Changes in the activities of key enzymes and the abundance of functional genes involved in nitrogen transformation in rice rhizosphere soil under different aerated conditions

Soil microorganisms play important roles in nitrogen transformation. The aim of this study was to characterize changes in the activity of nitrogen transformation enzymes and the abundance of nitrogen function genes in rhizosphere soil aerated using three different methods(continuous flooding(CF), continuous flooding and aeration(CFA), and alternate wetting and drying(AWD)). The abundances of amoA ammonia-oxidizing archaea(AOA) and ammonia-oxidizing bacteria(AOB), nirS, nirK, and nifH genes, and the activities of urease, protease, ammonia oxidase, nitrate reductase, and nitrite reductase were measured at the tillering(S1), heading(S2), and ripening(S3) stages. We analyzed the relationships of the aforementioned microbial activity indices, in addition to soil microbial biomass carbon(MBC) and soil microbial biomass nitrogen(MBN), with the concentration of soil nitrate and ammonium nitrogen. The abundance of nitrogen function genes and the activities of nitrogen invertase in rice rhizosphere soil were higher at S2 compared with S1 and S3 in all treatments. AWD and CFA increased the abundance of amoA and nifH genes, and the activities of urease, protease, and ammonia oxidase, and decreased the abundance of nirS and nirK genes and the activities of nitrate reductase and nitrite reductase, with the effect of AWD being particularly strong. During the entire growth period, the mean abundances of the AOA amoA, AOB amoA, and nifH genes were 2.9, 5.8, and 3.0 higher in the AWD treatment than in the CF treatment, respectively, and the activities of urease, protease, and ammonia oxidase were 1.1, 0.5, and 0.7 higher in the AWD treatment than in the CF treatment, respectively. The abundances of the nirS and nirK genes, and the activities of nitrate reductase and nitrite reductase were 73.6, 84.8, 10.3 and 36.5% lower in the AWD treatment than in the CF treatment, respectively. The abundances of the AOA amoA, AOB amoA, and nifH genes were significantly and positively correlated with the activities of urease, protease, and ammonia oxidase, and the abundances of the nirS and nirK genes were significantly positively correlated with the activities of nitrate reductase. All the above indicators were positively correlated with soil MBC and MBN. In sum, microbial activity related to nitrogen transformation in rice rhizosphere soil was highest at S2. Aeration can effectively increase the activity of most nitrogen-converting microorganisms and MBN, and thus promote soil nitrogen transformation.

Effects of Different Nitrogen and Phosphorus Synergistic Fertilizer on Enzymes and Genes Related to Nitrogen Metabolism in Wheat

In recent years,in order to improve nutrient use efficiency,especially nitrogen use efficiency,fertilizer valueadded technology has been developed rapidly.However,the mechanism of the effect of synergistic fertilizer on plant nitrogen utilization is not clear.A study was,therefore,conducted to explore the activities and gene expression of key enzymes for nitrogen assimilation and the gene expression of nitrogen transporters in wheat after the application of synergistic fertilizer.Soil column experiment was set up in Qingdao Agricultural University experimental base from October 2018 to June 2019.Maleic acid and itaconic acid were copolymerized with acrylic acid as cross-linking monomer to make a fluid gel,which was sprayed on the fertilizer surface to make nitrogen and phosphorus synergistic fertilizer.A total of 6 treatments was set according to different nitrogen and phosphorus fertilizer ratios:(1)100%common nitrogen fertilizer+100%common phosphate fertilizer(2)70%nitrogen synergistic fertilizer+100%phosphorus synergistic fertilizer;(3)100%nitrogen synergistic fertilizer+70%phosphorus synergistic fertilizer;(4)100%nitrogen synergistic fertilizer+100%phosphorus synergistic fertilizer;(5)70%nitrogen synergistic fertilizer+70%phosphorus synergistic fertilizer;(6)100%commercial nitrogen synergistic fertilizer+100%commercial phosphorus synergistic fertilizer.The results are as follows:(1)the enzyme activities of wheat plants under synergistic fertilizer condition were higher than those under ordinary fertilizer,except under the treatment that nitrogen and phosphorus synergistic fertilizer were both reduced;(2)the expression level of the genes under the treatment“100%nitrogen synergistic fertilizer+100%phosphorus synergistic fertilizer”was significantly higher than those in other treatments.Combined with the higher performance of nitrogen concentration in various parts of the plant under the condition of applying synergistic fertilizer,this study indicated that the application of synergistic fertilizer can improve the nitrogen metabolism of the plant by increasing the nitrogen level in the rhizosphere soil,inducing the expression of nitrogen transporter genes and key assimilation enzymes genes.

