Rice Storage Proteins:Focus on Composition,Distribution,Genetic Improvement and Effects on Rice Quality

Rice storage proteins(RSPs)are plant proteins with high nutritional quality.As the second largest type of storage substance in rice,it is the main source of protein intake for people who consume rice as a staple food.The content and type of RSPs affect the appearance,processing quality and eating quality of rice.These effects involve the distribution of RSPs in rice grains as well as the interactions of RSPs with other components such as starch in rice grains.In the past two decades,some progress has been made in the genetic improvement of RSPs.However,the determination mechanism of protein content and composition in rice is still unclear,and the mechanism of the effect of RSPs on rice quality has not been elucidated.In this review,the composition,biosynthesis and distribution of RSPs,and quantitative trait loci mapping and cloning of RSP genes are summarized,the research progress of the influence of RSPs and their components on rice quality are reviewed,and the research directions in the future are proposed.

Genetic Diversity of Seed Storage Proteins in Different Ecotype Varieties of japonica Rice and Its Application

One hundred and fifteen varieties （including cultivars and lines） with different ecotypes in japonica rice （Oryza sativa L.） were analyzed for endosperm storage proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） to estimate their genetic diversity for the purpose of genetic improving and variety identification. Nineteen types of profile were identified according to 1） presence/absence of 65 kDa bands, 2） staining intensity of 70, 60, 57, 37-39, 22-23, 13 and 10 kDa bands, 3） migration velocity of 35 kDa （α-4） and 4） band number at 57 kDa location. An unweighted-pair group average method with arithmetic mean （UPGMA） dendregram based on the cluster analysis of genetic similarity of the protein bands showed a small genetic variation among the tested materials, with the similarity coefficients varying between 0.75 and 1.00. Three distinct groups were identified from the cluster analysis of the rice varieties studied at the similarity coefficient level of 0.894. The first group included eight varieties with high amylose content, the second group contained fifteen varieties with high protein content, and the third group had the remaining ninety-two varieties, which accounted for 80% of the total materials. Clear relationship between ecotypes distinguished by maturity and groups revealed by cluster analysis was not found in this study. Only the group of high amylose linked with medium-maturity medium japonica ecotype. The bands of 70 kDa and 65 kDa can be used as protein markers to identify F1 seed purity of japonica hybrid rice Liuyanyou 422.

Analysis of Genetic Variation of Seed Proteins in the Genus Vigna and among Its Relatives Cultivated in China

The genetic variation of seed proteins was assayed by SDSPAGE for 24 cultivars belonging to 5 species in Vigna and 7 species in its 7 relative genera cultivated in China. There were 48 polymorphic subunit bands discriminated from electrophoretic profiles. Two dendrograms were constructed by UPGMA cluster analyses using PHYLIP3.6 respectively. Variation among genera or species was larger than that among lower taxonomic categories level. Little variation among cuhivars of yardlong bean （Vigna sesquipedalis ） and small variation of lablab （ Lablab purpureus）, pea （Pisum sativum）, or sword bean （Canavalia gladiata）, but large variation of soybean or rice bean in their origin of China were all revealed. The seed proteins profiles of traditionally regarded as typical species in Vigna such as yardlong bean, rice bean and small bean were more similar than mungbean （Vigna radiata） and black gram （Vigna mungo） were. Mungbean and black gram had distinct seed proteins pattern, they should be of two species.

Roles of HIV-1 auxiliary proteins in viral pathogenesis and host-pathogen interactions

Active host-pathogen interactions take place during infection of human immunodeficiency virus type 1 (HIV-1). Outcomes of these interactions determine the efficiency of viral infection and subsequent disease progression. HIV- infected cells respond to viral invasion with various defensive strategies such as innate, cellular and humoral immune antiviral mechanisms. On the other hand, the virus has also developed various offensive tactics to suppress these host cellular responses. Among many of the viral offensive strategies, HIV-1 viral auxiliary proteins (Tat, Rev, Nef, Vif, Vpr and Vpu) play important roles in the host-pathogen interaction and thus have significant impacts on the outcome of HIV infection. One of the best examples is the interaction of Vif with a host cytidine deaminase APOBEC3G. Although specific roles of other auxiliary proteins are not as well described as Vif-APOBEC3G interaction, it is the goal of this brief review to summarize some of the preliminary findings with the hope to stimulate further discussion and investiga- tion in this exhilarating area of research.

