Endogenous reference genes(ERGs) refer to a class of genes characterized by species specificity, low copies and allelic conservation. The specific PCR amplification of ERGs has important meaning for quantitatively detecting the foreign gene of transgenic crops. In this paper, after looking for present related article, 5 related endogenous reference genes (the rice root-specific gene(GOS9), the phospholipase D gene (PLD), the sucrose phosphate synthase gene (SPS), the rice starch branching enzyme gene (RBE4) and the Ubiquitin 5 gene(UBQ5)) and 2 exogenous reference genes(Cry1Ab and Cry1Ac/Cry1Ab) were selected. Transgenic rice KMD2 and TT51-1 were selected as template. By comparing the products of PCR of 5 related endogenous reference genes with the products of PCR of exogenous genes, the endogenous reference gene which PCR products are most close to exogenous gene PCR products was picked out. On the basis, more research on the selected endogenous reference gene was analyzed. Limitations in Real-time PCR applications to relative quantification of number of DNA targets had led to new developments such as the digital PCR(d-PCR) which allows accurate measurement of DNA copies without the need for a reference calibrator, so the ratio of copy number of KMD2/RBE4 and TT51-1/RBE4 were analyzed by digital PCR, Real-time PCR analysis on the amplification efficiency of two transgenic rice, the stability of PCR amplification and detection sensitivity. The results showed that compared to other endogenous reference genes, the amplification products of RBE4 was the most close to the amplification products of two exogenous genes, the ratio of copy number of two transgenic rice were close to 1∶1, 115.9% and 105.3%, respectively. The repeatability and stability of PCR system of RBE4 were also satisfied with the endogenous genes for quantify matrix reference materials of transgenic rice. In the case of the RBE4 TaqMan assay for two transgenic rice KMD2 and TT51-1, the limit of detection(LOD) were between 5 and 11 copies of TT51-1 and between 3 and 12 copies of KMD2 respectively, and the limit of quantitation(LOQ) are between 11 and 22 copies of TT51-1 and between 12 and 24 copies of KMD2, respectively. Therefore, RBE4 is as a proper endogenous gene for preparation of matrix reference materials of transgenic crops and quantitative detection of transgenic crops.

Rop(Rho-related GTPase of plant)is the plant-specific signaling small GTPase. It can start and terminate multiple cell behavious by converting its active guanosine triphosphate(GTP)-bound form and inactive guanosine diphosphate(GDP)-bound form. The interconversion of the two forms of Rop GTPases in plant is strictly regulated by three types of regulators: GTPase activating proteins (RopGAPs), guanine nucleotide exchange factors (RopGEFs) and guanine nucleotide dissociation inhibitors (RopGDIs). In this study, with the bait of CA-CaRop1 which is the GTP-bound form mutant of a Capsicum annuum L. Rop, CaRop1, we screened a regulator of Rho GTPase activating protein, designated as CaRopGAP3, from the prey library of the pepper seedling by ProQuestTM two-hybrid system. Blastp analysis showed that the deduced amino acid sequence of CaRopGAP3 not only had conserved domains of GAP but also had the Cdc42/Rac-interactive binding (CRIB) domain, which indicated that the CaRopGAP3 was a CRIB-containing RopGAP subfamily that was unique to the regulation of the Rop GTPase of plant. Yeast two-hybrid analysis showed that CaRopGAP3 could interact with constitutively active(CA)-CaRop1 but not with dominant negative(DN)-CaRop1, and the interaction was not obviously regulated by its CRIB-containing N-terminus. Subcellular localization analysis showed that the full-length CaRopGAP3 focused mainly on the cytomembrane while the truncated mutant that the CRIB-containing N-terminus was deleted dispersively distributed in the whole cytoplasm, which indicated that the CRIB-containing N-terminus may play important role in the membrane localization of CaRopGAP3. Real-time PCR analysis showed that the expression level of CaRopGAP3 in different tissues and organs were obvious different, the level in the young leaf was highest and was about 17-fold higher than that in the young root, 8-fold higher than that in the mature root and flower. The structure characterizations and the expression pattern of the CaRopGAP3 may be closely associated with the signaling pathway it involved, and may be important for clarifing the signaling regulation mechanism of the CaRop1 in pepper further.