Effects of Potassium-Solubilizing Bacteria on Growth, Antioxidant Activity and Expression of Related Genes in Fritillaria taipaiensis P. Y. Li

This study aimed to examine the effects of inoculating Fritillaria taipaiensis P.Y.Li leaves with different strains ofpotassium-solubilizing bacteria (KSB), or combinations thereof, focusing on aspects of photosynthesis and physiologicaland biochemical characteristics. At present, some studies have only studied the rhizosphere microbialcommunity characteristics of F. taipaiensis and have not discussed the effects of different microbial species on thegrowth promotion of F. taipaiensis. This paper will start from the perspective of potassium-solubilizing bacteria toconduct an in-depth study. Seed cultivation commenced at the base with three different KSBs in early October2022. The growth of F. taipaiensis leaves was observed after different treatments. Both single-plant and compoundinoculations were executed. A total of eight treatment groups were established, with aseptic fertilizer and sterilizedsoil functioning as the control group. The results reveal that intercellular CO_(2) concentration (Ci), stomatal conductance(Gs), and transpiration rate (Tr) were at their apex in the S7 group. Most treatment groups exhibited anincrease in leaf area, photosynthetic pigment content, soluble sugar, soluble protein, Superoxide Dismutase(SOD), Peroxidase (POD), Catalase (CAT) activities, and proline content. The expression levels of POD, SOD,and CAT genes were evaluated, following inoculation with different KSB. The highest was the S7 group. Theinoculation with various KSB, or combinations thereof, appears to bolster the growth and development of F. taipaiensis.The composite inoculation group S7, comprising Bacillus cereus, Burkholderia cepacia, and Bacillus subtilis,manifested the most favorable impact on the diverse indices of F. taipaiensis, thereby furnishing valuableinsights for the selection of bacterial fertilizer in the artificial cultivation of F. taipaiensis.

Cloning and Characterization of Genes Coding for Fructan Biosynthesis Enzymes (FBEs) in Triticeae Plants

Fructan is not only a carbon source for storage but also plays an important role as anti-stress agents in many plant species. Complex fructans having both β-（2,1）- and β-（2,6）-linked fructosyl units accumulate in Triticeae plants commonly. Three enzymes （sucrose： sucrose 1-fructosyltransferase, 1-SST, EC： 2.4.1.99; sucrose： fructan 6-fructosyltransferase, 6- SFT, EC： 2.4.1.10; and fructan： fructan 1-fructosyltransferase, 1-FFT, EC： 2.4.1.100） were involved in fructan biosynthesis in Triticeae plant species. We successfully isolated these genes from tetraploid wheat （Triticum turgidum, genotype： AABB）, common wheat （Triticum aestivum L., genotype： AABBDD） and three wild relatives of common wheat, Triticum urartu Thum. （the origin of the AA genome）, Aegilops speltoides （Tausch） Gren. （the putative source of the SS genome） and Aegilops tauschii Coss. （the source of the DD genome）. Sequence analysis revealed that all the FBEs （fructan biosynthetic enzymes） had three highly conserved functional motifs except 1-SST （EU981912） from tetraploid wheat species only with conserved DPNG. Low pI （isoelectric point） and potential N-glycosylation sites were predicted, which were crucial for protein compartmentation and post-translational process. Analysis on subcelluar localization signals showed that only 6-SFT had vacuolar-directed signal. Sequences alignment result showed that 1-SST and 1-FFT were more conservative and had closer relationship each other, while 6-SFT was more active during the evolution processing. According to the syntenic relationship between wheat and rice genome, FBEs were predicated to be located on the homeologous group 6 and group 2 chromosomes. Expression profile confirmed that expression of all the three FBEs were drought-stress induced. This study can assist to establish a useful theoretical platform for cold- or drought-tolerant improvement of wheat by modulating FBEs expression.