The Nucleocytoplasmic Transport of Viral Proteins

Molecules can enter the nucleus by passive diffusion or active transport mechanisms, depending on their size. Small molecules up to size of 50-60 kDa or less than 10 nm in diameter can diffuse passively through the nuclear pore complex (NPC), while most proteins are transported by energy driven transport mechanisms. Active transport of viral proteins is mediated by nuclear localization signals (NLS), which were first identified in Simian Virus 40 large T antigen and had subsequently been identified in a large number of viral proteins. Usually they contain short stretches of lysine or arginine residues. These signals are recognized by the importin super-family (importin α and β) proteins that mediate the transport across the nuclear envelope through Ran-GTP. In contrast, only one class of the leucine-rich nuclear export signal (NES) on viral proteins is known at present. Chromosome region maintenance 1 (CRM1) protein mediates nuclear export of hundreds of viral proteins through the recognition of the leucine-rich NES.

Advances of Studies on the Viral Proteins of PRRSV

Porcine reproductive and respiratory syndrome( PRRS) is one of viral diseases with severe reproductive obstacle of pregnant sows and respiratory tract symptoms and higher mortality of piglets as characteristics,which is caused by porcine reproductive and respiratory syndrome virus( PRRSV). PRRS has brought great threats to swine industry in the world. The advances of studies on the viral proteins of PRRSV were reviewed from the genome,non-structural proteins and structural proteins of PRRSV.

Preparation and application of monoclonal antibodies against hepatitis C virus nonstructural proteins

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Prokaryotical expression of structural and non-structural proteins of hepatitis G virus

AIM To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnositic reagents.METHODS Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or)pGEX. and expressed in E. coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins.``RESULTS One clone with HGV fragment from core to El(Gl). one from E2 (G31), three from NS3 (G6, G61, G7),one from NS5B (G821) and one chimeric fragment from NS3and NS5B (G61 821) could be expressed well and showed obvious immunoreactivity by Western blotting.One clone with I-KGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins Gl, G,31, G61, G821 and G61 821were detected in indirected ELISA as coating antigen respectively. Only recombinant Gl could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera.Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification.``CONCLUSION Core to El, E2. NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high. yield recombinant protein (Gl) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.

Suppression of Breast Cancer Proliferation and Induction of Apoptosis via AKT and ERK1/2 Signal Transduction Pathways by Synthetic Polypeptide Derived from Viral Macrophage Inflammatory Protein Ⅱ

SDF-1α,a ligand for the chemokine receptor CXCR4,is well known for mediating the migration of breast cancer cells.In a previous study we demonstrated that a synthetic 21-mer peptide antagonist of CXCR4（NT21MP） derived from the viral macrophage inflammatory protein Ⅱ could antagonize tumor growth in vivo by inhibiting cellular proliferation and inducing apoptosis in breast cancer cells.However,the role of SDF-1α in the signaling pathways underlying the proliferation of human breast cancer cells and associated signaling pathways and inhibiting signal pathways of NT21MP remained unclear.The present study investigated the mechanism of NT21MP on anti-tumor in breast cancer in vitro.The effect of NT21MP on the viability of cells was determined by the MTT assay.Annexin V-FITC and PI staining was performed to detect early stage apoptosisin SKBR3 cells treated with SDF-1α and AMD3100 or NT21MP.Western blotting techniques were used to assay the composition of phosphoproteomics and total proteins present in the SKBR3 breast cancer cells.RT-PCR and Western blotting technique were used to detect the effect of NT21MP and AMD3100 on Bcl-2 and Bax expression.The results indicated that SDF-1α prevented apoptosis and promoted the proliferation of SKBR3 human breast cancer cells.As compared with untreated SKBR3 cells,Treatment with SDF-1α significantly increased cell viability,and NT21MP abolished the protective effects of SDF-1α dose-dependently（P0.05）.There was a significant decrease in the percentage of apoptotic cells after SDF-1α treatment as compared with control group（2.7%±0.2% vs.5.7%±0.4%,P0.05）.But pretreatment of SKBR3 cells with NT21MP significantly attenuated the antiapoptotic effects of SDF-1α as compared with SKBR3 cells without NT21MP pretreatment.The proliferative and anti-apoptotic effects of SDF-1α in SKBR3 cells were associated with an increase in AKT and ERK1/2 phosphorylation as well as a decrease in Bax expression and an increase in Bcl-2 expression.These changes in intracellular processes were blocked by NT21MP in a dose-dependent manner（P0.05）.In conclusion,NT21MP efficiently inhibits SDF-1α-induced proliferation and antiapoptosis in SKBR3 cells by reducing the levels of phosphorylated AKT and ERK1/2,as well as decreasing the ratio of expression of Bcl-2 relative to Bax.