In order to establish a simple and efficient anti-serum preparation method for molecular biology research, in this research, porcine circovirus type Ⅱ capsid protein gene was selected to research on the possiblity of preparing high titer anti-serum by hydrodynamics gentic immunization. Endotoxin free plasmid of pcDNA-Cap, with would express porcine circovirus type Ⅱ capsid protein in the exkaryotic cell, was prepared using endotoxin free plasmid preparing kit and was delivered by hydrodynamics tail vein injection method for genetic immunization of mice(Mus musculus). After 5 times continuous immunization, the blood serum was collected. Using porcine circovirus type Ⅱ capsid protein which has been deleted its nuclear laction signal sequence at its N-terminal and expressed in Escherichia coli as antigen, the prepared anti-serum was tested by enzymeliked immuno sorbent assay(ELISA) and Western blot. The results showed that the anti-serum prepared by hydrodynamics tail vein gene delivery method had very high titer(1∶1 000 000) by ELISA and could detect at least 10 ng antigen after 5 000-fold dilution in Western blotting analysis. These results showed that hydrodynamics gene delivery method may become an efficient method for preparing antibody which will be used in porcine circovirus type Ⅱ related research. This study provides an alternative method for prevention the disease caused by porcine circovirus type Ⅱ.

The cannabinoid receptor type 1 (CNR1) is a key component of the endocannabinoid system, which has been reported to play a pivotal role in modulating feeding behavior and energy balance. In order to further study on gene function of CNR1, this study was conducted to construct and identify CNR1 gene small interfering(siRNA) expression vectors and screened the stable CNR1- interference positive L6 cell clones. Three pairs of CNR1-specific double-strand siRNAs were designed and inserted into the pYr-1.1vector. The CNR1 gene siRNA expression vectors were identified by restriction enzyme digestion and sequencing. After that siRNAs were transfected with L6 cells by LipofectamineTM(Lip)2000. Then, the transfection efficiency was detected by EGFP and FCM. CNR1 gene expression was determined by Real-time PCR and the stable transgenic L6 cell clones were screened by G418. The results revealed that the CNR1 gene siRNA expression vectors have been constructed successfully. The transient transfection efficiencies of L6 cells were 10.45%(P<0.01), 8.57%(P<0.01)and 8.71%(P<0.01) respectively, and the silencing efficacies of the transient transfected L6 cells were 39%(P<0.05), 64%(P<0.01) and 68%(P<0.01), respectively. The optimal selection concentration of G418 for stable transfected L6 cell clones was 800 μg/mL. The silencing efficacies of CNR-1-positive transgenic cell clones were 43%(P<0.05), 78%(P<0.01) and 91%(P<0.01), respectively. The results showed that CNR1-3 expression vector was optimal silencing vector and CNR1-3 stable transgenic cell clones were best silencing cell line. This study successfully provides CNR1 gene silencing method by siRNA and the screening of CNR1-interference positive L6 cell clones renders basic tools for further studying the functions of CNR1 gene.

BIO ORGANS(BIO)是百脉根中调控花器官形态和器官大小的基因，为深入研究其基因功能，利用根癌农杆菌介导法把BIO及其突变的截断基因bio定向导入烟草(Nicotiana tobacum)中，共获得18株转BIO基因烟草植株以及21株转bio烟草植株。转BIO及转bio植株表型具有一定的相似性：与野生型相比，转基因植株的生长周期显著缩短；接近一半的叶片出现缺刻或呈现一个叶片向两个叶片分裂的趋势；花冠颜色变浅，花器官大小、数目、形态等都表现出了一定的变化。不同的是，与野生型相比，转BIO植株明显矮化，器官变小；而转bio植株株高变高，器官变大。通过表型分析显示，BIO基因参与了植株形态建成的多个方面，包括植株的高度、叶片以及花器官的形态等，同时推测bio基因缺失的一段序列可能对调控器官大小起着重要的作用。对BIO及bio测序后进行序列比对，结果显示其基因序列及蛋白序列与豆科植物大豆(Glycine max)、蒺藜苜蓿(Nledicago truncatula)中的部分序列相似性均达到90%以上。该研究为下一步探索BIO基因的调控机理以及与其他基因的互作关系提供了实验依据。