Temperature Stress at Grain Filling Stage Mediates Expression of Three Isoform Genes Encoding Starch Branching Enzymes in Rice Endosperm

An early-maturity indica rice variety Zhefu 49, whose grain quality and starch structure are sensitive to environmental temperature, was subjected to different temperatures （32℃ for high temperature and 22℃ for optimum temperature） at the grain filling stage in plant growth chambers, and the different expressions of three isoform genes （SBEI, SBEIII and SBE/V） encoding starch branching enzyme （SBE） in the endosperms were studied by the real-time fluorescence quantitative PCR （FQ-PCR） method. Effects of high temperature on the SBE expression in developing rice endosperrns were isoform-dependent. High temperature significantly down-regulated the expressions of SBEI and SBEIII, while up-regulated the expression of SBEIV. Compared with SBEIV and SBEIII, the expression of SBEI gene in Zhefu 49 rice endosperms was more sensitive to temperature variation at the grain filling stage. This study indicates that changes in weather/climate conditions especially temperature stress influence rice grain formation and its quality as evidenced by isoform expression.

Contribution of Antioxidative Enzymes and Anoxia Responsive Genes to Submergence Tolerance in Rice: A Review

Flooding/submergence of rice fields is a severe problem in South and South-East Asia, affecting more than 20 million hectares of rice every year. Submergence creates hypoxic or anoxic condition causing poor germination, seedling establishment,and enormous yield loss. Standing water in the field from weeks to months also leads to significant yield losses when large part of aerial tissues is under water. For flash flooding, a rice variety FR1A3 with tolerant gene(SUB1A) was identified. SNORKEL1 and SNORKEL2 have been identified for their ability to survive deep-water flooding by rapid elongation. Submergence stress has also been reported to adversely affect cell division and damage cellular and organelle membranes. Research on antioxidative enzymes response and genes that confer tolerance to prolonged flooding is in progress. Here we review the different anoxia responsive genes and the potential involvement of antioxidative enzymes, such as superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase, which occur in cells of rice plant exposed to submergence stress.

Association of Polymorphisms in Angiotensin-converting Enzyme and Type 1 Angiotensin Ⅱ Receptor Genes with Coronary Heart Disease and the Severity of Coronary Artery Stenosis

To explore the relation of angiotensin-converting enzyme （ACE） and angiotensin Ⅱ type 1 receptor （AT1R） gene polymorphism with coronary heart disease （CHD） and the severity of coronary artery stenosis, 130 CHD patients who underwent coronary angiography were examined for the number of affected coronary vessels （≥75% stenosis） and coronary Jeopardy score. The insertion/deletion of ACE gene polymorphism and AT1R gene polymorphism （an A→C transversion at nucleotide position 1166） were detected by using polymerase chain reaction （PCR） and restriction fragment length polymorphism （RFLP） in CHD patients and 90 healthy serving as controls. The resuits showed that DD genotype and of ACE were more frequent in CHD patients than that in control group （38.5% vs 14.4%, P〈0.001）. The frequency of the ATIR A/C genotypes did not differ between the patients and the controls （10% vs 13.1%, P〉0.05）. The relative risk associated with the ACE-DD was increased by AT1R-AC genotype. Neither the number of affected coronary vessels nor the coronary score differed among the ACE I/D genotypes （P〉0.05）. But the number of affected coronary vessels and the coronary score were significantly greater in the patients with the AT1R-AC genotype than in those with the AA genotype （P〈0.05）. In conclusion, DD genotype may be risk factor for CHD and MI in Chinese people, and is not responsible for the development of the coronary artery stenosis. The AT1R-C allele may increase the relative risk associated with the ACE-DD genotype, and may be involved in the development of the stenosis of coronary artery.