Hepatocellular transport proteins and their role in liver disease

MOLECULAR PHYSIOLLGY OF HEPATOCELLULAR TRANSPORT PROTEINS Basolaferal transport systems Na+-dependent bile salt uptake Uptake of bile salts into the liver was first isolated perfused rat liver[1],isolated hepatocyte cultures and basolateral plasma membrane vesicles [2,4].

Genetic Polymorphism of Milk Protein and Their Relationships with Milking Performances in Chinese Yak

The milk protein polymorphisms were typed by polyacrylamide gel electrophoresis (PAGE)from 109 Maiwa and 100 Jiulong yaks, and the relationships among milk protein polymorphisms,milking traits and milk protein compositions were studied. The results showed thatβ-CN,κ-CN andα-La were monomorphic,αs1-CN andβ-Lg were polymorphic, the dominantgenes were αs1-CN D and β-Lg E,respectively. The frequencies of αs1-CN D were 0.8073and 0.6000 and β-Lg E were 0.9770 and 0.9700 in two populations respectively.The meanheterozygosities were 0.1021 and 0.1867 in two populations. No significant effects onmilking traits and milk protein compositions were observed except for αs1-CN locus onfat percentage in Jiulong yak.

Reverse genetics: Unlocking the secrets of negative sense RNA viral pathogens

Negative-sense RNA viruses comprise several zoonotic pathogens that mutate rapidly and frequently emerge in people including Influenza, Ebola, Rabies, Hendra and Nipah viruses. Acute respiratory distress syndrome, encephalitis and vasculitis are common disease outcomes in people as a result of pathogenic viral infection, and are also associated with high case fatality rates. Viral spread from exposure sites to systemic tissues and organs is mediated by virulence factors, including viral attachment glycoproteins and accessory proteins, and their contribution to infection and disease have been delineated by reverse genetics; a molecular approach that enables researchers to experimentally produce recombinant and reassortant viruses from cloned cD NA. Through reverse genetics we have developed a deeper understanding of virulence factors key to disease causation thereby enabling development of targeted antiviral therapies and well-defined live attenuated vaccines. Despite the value of reverse genetics for virulence factor discovery, classical reverse genetic approaches may not provide sufficient resolution for characterization of heterogeneous viral populations, because current techniques recover clonal virus, representing a consensus sequence. In this review the contribution of reverse genetics to virulence factor characterization is outlined, while the limitation of the technique is discussed withreference to new technologies that may be utilized to improve reverse genetic approaches.

Genetic Analysis of the P1 Region of Human Enterovirus 71 Strains and Expression of the 55 F StrainVP1 Protein

Enterovirus 71 (EV71) is a member of the Entero-virus genus of the Picornaviridae family and is the major cause of Hand, foot, and mouth disease (HFMD) in children. Different strains from Gansu were cloned and the P1 protein was sequenced and analysed. Results indicate that there are three kinds of EV71 infections prevalent in Gansu. The VP1 protein from one of these strains, 55F, was expressed. The recombinant protein was expressed with high level and reacted specifically with the EV71 patient antibody, the recombinant protein was also applied to raise antiserum in rabbits and after the fourth injection a high titer of antiserum was detected by ELISA assay. These data are useful for further clarification of prevalent EV71 strains in the north of China at the molecular level and provide a basis for EV71 diagnosis.

Efficient and Soluble Expression of α Protein of Clostridium perfringens and Preparation of Genetic Engineering Subunit Vaccine

[Objective] The paper was to develop genetic engineering vaccine that can express α exotoxin antigen protein efficiently without destroying its immunogenicity for preventing and controlling the diseases caused by Clostridium perfringens. [Method] Efficiently expressed soluble recombinant α protein was obtained from Escherichia coli expression system by optimizing codon,removing signal peptide,selecting sequences with better hydrophilicity and antigenicity,and optimizing expression conditions. [Result] Mice obtained higher serum antibody level when immunized by α protein,and the immune protection rates against type A,type B,type C and type D C. perfringens were 100%,90%,85% and 90%,respectively. The antibody titer of mice within 7-14 d after the third immunization reached the peak. [Conclusion]The α protein has good immunogenicity,and can be further used to develop genetic engineering subunit vaccines for preventing C. perfringens.