Growth hormone is one of the most important endocrine hormones that can affect animal growth. In order to explore the influence of exogenous growth hormone on growth, skeletal muscle hyperplasia and hypertrophy of Nile tilapia (Oreochromis niloticus), the same batch of young fish were seperated into two groups: The experimantal group which was injected with exogenous recombinant human growth hormone(hGH) in muscle once a week, and the control group which was injected with phosphate buffer(PBS) only. The body length and weight of the fish were measured weekly, skeletal muscle fibers number and area in the first dosal myomere were analysed by means of paraffin tissue sections once every two weeks. The results showed that: (1) Body length and weight of the experimental group were bigger than that of the control group since the first week, but the difference was not significant(P＞0.05). however, significant variation were observed from the 5th and 7th week for body weight and body length(P＜0.01 or P＜0.05), respectively. (2) Fibers number and area in the experimantal group were also bigger than that in the control group since the first week, exogenous hyperplasia and hypertrophy were detected obviously in the experimental group from the 5th week (P＜0.01). These results suggested that exogenous growth hormone can promote growth in body length, body weight, as well as in skeletal muscle, hypertrophy growth is more dominant than hyperplasia growth in skeletal muscle growth of Nile tilapia.

Streptococcus pneumoniae is the importance germ to influence macaques's health. In order to investigate the expression and distribution of secretory immunoglobulin A(sIgA) protein and mRNA in lung and trachea of macaque infected Streptococcus pneumoniae spontaneously, immunohistochemistry and in situ hybridization were used. Results showed that the positive area of sIgA protein in alveolar septum were extremely higher than that of normal group (P<0.01), while in pulmonary vascular wall the difference were significantly higher (P<0.05), the optical density and positive area of sIgA in tracheal epithelium and lamina propria were higher than that of normal group (P<0.05). The expression of sIgA mRNA in alveolar septum, pulmonary intravascular and vessel wall were extremely higher than that of normal group (P<0.01), while in tracheal epithelium was significantly higher (P<0.05); compared with normal group, the optical density in tracheal epithelium and muscular layer were significantly higher than that of normal group (P<0.01), and it was higher in lamina propria of trachea (P<0.05). It can be concluded that sIgA participated the immunomodulation process of Streptococcus pneumoniae infection in lung and trachea. The results give us a new ideas to establish directional treatment and immune way.

To investigate immuno-enhancement activity of chicken interleukin-2 (IL-2) to Newcastle disease virus (NDV) F protein, NDV F gene was linked with chicken IL-2 via 7-amino acid glycine-rich linker(G2SG3S) by SOE-PCR(splicing by overlap extension-PCR) technique and then inserted into pcDNA3.1(+) and constructed the recombinant plasmid pcDNA-IL2-F which was transfected into chicken embryo fibroblast cells with liposome. The recombinant poteins were expressed correctly with molecular mass of 76.0 kD and 59.6 kD. The results of Western blotting analysis indicated that the interest proteins(pcDNA-IL-2-F and pcDNA-F) were recognized specifically by antisera against NDV. SPF chickens(Gallus domedticus), and were divided into 5 groups and immunized by recombinant plasmid pcDNA-IL-2-F, pcDNA-IL-2 mixing pcDNA-F, pcDNA-F, Lasota vaccine and plasmid pcDNA-3.1(+), respectively. The sera of each group were collected weekly for detecting antibodies of immunized chickens by ELISA. The results suggested that SPF chickens immunized pcDNA-IL-2-F exhibited stronger antibody response(P <0.05) than that of both the group immunized mixing pcDNA-F with pcDNA-IL-2 and the group immuned pcDNA-F. The SPF chickens were challenged with NDV F48E9 on the 20th day post immunization and the protective rate of the group immunized pcDNA-IL-2-F was higher than that of other plasmid groups. These results revealed that expression of tandem genes of NDV F and Chicken IL-2 could provide an optional immune response that in turn protected chickens from NDV challenge than that of sigle NDV F gene. In general, the vaccine strategy described in this study can provide a good platform for future desire of virus vaccine.