Gene Expression and Activity of Enzymes Involved in Sugar Metabolism and Accumulation During “Huangguan” and “Yali” Pear Fruit Development

Since the carbohydrate content affects pear flavor during the process of growth, it is necessary to determine the sugar components that accumulate in the fruit. We analyzed the fruit carbohydrate content, and the gene expression and activity ofacid invertase(AI), neutral invertase(NI), sucrose synthase(SS), and sucrose phosphate synthase(SPS) during the development of "Huangguan" and "Yali" pears. The results demonstrate that during development, the fruit sugar metabolism of the "Huangguan" pear follows a typical sorbitol–starch-soluble sugars middle model, whereas the "Yali" pear fruit follows a typical sorbitol–sucrose–starch-soluble sugars middle model. In the "Huangguan" pear, we found the AI and NI gene expressions, as well as AI( P < 0.05) and NI( P < 0.01) enzyme activities, to be positively correlated, whereas we found the NI gene expression and NI enzyme activity of "Yali" pear to be negatively correlated( P < 0.01). We observed the high levels oflate-stage AI and early-stage SS during development to roughly correspond with the gene expression found in the late and early stages, respectively, suggesting their potential regulatory roles in "Huangguan" pear fruit development. Our results indicate that the primary function of SPS during the early developmental stage is to accumulate sucrose, whereas the primary function of AI is to promote hexose accumulation during the late developmental stage ofmature "Yali" pear fruit.

Angiotensin-converting Enzyme Gene Insertion/Deletion Polymorphism in Children with Henoch-Schonlein Purpua Nephritis

This study investigated the relationship between angiotensin-converting enzyme (ACE) gene insertion/deletion polymorphism and the occurrence, severity, prognosis of HSPN. The polymorphism of ACE gene in 103 HSPN cases and 100 healthy children was studied by using the polymerase chain reactions (PCR). Its relation to the clinical manifestation, pathological classification and prognosis of HSPN was analyzed accordingly. The results showed that: (1) there was a significantly higher frequency for DD genotype in HSPN children (P<0.01); (2) DD genotype was more frequently seen in HSPN children with gross hematuria and massive proteinuria (P<0.05), while DI genotype was more common in HSPN children group with renal insufficiency (P<0.05); (3) although mesangial proliferative lesion was most frequently observed in 21 biopsied HSPN children, and DD genotype frequency was still higher in children with severe pathology (Class Ⅲ Ⅳ); (4)II genotype was significantly frequent in HSPN children with complete remission in the follow-up of 32 HSPN children. It was concluded that the deletion allele of ACE gene might play a role, at least to some extent, in the occurrence, deterioration and progression in juvenile HSPN.

Angiotensin-converting enzyme and bradykinin gene polymorphisms and cough:A meta-analysis

AIM:To evaluate the association between genetic polymorphisms and angiotensin converting enzyme in-hibitor (ACEI)-related cough,and the race-or ethnicity-related difference in the prevalence of cough attributed to ACEI therapy.METHODS:We conducted a search in PubMed,EM-BASE,Cinahl,and the Cochrane Database without language limitation.A database of 11 studies on ACEI-related cough,with detailed information regarding ACE I/D or bradykinin B 2 receptor polymorphisms,was created.Eligible studies were synthesized using meta-analysis methods,including cumulative meta-analysis.A subgroup analysis was also performed using ethnicity.RESULTS:Six studies were included on ACE I/D poly-morphism (398 Caucasians,723 East Asians),and three studies were included on bradykinin B 2 receptor poly-morphism (300 East Asians).The distribution of ACE genotypes showed significant differences in the entire population (P=0.004) and in East Asians (P=0.005)but not in Caucasians (P=0.23).Allelic frequencies of ACE showed significant differences in East Asians [odds ratio (OR)=1.49 (1.11-2.02)].The meta-analysis with a random effects model showed a significant associa-tion between ACE allele I/D and ACEI-related cough [random effects (RE) OR=1.49 (1.11-2.02),P=0.009] in East Asians,but not in Caucasians [RE OR=0.90 (0.60-1.35)].The allelic frequencies of the bradykinin B 2 receptor gene were significantly different [OR=2.25 (1.42-3.57)].The distributions of the T/C genotypes of the bradykinin B 2 receptor gene were significantly dif-ferent (χ 2=8.366,P=0.015).The meta-analyses re-vealed that there was a significant association between the bradykinin B 2 receptor allele and ACEI-related cough in East Asians [RE OR=2.29 (1.42-3.69),P=0.001].CONCLUSION:ACE I/D and Bradykinin B 2 receptor polymorphisms contributed to the risk of ACEI-related cough in East Asians,but a negative association be-tween ACE I/D polymorphism and ACEI-related cough was observed in Caucasians.

