RNAi Plasmid Construction of PttGA 20-oxidase Gene

[Objective] Genetic Engineering technology was used to regulate the expression of PttGA20-oxidase gene thus restrained plant height growth and internode elongation for cultivating dwarfed plant.[Method] Based on the RNAi principle,the gene specific sequences of PttGA20-oxidase in the antisense and sense orientations interrupted by a gene sequence from GUS were cloned into a binary vector pBI121.The selection marker gene npt Ⅱ was replaced with bar gene to RNAi plasmid.[Result] After undergone different endonuclease restrictions,the constructed constraint vector released different segments whose sizes were similar to that of target segment,which demonstrated that the RNAi plasmid of PttGA20-oxidase gene was successfully constructed.[Conclusion] The experiment provided a new way for culturing dwarfed plant.

Effects of L-lysine·H2SO4 product on the intestinal morphology and liver pathology using broiler model

Background: Lysine is used widely in livestock production due to the shortage of feed protein resources.Llysine·H2SO4 contains L-lysine sulphate as well as fermentation co-products which contain other amino acids and phosphorus.However,there are few articles about L-lysine·H2SO4 product regarding intestinal morphology and liver pathology of broiler chickens.In this article,we focus on the absorption and metabolism of L-lysine·H2SO4 revealed in the variation of intestinal morphology and liver pathology to determine the tolerance of chicks for L-lysine·H2SO4.Methods: To evaluate the tolerance of broilers for L-lysine·H2SO4,240 one day old broilers were allocated randomly to one of five dietary treatments which included corn-soybean diets containing 0,1%,4%,7% or 10% L-lysine·H2SO4(L-lysine content = 55%).Results: Supplementation of 1% L-lysine·H2SO4 in the diet had no negative effects.However,4%,7% or 10% Llysine·H2SO4 supplementation produced negative responses on broiler performance,carcass characteristics,blood biochemistry,and particularly on intestinal morphology and liver pathology compared with broilers fed the control diet.Conclusion: Our results show that supplementation with 1% L-lysine·H2SO4 had no negative effects on performance,carcass characteristics,blood biochemistry,intestinal morphology and liver pathology in broilers,but supplementation with 4%,7% or 10% L-lysine·H2SO4 produced a negative response,particularly with respect to intestinal morphology and liver pathology.

Status and Advances of Researches on GA 20-oxidases

GA 20-oxidase, the most important limiting enzyme, can catalyze a series of oxidization of GA biosynthesis pathway from GA12 to GA9 and from GA53 to GA20 in the higher plants. This paper reviews the studies on the characters of GA 20-oxidase, the gene and the protein of GA 20-oxidase and the regulation of GA 20-oxidase gene expression in recent years. At the same time, the prospects for the gene transformation of GA 20-oxidase in agriculture, forestry and horticulture are also discussed.

Characterization and Expression Profiling of Genes Encoding the Gibberellin 3-oxidase in Dasypyrum villosum Dwarf Mutant

Gibberellin 3-oxidase catalyzes the conversion of inactive gibberellin(GA) species into GAs with biological activity and it is subjected to strict developmental controls in the life cycle of a plant. In this study, 33 gene sequences, encoding the gibberellin 3-oxidase(GA3ox) from Dasypyrum villosum and its dwarf mutant, were obtained. Each contained a 1 107 bp coding sequence(CDS) that encoded a putative protein containing 369 amino acids. The GA3ox protein showed 77% to 97% homology and shared the major conserved structural domains of GA3ox proteins with rice, sorghum bicolor, oat, barley, and wheat. Sequence alignment showed that there were 20 single nucleotide polymorphisms(SNPs) and 22 Insertion/deletions(In Dels) among these sequences, which could be divided into 2 haplotypes, haplotypes Ⅰ and Ⅱ. Haplotype Ⅰ was found in the wild type and was1 495 bp in length, and haplotype Ⅱ was found in the dwarf mutant and was 1 485 bp in length. The Q-PCR results showed that GA3ox was expressed in the leaves, roots, internodes, and stem nodes, and that there was a significant difference in the transcript level of the GA3ox between the wild type and dwarf mutant. The transcript levels of GA3ox in the leaves at the seedling stage, stem elongation stage and the heading stage, in the root and stem nodes at the stem elongation stage and in the internodes at the heading stage of the wild type, were significantly higher than those in the dwarf mutant. However, GA3ox expression in the rest of the wild type tissues at the 3 stages was slightly higher than or not different from the dwarf mutant.The results suggested that the wild type and mutant allele sequences of GA3ox in D. villosum showed 2 amino acid changes in exons and variations in the lengths of introns or the SNPs in introns, which most probably impaired the function of the enzyme,affected the GA3ox expression level, and eventually gave rise to dwarfing.