Intracellular Transport of HIV-1 Matrix Protein Associated with Viral RNA

HIV-1 matrix protein (MA) is a multifunctional structural protein localized on N terminus of Gag precursor p55. MA participates in HIV-1 assembly as membranotropic part of Gag precursor as well as an individual protein spliced from Gag early in infection. MA is found in the nuclei of infected cells and in plasma membrane, the site of virus assembly, in association with viral genome RNA. MA mutated variant M4 which contains two changed amino acids in N-terminal regions is also associated with viral RNA, but it is localized in the nuclear and cytoskeleton fractions but not in the plasma membrane suggesting that the mutant is deprived of membranotropic signal and “sticks” in the nuclei an d cytoskeleton, its previous location sites. These data allow suggesting that MA involved into transmission of viral RNA is transported to plasma membrane by cytoskeleton.

COVID-2019 Genome Sequence Analysis: Phylogenetic Molecular Evolution and Docking of Structural Modelling of Receptor Binding Domain of S Protein in Active Site of ACE2

Meanwhile the outbreak of the Covid-19 since December, 2019 in China, it has killed more than a hundred thousand of people of all ages and sex across the globe in a short span of time. On the bases of this study the nearest family member of the virus and its receptor binding domain of S protein including its model structure and function of its active sites were naked through Multiple Sequence Alignment, modelling and molecular docking software accordingly its repository genome databases. The virus was genetically associated and molecular evolutionary related with (RaTG13) and it scores 96.12% homology with 99% query coverage followed by bat-SL-CoVZC45 and bat-SL-CoVZXC21 notch 89.12% and 88.65% respectively. However, SARS and MERS corona type virus those outbreak earlier respectively less likely family members of 2019-nCoV. Though the virus has a close genetic association with those previous SARS coronaviruses, and certainly the spike protein used as a binding receptor to fight against human receptor protein of ACE 2, but on the basis of FRODOC and HDOCK server analysis multi favorable active sites of S protein was discovered such GLN493 shown as a finest key in both model and possessed a unique traits on it resulting unexpected rate of transmission and number of people died while compared to the previous one. TYR500, ASN501, GLN498 and others residues preferably contemplate site also. In particular, the diversity of the virus in the world may be due to the genome structure of the virus and S gene changed over the time, across the world against to host of human genetic diversity, which may be more robust, and may be a new and unique feature. This is because it is characterized close to contact with distance divergence between wild type novel coronavirus which was risen from China against to the genomes from Lebanon, India, Italy, and USA and so on. Thus, the World Health Organization and its researchers should focus on immunologic research and effective drug and vaccine development that will help to address the epidemiology of the virus, which can provide a long-term solution.

Genetic Characteristics of <i>Citrus Tristeza Virus</i>Isolates from Cultivated Citrus in China Based on Coat Protein Gene

Citrus tristeza virus (CTV) is an important citrus pathogen causing considerable economic loss to citrus production. Knowledge on genetic evolutionary of the CTV population in China remains limited. In this study, 1439 samples were collected from nine citrus-producing areas of China. The coat protein (CP) genes of CTV were amplified by RT-PCR, and sequenced to analyze the genetic evolution. Analysis of the base composition showed an AU preference pattern, with the GC content was lower than AU content. Nine CTV populations were clustered into one clade in neighbor-joining (NJ) tree, indicative of a close phylogenetic relationship among the populations in China. Analysis of molecular variation (AMOVA) revealed that 77.72% genetic variations of CTV populations were observed among populations, with an FST value of 0.223. The values of dN/dS and neutrality test of CP gene were ranged from 0.016 to 0.082 and -1.377 to 1.456, respectively, the results suggesting that all of nine CTV populations were relatively constantly maintained under purifying selection. Our study demonstrated the genetic characteristics and molecular evolution relationship of CTV populations in China, and provided a theoretical basis for scientific control of CTV.