Correlation of angiotensin converting enzyme gene polymorphism with perioperative myocardial protection under extracorporeal circulation

Objective:To observe the expression of angiotensin converting enzyme(ACE),angiotensinⅡ(AngⅡ),cardiac troponin 【cTnⅠ),creatine kinase isozymes(CK-MB) and muscle red protein(Myo) after cardiopulmonary bypass(CPB),and to investigate the association of polymorphisms in angiotensin converting enzyme genes and myocardial injury.Methods:Sixty-three patients suffered from rheumatic mitral stenosis and scheduled for mitral valve replacement with CPB, were randomly divided into three groups according polymorphisms in angiotensin converting enzyme genes:typeⅡ,type ID,type DD(each=21).Blood samples were withdrawn from artery before operation(T1),at the beginning of CPB(T2),30 min after CPB(T3),(T4) at the end of CPB(T5), 2 h after CPB(T6),6 h after CPB(17) to measure the expression of ACE,AngⅡ,cTnⅠ,CK-MB, Myo.Results:The level of ACE during and after CPB were significantly higher than those before CPB(P【0.05).As extension of CPB time,the expression of ACE was increased.The level of cTnⅠ, CK-MB,Myo after CPB were significantly higher than those before CPB(P【0.05).The level of cTnⅠ,CK-MB and Myo were highest at T7,T6 and T5 and T7,respectively.The level of ACE,AngⅡ,cTnⅠin patients with DD genotype was significantly higher than the ID andⅡgenotype(P【 0.05).Besides,the level of ACE,AngⅡin patients with ID genotype was significantly higher than the II(P【 0.05).Conclusions:There is certain correlation between CPB perioperative midterm ACE and cTnⅠ,Myo,CK-MB.ACE DD genotype is a susceptibility gene of the CPB perioperative myocardial injury.

Effects of Abscisic Acid Treatment on Berry Coloration and Expression of Flavonoid Biosynthesis Genes in Grape

In order to enhance berry coloration of bright-red grape cultivars, the effects of abscisic acid (ABA) treatment on the quantity and composition of anthocyanins as well as the expression of genes related to flavonoid biosynthesis in the berry were examined. Exogenous ABA treatment increased anthocyanin content, especially petunidin- and malvidin-type anthocyanins. Quantitative real-time PCR analysis revealed that ABA treatment around véraison resulted in the upregulation of genes encoding enzymes responsible for both general flavonoid and anthocyanin biosynthesis. On the other hand, the gene expressions of enzymes involved in proanthocyanidin synthesis were drastically decreased at véraison and remained extremely low even with ABA treatment. Thus, increases in the total amount and composition ratios of petunidin- and malvidin-type anthocyanins were mainly caused by ABA-induced upregulation of uridine diphosphate glucose flavonoid glucosyl transferase, glutathione S-transferase 4, O-methyl transferase and flavonoid 3’, 5’ hydroxylase expression, resulting in the deep coloration of berry of skin.