Sucrose phosphate synthase (SPS, EC 2.4.1.14) is a key enzyme for sucrose metabolism in plant. In sugarcane, at least 5 genes were classified into 4 subfamilies of SPS. Of the 5 SPS genes, SPSⅢ has high-level expression in the mature stem tissue of sugarcane, which is considered to be a Poaceae-specific member. Therefore, the study of binding protein genes of Cis-element in promoter region of SPSⅢ would provide initial insight for regulation of sucrose metabolism. In this study, to specific analyze the function of ATCT-motif and CAT-box from SPSⅢ in sugarcane (Saccharum spp.) for potential Cis-element of light response ATCT-motif and meristem-specific, the fragment containing ATCT-motif and CAT-box from SPSⅢ promoter region (from －1 320 to－1 210 bp) were cloned into T-vector, then, yeast one-hybrid bait vector pAbAi-SPSⅢ-AC were constructed and transformed to yeast. Subsequently, the ds-cDNA from mature leave of sugarcane were transformed to the yeast one-hybrid bait vector for constructing the Yeast One-hybrid Library System, and the insert sizes of the library were identified to be ranged from 0.25 kb to 1.5 kb. Fifty four positive clones of the candidate binding protein cDNA sequences were obtained from the Yeast One-hybrid Library, of them, 14 sequences were observed to be unique based on sequencing. Blast analysis showed that, besides E1-3, E9-1, and E0-3, the other 11 cDNA sequences had high homology to the close relatives of sugarcane with identities above 90%. E0-3 was found no Blast hit in the NCBI, which could be undiscovered gene or sugarcane specific gene. Furthermore, the potential Cis-element sequences were analyzed through SMART and SBASE. The results showed that five of the sequences, E0-3, E2-3, F2-1, F4-2 and G8-2, contained regulated motif domain. E2-3 was assumed to be a light-regulated element for SPSⅢ-AC due to the photo morphogenesis relative domain transcript presented in the corresponding amino acid sequence. BURP domain was proved to take part in the regulation of photosynthesis in bacterial. Based on the analysis of conserved domain by Blastx, a BURP domain with CH-X(10)-CH-X(25-27)-CH-X(25-26)-CH motif was found in sequence F2-1. Both of E0-3 and F4-2 contained AT-hook motif, which is DNA binding element in plant and was demonstrated to play roles in photosynthesis and the regulation of relative target genes. G8-2 belongs to super gene families of P-loop_NTPase with a conserved phosphate binding motif. The other ten sequences were found no homologous sequence relative to transcript factors. This study provides a frame for further investigating sucrose mechanism and gene engineering for sugar improvement of sugarcane.

Defensins are antimicrobial peptides which play important roles in the innate immune system of mammals. β-defensin-1(pBD-1) and β-defensin-2(pBD-2) are two defensins of great importance in swine. The information on defensin activity and function is very scarce. To study their antimicrobial and antioxidant activities, pBD-1 and pBD-2 were chemically synthesized, and their antimicrobial activities against eight gram-negative bacteria and three gram-positive bacteria were determined by minimal inhibitory concentration(MIC). The mechanisms of antimicrobial peptide activity were preliminary inquiried by transmission electron microscope, and the antioxidant activities in vitro were also studied. The results showed that pBD-1 had strong inhibition against Escherichia coli EPEC O78:K80 and Bacillus subtilis ATCC 6633, which MIC were both 32 μg/mL and minimal bactericide concentration(MBC) were 32 μg/mL and 128 μg/mL, respectively. But pBD-2 had strong inhibition just against Pseudomonas.aeruginosa CMCC 10104, which MIC was 8 μg/mL and MBC was 32 μg/mL. Transmission electron microscopy results showed that pBD-1 and pBD-2 might kill bacteria through the formation of transmembrane pore. pBD-1 and pBD-2 showed radicals scavenging activities and reducing power. Furthermore, the antioxidative activities of them were stronger when the concentrations were higher within certain concentration ranges. In conclusion, the research results indicated that pBD-1 and pBD-2 not only had antimicrobial activities but also had antioxidant activities in certain extent，and this may provide data base to clarify their biological functions and their development and application in animal husbandry.

