weight of single fruit with a R2 range of 6.25%~14.22%, 4 for 1000-seed weight with a R2 range of 5.63%~10.50%, and 14 for yield per plot with a R2 range of 47.4%~16.08%. As for some SSR markers, each co-associated with two or more traits. Positioning of some markers in association map were in agreement with the results of QTL mapping in reported genetic maps, whereas some markers were complementary to those located in the genetic maps. From the present study, the association loci mentioned above as well as their adjacent genome regions could contain numerous QTLs that influence melon yield and the related traits. The marker-trait association loci developed in present study will offer an important information for molecular improvement of melon yield.

The microbial DNA of goat (Capra hircus) rumen was extracted and a metagenomic library of uncultured microorganism was constructed, which contained 12 100 clones. Functional screening of the library led to the isolation of ten clones expressing β-glycosidase activity and a clone designated pET-6 was subcloned. Sequencing analysis showed that there was a β-glucosidase gene umcel 6X with an ORF of 1 836 bp. The encoded product shared highest homology with a Dictyoglomus thermophilumH-6-12 (GenBank No.: YP_002251384) at 55% identity and 65% similarity. The umcel 6X was expressed in Escherichia coli though IPTG and the size of the translated product Umcel 6X on SDS-PAGE was in agreement with the predicated molecular mass. Zymogram analysis revealed that Umcel 6X exhibited β-glucosidase activity, confirming that the ORF encoded a β-glucosidase. The Umcel 6X, purifed with Ni-NTA column, showed optimal activity at pH7.5 and at 50℃. Ca2+, Na+ and Mg2+ had significant positive effect on the activity of Umcel 6X, while Cu2+, Fe2+ and Hg+ gave significant inhibitory effect on the enzyme. The Ni-NTA purified recombinant β-glucosidase Umcel 6X had a specific activity of 33.6 IU/mg at pH 5.5, 35℃. Therefore, the cellulase gene from rumen would provide materials for research of degradation crude fiber in vitro.

Maize(Zea mays L.) is the crop that has the highest area and yield in China since 2012. The incidence and prevalence of corn diseases affecting maize yield, especially southern corn rust and rough dwarf disease which broadly distribute southeast of China and Huanghuaihai maize zone, cause great losses. It is important to use the resistant germplasms for maize breeding. The bacterial arti?cial chromosome (BAC) cloning system is an invaluable tool in the cloning of disease resistance genes and in conducting structural and functional analyses. A BAC library for Qi319, the key source for disease-resistant maize breeding in China, was constructed in this study. Based on the modifications consisted of using etiolated cotyledon from 7 to 9 d growing plants as the DNA preparation source and addition of PVP-40 and β-mercaptoethanol in the extraction-washing buffer, we used 100 to 200 kb partial digested DNA fragments for 233 transformations. The fragments were ligated into CopyControl pCC1. The library contains 270 720 clones with an average insert size of 90 kb. Up to 1.33% of clones had no insert. Based on a haploid genome size of 2 300 Mb, the coverage of the library is about 10.43 genome equivalents, providing 99.99% possibility to isolate any maize gene or sequence in the library. No visible changes from the EcoRⅠ restriction patterns of four BAC clones between 20 and 100 generations indicated the stability of the BACs. In the future, the BAC library will serve as both a giant gene resource and an invaluable tool for map-based gene isolation, physical mapping and comparative genome analysis.

microRNA-124(miR-124), high expression in brain, regulate neuronal differentiation and the proliferation of cancer cells. The porcine miR-124 precursor is transcribed from chromosome 4 and 14. In this study, the miR-124 precursor was amplified by PCR from landrace genomic DNA, ligated into T vector, then digested by EcoR Ⅰand NotⅠ, the digested fragment were connected into PiggyBac(PB) transposon vector, then the recombinant vector (PB-miR-124a1, PB-miR-124a2) was transfected to procine kidney epithelial cells(PK15). The transfection efficiency was detected by expression of green fluorescent protein. The stable transfection cells were screened by puromycin, and the expression of miR-124 was determined by qRT-PCR. The results showed that the PB transposon recombinant vector (PB-miR-124a1, PB-miR-124a2) were constructed successfully with high transfection efficiency. The best stability screening concentration of puromycin was 2 μg/mL. qRT-PCR results showed that the expression of miR-124 in transfected PB-miR-124a2 group was increased significantly compared with control groups (P＜0.01), but in transfected PB-miR-124a1 group, the expression of miR-124 increased not significantly (P=0.06). These results demonstrated that the basal level of expression of miR-124 mainly transcribed from ssc-mir-124a2 gene. The study provides a foundation material for porcine miR-124 function research.