Association of Angiotensin Converting Enzyme Gene I/D Polymorphism With Type 2 Diabetes Mellitus

Objective To investigate the association of angiotensin converting enzyme （ACE） gene insertion/deletion （I/D） polymorphism with type 2 diabetes mellitus （T2DM）. Methods Two hundred and nine patients with T2DM diagnosed based on the criteria for diabetes mellitus in 1999 by WHO and 221 controls were recruited from general population of Dongcheng District in Beijing. All subjects were genotyped for the I/D polymorphism of ACE gene by PCR-fragment length polymorphism （FLP） assay. Blood pressure, levels of plasma glucose, lipids and serum insulin were determined. Body mass index （BMI）, waist-trip ratio （WHR） and homeostasis model assessment-insulin resistance index （HOMA-IR） were calculated. Results The genotype frequencies for ACE genes DD, ID, and II were 19.1%, 42.1%, and 38.8% in patients, respectively, and 9.6%, 49.4%, and 41.0% in controls, respectively. The ACE DD genotype frequency was significantly higher in patients than in controls （χ^2=7.61, P=0.022）. Multivariate logistic regression analysis showed that the ACE DD genotype was a risk factor for T2DM, with the OR of 2.35 （95% CI 1.17-4.71） adjusted for age, sex, BMI, WHR, blood pressure, and serum cholesterol levels. Conclusion The ACE DD genotype is associated with the increased susceptibility to type 2 diabetes mellitus.

cDNA Cloning and Sequence Analysis of Rice Sbe1 and Sbe3 Genes

Two starch-branching enzyme (SBE) in rice, is known to be a key enzyme in amylopectin biosynthesis. The cDNA of two SBE(starch-branching enzyme) genes Sbe1 and Sbc3 encoding SBE Ⅰ and SBE Ⅲ (two major isoforms in rice) were cloned by an improved RT-PCR technique, from a template cDNA library derived from the total mRNAs extracted from the immature seeds of a japonica rice Wuyunjing 7. DNA sequence analysis showed that the size of the cloned Sbe1 and Sbe3 cDNAs were 2490 and 2481 bp long, respectively, including their entire coding sequences. Comparison analysis indicated that the nucleotide sequence of Sbe3 was the same as that of sbc3 (Genbank Accession No. D16201) as reported previously. There were only four base-pairs difference, which resulted in changes of two deduced amino acids between the cloned Sbel cDNA and the reported sbe1 (Genbank Accession No. D11082). The cloned Sbe1 and Sbe3 cDNAs make it possible to improve rice starch quality through genetic engineering

Interaction and Relationship Between Angiotensin ConvertingEnzyme Gene and Environmental Factors Predisposing toEssential Hypertension in MongolianPopulation of China

Objective To investigate the association of specific functional gene ACE (I/D) variants of the renin-angiotensin system with essential hypertension (EH) and interaction between ACE (I/D) gene and risk factors for EH in a genetically homogenous Mongolia rural population of China. Methods Individuals (n=1099) were recruited from general population of Kezuohouqi Banner in Inner Mongolian Autonomous Region. Results The association was found between ACE genotype DD plus ID and EH, with an interaction between ACE genotype DD plus ID and cigarette smoking in an additive model. Cigarette smoking index and ACE gene showed a low exposure-gene (LEG) effect on EH, with interaction indices from 7.10 to 1.16. Interaction between ACE genotype DD plus ID and alcohol drinking on EH appeared an additive model. Alcohol drinking index and ACE gene showed a low exposure-gene (LEG) effect on EH, with interaction indices from 1.66 to 1.09. BMI and ACE gene showed a low exposure-gene (LEG) effect on EH, with interaction indices from 6.15 to 2.49. Interactions between ACE genotype and WHR on EH showed a multiplicative model. In a short, there was an interaction between ACE gene and cigarette smoking, alcohol drinking and BMI on EH, especially in a low dose-exposure effect Conclusion It is important for individuals who carry ACE D allele gene to prevent EH, and furthermore, to prevent and control coronary heart disease, in a view of population-based prevention.