Genetic polymorphisms of MAFK, encoding a small Maf protein, are associated with susceptibility to ulcerative colitis in Japan

AIM To investigate whether single nucleotide polymorphisms in maf protein K(MAFK), which encodes the MAFK, lead to increased susceptibility to ulcerative colitis in the Japanese population. METHODS This case control study examined the associations between MAFK single nucleotide polymorphisms(rs4268033 G>A, rs3735656 T>C and rs10226620 C>T) and ulcerative colitis susceptibility in 174 patients with ulcerative colitis(UC) cases, and 748 subjects without no lower abdominal symptoms, diarrhea or hematochezia(controls). In addition, as the second controls, we set 360 subjects, who have an irregular bowel movement without abnormal lower endoscopic findings(IBM controls).RESULTS The genotype frequency of rs4268033 AA and allelic frequency of the rs4268033 A allele were significantly higher in the UC cases than in both controls(P = 0.0005 and < 0.0001, P = 0.015 and 0.0027 vs controls and IBM controls, respectively). Logistic regression analysis after adjustment for age and gender showed that the rs4268033 AA and rs3735656 CC genotypes were significantly associated with susceptibility to UC development(OR = 2.63, 95%CI: 1.61-4.30, P = 0.0001 and OR = 1.81; 95%CI: 1.12-2.94, P = 0.015, respectively). Similar findings were observed by the comparison with IBM controls. In addition, the rs4268033 AA genotype was significantly associated with all phenotypes of UC except early onset. There was no significant association between rs10226620 and ulcerative colitis. CONCLUSION Our results provide the first evidence that MAFK genetic polymorphisms are significantly associated with susceptibility to UC development. In particular, rs4268033 is closely associated with an increased risk for the development of UC.

Investigation of Aegilops umbellulata for stripe rust resistance,heading date,and the contents of iron,zinc,and gluten protein

Aegilops umbellulata(UU)is a wheat wild relative that has potential use in the genetic improvement of wheat.In this study,46 Ae.umbellulata accessions were investigated for stripe rust resistance,heading date(HD),and the contents of iron(Fe),zinc(Zn),and seed gluten proteins.Forty-two of the accessions were classified as resistant to stripe rust,while the other four accessions were classified as susceptible to stripe rust in four environments.The average HD of Ae.umbellulata was significantly longer than that of three common wheat cultivars(180.9 d vs.137.0 d),with the exception of PI226500(138.9 d).The Ae.umbellulata accessions also showed high variability in Fe(69.74-348.09 mg kg^(-1))and Zn(49.83-101.65 mg kg^(-1))contents.Three accessions(viz.,PI542362,PI542363,and PI554399)showed relatively higher Fe(230.96-348.09 mg kg^(-1))and Zn(92.46-101.65 mg kg^(-1))contents than the others.The Fe content of Ae.umbellulata was similar to those of Ae.comosa and Ae.markgrafii but higher than those of Ae.tauschii and common wheat.Aegilops umbellulata showed a higher Zn content than Ae.tauschii,Ae.comosa,and common wheat,but a lower content than Ae.markgrafii.Furthermore,Ae.umbellulata had the highest proportion of γ-gliadin among all the species investigated(Ae.umbellulata vs.other species=mean 72.11%vs.49.37%;range:55.33-86.99%vs.29.60-67.91%).These results demonstrated that Ae.umbellulata exhibits great diversity in the investigated traits,so it can provide a potential gene pool for the genetic improvement of these traits in wheat.

牛病毒性腹泻病毒E2蛋白的真核表达及间接ELISA抗体检测方法的建立

为建立检测牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)抗体的间接ELISA方法,利用昆虫细胞真核表达系统成功表达E2蛋白,将纯化后的E2蛋白作为包被抗原,用方阵滴定方法对影响ELISA的各个因素进行优化,并进行特异性、敏感性和重复性试验。结果表明,在昆虫细胞中表达了BVDV E2蛋白,Western blot证实目的蛋白可与BVDV阳性血清发生特异性反应。ELISA优化结果显示,E2蛋白最佳包被浓度为0.5μg/mL,最佳封闭液为1%明胶,最佳血清稀释度为1∶400,最佳血清作用方式为37℃作用30 min,酶标抗体的最佳作用方式为1∶2000稀释、37℃作用30 min,最佳底物作用时间为室温20 min,阳性临界值为OD 450≥0.423。与血清中和试验法进行比较,总符合率为97.8%,板内和板间重复性试验的变异系数均小于10%。该方法与牛常见病毒阳性血清均无交叉反应。说明建立的间接ELISA抗体检测方法特异性、敏感性和重复性良好,可用于大批量样本的临床检测和流行病学研究。