The dehydration responsive element binding protein(DREB) transcription factors play important roles in improving plant tolerance of abiotic stresses through regulating the expressions of the stress-related genes. To study the characteristics of drought resistance of the transgenic plant，the expression patterns of transcription factor W16 were analyzed in wild type and backcross lines of transgenic wheat(Tritium aestivum) cultivated in pots under different water stress treatments from tillering to heading stages, and the physiological and biochemical indexes related to drought tolerance were investigated. Semi-quantative RT-PCR analysis revealed that the expression of W16 was weak under no water stress condition, and its expression was up-regulated along with the enhancement of the water stress and reached the peak(12 h), then its expression dropped rapidly at serious water stress, which displayed a curve of "up-max-down" in wild type. Whereas the expression of W16 under the control of Ubiquitin promoter was strongand stable in transgenic wheat during the whole water stresses. The analysis on the physiological and biochemical indexes related to drought tolerance indicated that the changes of cholorophyll content, proline content, soluble protein content and water use efficiency of the transgenic lines were significantly higher than that of wild type under different water stresses, having significantly more dissimilarities especially under severe drought stress(SD), exceeding maximally 8.48% in chlorophyll content, 124.72% in proline, 37.74% in soluble protein content and 32.75% in water use efficiency of the controls and being more sensitive to water in jointing stage. Soluble protein content increased under moderate drought stress (MD) and decreased under severe drought stress (SD). The yield of transgenic lines were higher than that of wild type under different water stress, increasing more significantly under drought stress. Respectively, the yields of the strains A29-15 and A31-8 increasd 4.00% and 6.22% under moderate drought stress (MD), and that of the strains A29-15 and A31-8 increasd 11.16% and 16.27% under serious water stress, and their drought tolerant index was in the category of strong drought tolerance. Our results showed that over expression of W16 improves physiological and biochemical characteristics related to drought, and improves drought resistance in the backcross lines of transgenic wheat, achieving the goal of increasing production.

Trehalose-6-phosphate Synthase(TPS) is a key enzyme in the synthesis of trehalose in plants. At present, researches about TPS have mainly focused on bacteria and fungi but little about plants. An EST, having high homology with TPS gene from other organisms, was screened from the whole organic transcriptomic library of tea (Camellia sinensis (L.) O. Kuntz) and amplified through RACE technology to obtain the cDNA full-length of trehalose-6-phosphate synthase gene, named CsTPS (GenBank accession number: JQ742017). The cDNA full-length of CsTPS was 3 125 bp with a single 2 799 bp opening reading frame that predicted to encoded a 932 animo acid, which contained two obvious structure domains, TPS and TPP, through multiple sequences alignment. Phylogenetic tree indicated that the deduced animo acid sequence of CsTPS gene had a very high identity with TPS genes from other organisms such as Arabidopsis thaliana, Nicotiana tobacum and Solanum lycopersicum. And the holomogy between CsTPS and AtTPS1 genes was higher than that of AtTPS2 and AtTPS3. Quantitative Real-time PCR analysis showed that differential expression level of CsTPS gene existed in different organs and upregulation of CsTPS gene induced by low temperature in mature leaf and immature leaf were obviously higher than that in roots, which revealed that CsTPS gene may involve in cold-resistant mechanism of tea plant.