Escherichia coli Protoplast Fusion and Screening of High L-Lysine-Producing Strain

[Objectives]Protoplast fusion of two parental strains with weak L-lysine production ability was carried out to obtain new fusion strains with strong L-lysine production ability.[Methods]The effects of bacterial age,lysozyme concentration,enzymolysis temperature and time on the protoplast formation rate and regeneration rate were investigated by single factor experiments.On this basis,with the protoplast formation rate as an index,the protoplast preparation process was optimized by an orthogonal experiment.[Results]Bacterial age and enzymolysis time had a greater impact on the protoplast formation rate,followed by enzymolysis temperature and lysozyme concentration.The optimal process for the preparation of L-lysine-producing Escherichia coli protoplasts was to prepare parental protoplasts from bacterial cells cultured for 15 h in the late logarithmic growth phase by enzymolysis with 0.8 mg/ml lysozyme at 37℃for 180 min and promote fusion with PEG6000.In order to facilitate the screening of fusion protoplasts,the empty plasmids p ET-28a and p ET-Duet were transformed into L-lysine-producing E.coli,respectively,and strains p ET-28a-lys01 and p ET-Duet-lys01 were obtained.Fusion strains were then obtained through protoplast fusion.Double-resistance KA1-10were screened on plates containing kanamycin and ampicillin,and a high-yielding fusion strain KA8,which produced L-lysine,was screened by fermentation experiments finally.[Conclusions]The results of this study provide a reference method for further improving the yield of L-lysine and other amino acid strains.

Effect of L-lysine in culture medium on nodule formation by bone marrow cells

The aim of this study was to estimate the effect of L-lysine on nodule formation by rat bone marrow cells in vitro. In this study, L-lysine was added to medium for mesenchymal stem cell culture to promote proliferation and differentiation of the cells, and then nodule formation was estimated in an in vitro rat bone marrow cell culture. Bone marrow cells from the bone shafts of the femora of Fischer 344 rats were cultured in minimum essential medium with 20 μl of L-lysine solution at 10﹣4, 10﹣5, 10﹣6, 10﹣7 or 10﹣8 M. Dexamethasone was also added to the medium at 10 nM for differentiation of stem cells from bone marrow into osteoblast progenitor cells. The subculture was performed for 2 weeks. The quantity of osteocalcin in rat bone marrow cell culture with dexamethasone was 392 ng/ml. In the medium with dexamethasone and 10﹣8 M L-lysine, the quantity of osteocalcin was 437 ng/ml. Nodules only formed upon addition of 20 μl of L-lysine at 10﹣8 M. It was indicated that 10﹣8 M L-lysine should be the optimal concentration for calcification. For nodule formation by rat bone marrow cells in vitro, the optimum concentration of L-lysine in culture medium might be 20 μl of 10﹣8 M. L-lysine could play an important role in matrix production for bone formation in vitro.

Dissolution of highly molecular weight cellulose isolated from wheat straw in deep eutectic solvent of Choline/L-Lysine hydrochloride

Green solvents for cellulose dissolution is a key topic for green chemistry,especially natural cellulose with high molecular weight,and there are scarce solvents reported.Deep eutectic solvent(DES)is a typical kind of green solvent that has been attracted much attention recently.Here,high molecular weight natural cellulose(DP>3000)was first isolated from wheat straw and then be directly dissolved in the choline/L-lysine(Ch/Lys)DES.The solution owns excellent stability,and the solubility reaches^5%.Rheological studies revealed that the natural cellulose can be well dispersed in the DES solution and showed gelation at high concentrations.The dissolved cellulose can be regenerated when the dilute acid aqueous solution was added into the solution.It provides an energy conversation and an environmentally friendly route to prepare a cellulose solution,which makes it possible to convert cellulose to valuable chemicals and materials in its homogeneous solution.