Angiotensin-converting enzyme gene polymorphism and middle cerebral artery stenosis in a Chinese Han population

The angiotensin-converting enzyme gene is a candidate gene of stroke. The present study involved 62 healthy volunteers and 148 patients with middle cerebral artery stenosis as confirmed by brain color ultrasound from a Han population in North China, and determined the peripheral blood angiotensin-converting enzyme genotype using PCR-restriction fragment length polymorphism analysis. The results showed that the frequencies of the DD genotype and D allele were increased in patients with middle cerebral artery stenosis, but the difference was not statistically significant compared with healthy controls. The findings of this study on the relationship between stroke genes and middle cerebral artery stenosis indicate no significant correlation between the frequencies of the DO genotype and D allele of angiotensin-converting enzyme and middle cerebral artery stenosis in this Han population from North China. In the future, studies will be carried out to investigate correlations between multiple stroke candidate gene synergy and middle cerebral artery stenosis to provide a foundation for the development of gene therapy.

Cloning and Characterization of a Galactomannan-degrading Enzyme Gene in Pichia pastoris

[ Objective ] This study aimed to obtain the recombinant Pichia yeast strain which can efficiently degrade guar gum. The properties of the recombinant enzyme were studied preliminarily. [ Method ] A positive clone that could hydrolyze guar gum was obtained through the construction and functional screening of a soil genome library. Sequence analysis indicated that the 1485-bp clone encodes a 494-amino acid protein with a relative molecular mass of 53 949 kD, containing a cellulose-binding domain. The recombinant plasmid pHBM731 was generated by inserting the optimized target gene into a Pichia pastoris expression vector pHBMg05 that was transformed into three Pichia pastoris strains, GS115, KM71 and SMD1168. The biochemical properties of the enzyme were assessed. [ Result] The cloned galactonumnan （GM）-degrading enzyme was expressed and secreted by Pichia pastoris GSll5. High cell density fermentation was induced in recombi- nant Pichia pastoris at 25 and 28 ~C ; a higher enzyme activity was observed at an induction temperature of 28 ~C. The optimal temperature for the recombinant en- zyme is 60 ~C, and the optimal pH is 6.6. The enzyme activity was 38.61 U under optimal conditions. Over 50% of the enzyme activity was maintained under the optimal conditions after 9 h. Under the optimal conditions, the effect of metal ions on enzyme activity was analyzed. Ca2 ＋ , Fe2 ＋ and Li ~ slightly enhanced enzyme activity, while Mn2＋ and Co2＋ had little effect. Enzyme activity was modestly suppressed by Mg2~ , K~ and Na＋ , but considerably suppressed by Ag2~ and Zn2~ , with Cu2 ＋ showing the strongest inhibitory effects. [ Conclusion] A novel GM-degrading enzyme expressed by soil yeast was cloned, which can potentially be used in industrial applications to obtain eommereially useful guar gum-degradation products.

Comparison of Five Endogenous Reference Genes for Specific PCR Detection and Quantification of Rice

Endogenous reference genes (ERGs) provide vital information regarding genetically modified organisms (GMOs). The successful detection of ERGs can identity GMOs and the source of genes, verify stability and reliability of the detection system, and calculate the level of genetically modified (GM) ingredients in mixtures. The reported ERGs in rice include sucrose-phosphate synthase (SPS), phospholipase D (PLD), RBE4 and rice root-specific GOS9 genes. Based on the characteristics of ERGs, a new ERG gene, phosphoenolpyruvate carboxylase (PEPC), was selected, and further compared with the four existing genes. A total of 18 rice varieties and 29 non-rice crops were used to verify the interspecies specificity, intraspecies consistency, sensitivity, stability and reliability of these five ERGs using qualitative and quantitative PCR. Qualitative detection indicated that SPS and PEPC displayed sufficient specificity, and the detection sensitivity was 0.05% and 0.005%, respectively. Although the specificity of both RBE4 and GOS9 were adequate, the amplicons were small and easily confused with primer dimers. Non-specific amplification of the PLD gene was present in maize and potato. Real-time quantitative PCR detection indicated that PLD, SPS and PEPC displayed good specificity, with R2 of the standard curve greater than 0.98, while the amplification efficiency ranged between 90% and 110%. Both the detection sensitivities of PLD and PEPC were five copies and that of SPS was ten copies. RBE4 showed typical amplification in maize, beet and Arabidopsis, while GOS9 was found in maize, tobacco and oats. PEPC exhibited excellent detection sensitivity and species specificity, which made it a potentially useful application in GM-rice supervision and administration. Additionally, SPS and PLD are also suitable for GM-rice detection. This study effectively established a foundation for GMO detection, which not only provides vital technical support for GMO identification, but also is of great significance for enhancing the comparability of detection results, and the standardization of ERG testing in GM-rice.