Brown cotton processes the natural brown fiber color, without dyeing during spinning and processing process, which is really the "ecological", "environmental protection" cotton. Molecular cloning and understanding of the brown pigment synthesis-related gene in cotton fiber is very important to reveal molecular mechanism in the formation and accumulation of brown pigment. The brown fiber Asian cotton (Gossypium arboreum) Cixizimian and white fiber Yuyaozhongmian were selected in this study. Cotton fiber total RNA was extracted from the cotton fiber different development period. Ultimately, only one expressed fragment of 500 bp was obtained from the cDNA of brown cotton fiber. With sequencing and bioinformatics analysis, one gene named GaTT12a (GenBank accession number: JX013908) was cloned using the RT-PCR method. The sequence of the gene showed 75% homologous with testa brown pigment synthesis TT12 gene of Arabidopsis thaliana in amino acid sequence. The gene encoded 490 amino acid residues with the molecular weight of 52.7 kD, belonging to MATE super-gene family. Real-time quantitative PCR analysis of the gene showed that the gene presented super-dominant expression both in the brown cotton fiber, testa of brown and white cotton, while which not detected in white fiber. The result indicated that The GaTT12a gene was involved in the formation of brown pigment in cotton fiber, the result further suggested that there was the same synthesis metabolic pathway of the brown pigment in both testa and fiber. The above result gives the fundamental information for further understanding its function participating in the formation of brown pigment of cotton, the relationship of brown pigment of cotton fiber and brown pigment of seed coat.

Wnt3a is an important secreted protein of the canonical Wnt pathway and its activation can promote cell proliferation and division. In order to reveal the roles of Wnt3a in regulating the proliferation and differentiation towards insulin-secreting cells of porcine pancreatic stem cells (PSC), the full length of Wnt3a cDNA fragment was amplified from pGKS2P(+)-Wnt3a plasmid by RT-PCR. The recombinant plasmid pIRES2-AcGFP1-Wnt3a was constructed by inserting Wnt3a fragment sequenced correctly into eukaryotic expression vector pIRES2-AcGFP1. After identifying with double digestion of EcoRⅠ/BamHⅠ, we transfected pIRES2-AcGFP1-Wnt3a into porcine PSC by lipofectamin 2000. We obtained the stably transfected pocine PSC strain after screening culture by G418 for 3 weeks. Almost all cells expressing green fluorescence protein were observed by fluorescence microscope. The expression of Wnt3a mRNA and protein obviously increased in stable transfection group of Wnt3a compared to control group when detected by RT-PCR and Western blot. These results indicated that we successfully established the porcine PSC strain stably expressing Wnt3a. This study provide basal data for further exploring the mechanism of Wnt3a in porcine PSC proliferation and differentiation.

To find a suitable feeder layer for successful culture conditions of bovine embryonic stem cells is important. SIM mouse(Mus nusculus) embryo-derived thioguanine and ouabain resistant(STO) cells were treated by domestic mytomycin in different concentrations, which were used as feed layer cells to culture bovine embryonic stem cell. STO cells were treated with domestic mytomycin under 10, 15, 20, 30 and 40 μg/mL for 1.5, 3.0 and 4.0 h, respectively, and growth states of STO cells were observed in difference generations. Bovine embryonic stem cell cultured in the STO feeder layers were detected, including expression of alkaline phosphatase(AKP), octamer-binding transcription factor 4(OCT-4) and stage specific embryonic antigen 1 (SSEA-1), and formation of embryoid bodies in vitro. The results indicated that domestic mytomycin (under 15 μg/mL for 3.0 h) could inhibit effectively the division of STO cells, but not affect vitality of STO cells. Compared with conducts of Sigma, there were have the same effects, and there we not only could content the demand of preparation of feeder layers, but also could reduse the experiment costs. At the same time this research found that STO cells could be passaged proliferation in vitro long-term. However, STO cells affect the morphological structure which passed on long-term, eventually leading to the decline in the quality of feeder layers. The morphological changes of STO cells after continuous passaging indicated that of the STO cells was the better growth states than that of others generations before five generations, specially the fifth generation. Spreaded to the tenth generations, the cells occured degenerative changes and STO cells of dead rate obvious reaching to fourteenth generations. From the first to the tenth generations could been best used for feeder layers. Representative immuonofluorescence staining in the eighth passage of bovine embryonic stem cells were used. Results showed bovine embryonic stem cells cultured on STO feeder layer cells from the fifth generation to the tenth generation were in good morphology, and expressed AKP, antigen of OCT-4 and SSEA-1 in positive strongly fromed embryoid bodies. All results suggest that domestic mytomycin could inhibit effectively the division of the STO cells, and the STO cells can be used as conventional feeder layer for bovine embryonic stem cells culture.