A NEW BIFUNCTIONAL CHELATING AGENT α,ε-N,N'-BIS(L-CYSTEINYL)-L-LYSINE FOR RADIOLABELLING OF MONOCLONAL ANTIBODIES WITH TECHNETIUM-99M

α,ε-N,N'-bis(L-cysteinyl)-L-lysine was synthesized and char- acterized for the first time.It was then employed as a bifunctional chelating agent to chelate technetium-99m and subsequently conjugated to fragment F(ab')_2 of anti-gastric tumor monoclonal antibody 3G9.The radiolabelled antibody was satisfactorily stable and immunoreactive.

GA2-oxidase氧化酶基因的研究进展

GA2-oxidase氧化酶(GA2ox)是影响赤霉素合成和代谢的关键酶之一,其可以将具有生物活性的赤霉素或前体分解成没有生物活性的物质,在植物的生长发育中起着重要的作用。目前已经在拟南芥、杨树、水稻和葡萄等多种植物中克隆表达,其功能在不同物种之间也存在着差异。本研究主要从GA2ox基因的表达模式和基因功能进行阐述,重点阐述了GA2ox基因的时空表达情况,环境和激素对GA2ox基因的表达影响;GA2ox基因对植物的株高、花、果实和种子性状的改变,及与抗性的相互作用,更好地为了解GA2ox基因的作用机制提供参考。

植物赤霉素生物合成关键基因GA3-oxidase的研究进展

在植物赤霉素合成途径中,GA3ox将无活性的GA12和GA53转变为有活性的GA4和GA1,进而实现了调控植物生长发育。GA生物合成及其信号传导途径相关基因的研究备受关注。本研究对高等植物体内GA3-oxidase基因的克隆、组织表达、调控表达、功能鉴定等方面的研究进行综述,并对进一步深入研究GA3ox的互作、抗逆性等方面进行了展望,揭示GA3-oxidase氧化酶信号网络系统及其作用机制。

Activation of gibberellin 2-oxidase 6 decreases active gibberellin levels and creates a dominant semi-dwarf phenotype in rice (Oryza sativa L.)

Gibberellin （GA） 2-oxidase plays a key role in the GA catabolic pathway through 2β-hydroxylation.In the present study,we isolated a CaMV 35S-enhancer activation tagged mutant,H032.This mutant exhibited a dominant dwarf and GA-deficient phenotype,with a final stature that was less than half of its wild-type counterpart.The endogenous bioactive GAs are markedly decreased in the H032 mutant,and application of bioactive GAs （GA3 or GA4） can reverse the dwarf phenotype.The integrated T-DNA was detected 12.8 kb upstream of the OsGA2ox6 in the H032 genome by TAIL-PCR.An increased level of OsGA2ox6 mRNA was detected at a high level in the H032 mutant,which might be due to the enhancer role of the CaMV 35S promoter.RNAi and ectopic expression analysis of OsGA2ox6 indicated that the dwarf trait and the decreased levels of bioactive GAs in the H032 mutant were a result of the up-regulation of the OsGA2ox6 gene.BLASTP analysis revealed that OsGA2ox6 belongs to the class III of GA 2-oxidases,which is a novel type of GA2ox that uses C20-GAs （GA12 and/or GA53） as the substrates.Interestingly,we found that a GA biosynthesis inhibitor,paclobutrazol,positively regulated the OsGA2ox6 gene.Unlike the over-expression of OsGA2ox1,which led to a high rate of seed abortion,the H032 mutant retained normal flowering and seed production.These results indicate that OsGA2ox6 mainly affects plant stature,and the dominant dwarf trait of the H032 mutant can be used as an efficient dwarf resource in rice breeding.