CATIONIC AMPHIPHILE-MEDIATED TRANSFER OF THEENZYME/PRODRUG GENE INTO C_6 GLIOMA CELLS-AN EFFECTIVE IN VITRO CHEMOSENSITIVITY SYSTEM

Objectivs Enzyme/prodrug gene therapy provides a potential strategy for the treatment of glioma.Because of the limitations of using viral vectors for clinical application, we investigated the feasibility of cationicamphiphile-mediated enzyme/prodrug gene transfer into C6 glioma cells. Methods Rat C6 glioma cells weretransfected with pUT599plasmid encoding the herpes simplex virus thymidine kinase (HSV-tk) gene via DOTAPand tested for chemosensitivity of prodrug ganciclovir (GCV). To demonstrate in vitro bystander effect, HSV-tkpositive cells were co-cultured with HSV-tk negative cells at varying proportions. Results DOTAP mediatedHSV-tk gene transfer into C6 cells showed 30%-40% of transfection efficiency. HSV-tk infected C6 glioma cellswere rendered sensitive to concentrations of GCV that were 3-4 logs lower than uninfected cells, with an IC05 of0.087μmol/L. In terms of the bystander effect, the viability of co-cultured cells decreased with increasingpopulations of HSV-tk positive cells after GCV treatment. Conclusion C6 cells were successfully transfected withthe HSV-tk gene via cationic amphiphile and displayed a strong bystander effect after GCV treatment. Cationicamphiphile - mediated HSV- tk/GCV chemosensitivity System may have promise as an intratumoral treatment forglioma.

Role of angiotensin converting enzyme and angiotensinogen gene polymorphisms in angiotensin converting enzyme inhibitor-mediated antiproteinuric action in type 2 diabetic nephropathy patients

AIM To investigate the role of genetic variants of angiotensin converting enzyme(ACE) and angiotensinogen(AGT) genes in the antiproteinuric efficacy of ACE inhibitor therapy in diabetic nephropathy(DN) patients.METHODS In the present study, 270 type 2 diabetes mellitus patients with nephropathy were enrolled and treated with ACE inhibitor(ramipril) and followed at 6 mo for renal function and albumin excretion by estimating serum creatinine, end stage renal disease, and albumin/creatinine ratio(ACR) in urine. Genotyping of ACE I/D and AGT M235 T polymorphisms were performed by using primer specific polymerase chain reaction(PCR) and PCR-RFLP techniques, respectively. RESULTS Forty-eight percent of DN patients(responders) benefited with respect to proteinuria from ACE inhibitor therapy at 6 mo follow-up. A significant reduction in ACR was observed after 6 mo treatment with ACE inhibitor irrespective of whether DN patients were micro-albuminuric(≥ 30 and < 300 mg/g creatinine) or macro-albuminuric(≥ 300 mg/g creatinine) at the time of enrollment. However, macro-albuminuric patients(55%) showed better response to therapy. A reduction in urinary ACR was found independent of genotypes of ACE I/D and AGT M235 T polymorphisms although macro-albuminuric patients having TT genotype showed statistically insignificant increased response(72%). CONCLUSION ACE inhibitor therapy reduced urinary ACR by ≥ 30% in 50% of DN patients and the response is independent of ACE I/D and AGT M235 T polymorphisms.