ε-Poly(L-lysine)-based Hydrogels with Fast-acting and Prolonged Antibacterial Activities

Bacterial infections and the associated morbidity and mortality due to bacterial pathogens in wounds and medical implants have been increasing as most of current coatings cannot fulfill all the requirements including excellent intrinsically antibacterial activity, low cytotoxicity, and favorable physical properties. Herein, we present a kind of antibacterial bydrogel based on e-poly（L-lysine） （EPL） grafted carboxymethyl chitosan （CMC-g-EPL） as the inherently antibacterial matrix and the surplus EPL as highly efficient antimicrobial agent. Such hydrogels possess tunable swelling abilities with water absorption percentages of 800%-2000% and modulus varying from 10 kPa to 100 kPa, and exhibit two-stage excellent antibacterial behavior. First, the free EPL can be released from the hydrogel network for quick and highly efficient bacteria killing with 99.99% of efficacy; second, the grafted EPL endows bydrogel matrix with prolonged intrinsically antibacterial activity, especially when most of free EPL is released from the hydrogel. Overall, we provide a new insight for preparing highly effective antibacterial hydrogels.

A study on α-ketoadipic aciduria by gas chromatographic-mass spectrometry

INTRODUCTIONα-ketoadipate(α-KA),an intermediate in thecatabolism of L-lysine,hydroxylysine,and L-tryptophan,undergoes oxidative deearboxylation toform glutaryl-CoA and then dehydrogenates to formcrotonyl-CoA,the latter undergoes furtherdegradation and enters in TCA cycle,as shown inFigure 1.α-ketoadipic aciduria (Mckusick 245130)is a rare inborn error in the metabolism of α-KA

Green synthesis of gold nanoclusters using papayajuice for detection of L-lysine

Gold nanoclusters were rapid synthesized within 3 min at 120 ℃ by using papaya juice as a capping and reducing agent(P-AuNCs). The properties of the fluorescent probe were characterized by fluorescent spectroscopy, UV-vis spectroscopy, dynamic light scattering and transmission electron microscope.Based on the surface electron density increase-induced fluorescence enhancing principle, a high selective method for detection of L-lysine was developed with the as-prepared P-AuNCs coupling the fluorescence emission at 440 nm. The fluorescent probe showed high stability and good biocompatibility. Its fluorescence intensity was found to be linearly dependent on the L-lysine concentration in the range of 10.0μmol/L to 1000.0 μmol/L(R^2=0.969) with a limit of detection of 6.0μmol/L. Furthermore, the PAuNCs based approach was applied for monitoring the urine L-lysine contents, demonstrating great potential of fluorescent probes in real samples analysis.

Syntheses and Characterization of Metal Complexes of L－Lysine－N，N，N＇，N＇－tetraacetic Acid and L－Lysine－N，N，N＇，N＇－tetramethylenepho sphonic Acid

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L-Lysine/imidazole-catalyzed Multicomponent Cascade Reaction： Facile Synthesis of C5-substituted 3-Methylcyclohex-2-enones

A facile and simple route for the direct preparation of substituted 3-methylcyclohex-2-enone via Aldol- Robinson cascade reaction of aldehydes and acetones catalyzed by the new catalytic system of L-lysine/imidazole in n-heptane with 0.5% water was reported. A variety of substrates can participate in the process efficiently. The merits of this method included inexpensive and easily available starting materials and catalyst, the good yield of products and the straightforward work-up.

Interfacial synthesis and properties of L-lysine-derived optically active poly(ester-imide)s

L-Lysine hydrochloride was transformed to ethyl L-lysine dihydrochloride.This salt was reacted with trimellitic anhydride to yield the corresponding diacid（1）.Intertacial polycondensation results novel poly（ester-imide）s（PEI_（a-i））.These polymers have inherent viscosities in the range of 0.23-0.47 dl g^（-1）,display optical activity,and are readily soluble in polar aprotic solvents.They start to decompose（T_（10%）） above 350℃and display glass-transition temperatures at 100.42-172.81℃.All of the above polymers were fully characterized by UV,FT-IR and ~1H NMR spectroscopy,elemental analysis,TGA,DSC,inherent viscosity measurement and specific rotation.

Composite Eu-MOF@CQDs“off&on”ratiometric luminescent probe for highly sensitive chiral detection of l-lysine and 2-methoxybenzaldehyde

The high amount of l-lysine can increase the potential risk of cardiovascular disease.Additionally,2-methoxy benzaldehyde(2-MB)has high toxicity and can easily pollute the environment.In this work,carbon quantum dots(CQDs)can be encapsulated into Eu-BTB(H_(3)BTB=1,3,5-tri(4-carboxyphenyl)benzene),forming the multi-emission composite material Eu-BTB@CQDs.It has two emissions peaks(617 nm for Eu and 470 nm for CQDs).Eu-BTB@CQDs can be applied as bi-functional ratiometric“off&on”luminescent sensor for l-lysine and 2-MB with high sensitivity and selectivity,the low limit of detection(LOD)for l-lysine is 3.68μmol/L and for 2-MB is 0.54μmol/L,respectively.Additionally,Eu-BTB@CQDs can quantitatively discriminate l-lysine in the mixed d-and l-lysine water solutions(five different concentrations ratio of l/d-lysine has been set)makes the chiral detection of l-lysine are more meaningful.On the other hand,Eu-BTB@CQDs also can detect 2-MB over 4-methoxybenzaldehyde(4-MB)with high selectivity.Further the detection of 2-MB and l-lysine in the lake water real samples with the reasonable recovery rate.Finally,the detection mechanisms for l-lysine and 2-MB were also investigated and discussed in detail.

From model to alfalfa:Gene editing to obtain semidwarf and prostrate growth habits

Alfalfa(Medicago sativa L.)is a nutritious forage crop with wide ecological adaptability.The molecular breeding of alfalfa is restricted by its heterozygous tetraploid genome and the difficult genetic manipulation process.Under time and resource constraints,we applied a more convenient approach.We investigated two MtGA3ox genes,MtGA3ox1 and MtGA3ox2,of Medicago truncatula,a diploid legume model species,finding that MtGA3ox1 plays a major role in GA-regulated plant architecture.Mutation of neither gene affected nitrogenase activity.These results suggest that MtGA3ox1 can be used in semidwarf and prostrate alfalfa breeding.Based on the M.truncatula MtGA3ox1 sequence,MsGA3ox1 was cloned from alfalfa,and two knockout targets were designed.An efficient CRISPR/Cas9-based genome editing protocol was used to generate msga3ox1 mutants in alfalfa.We obtained three lines that carried mutations in all four alleles in the T0 generation.Fifteen clonal plants were vegetatively propagated from each transgenic line using shoot cuttings.The plant height and internode length of msga3ox1 null mutants were significantly decreased.The number of total lateral branches,leaf/stem ratio and crude protein content of aerial plant parts of msga3ox1 mutants were significantly increased.Thus,we obtained semi-dwarf and prostrate alfalfa by gene editing.

Core-Corona Structure Formed by Hyaluronic Acid and Poly(L-lysine)via Kinetic Path

The structure and kinetics of the complex formed by hyaluronic acid(HA) and poly(L-lysine)(PLL) were studied by timeresolved laser light scattering, TEM, and AFM. Because HA has a hydrophilic backbone, the complexes formed by HA and PLL are different from those formed by oppositely charged polyelectrolytes both having hydrophobic backbones. Instead of forming strong aggregates and even precipitates, the complex in the presence of excess HA is stable in the studied time period. More importantly, the complex spontaneously forms core-corona structure by the rearrangement of HA chains. The core is composed of complex rich of PLL and the corona is mainly HA. Further analysis shows that the hydrogen bond formed by HA creates a barrier hindering the further relaxation of HA chains. The automatic formation of core-corona structure by PLL/HA is helpful not only to understand the relaxation of polyelectrolyte in complex, but also to develop drug carriers with desirable properties.