Cell-wall Invertases from Rice are Differentially Expressed in Caryopsis during the Grain Filling Stage

Cell-wall invertase plays an important role in sucrose partitioning between source and sink organs in higher plants. To investigate the role of cell-wall invertases for seed development in rice （Oryza sativa L.）, cDNAs of three putative cell- wall invertase genes OsCIN1, OsCIN2 and OsCIN3 were isolated. Semi-quantitative reverse transcription-polymerase chain reaction analysis revealed different expression patterns of the three genes in various rice tissues/organs. In developing caryopses, they exhibited similar temporal expression patterns, expressed highly at the early and middle grain filling stages and gradually declined to low levels afterward. However, the spatial expression patterns of them were very different, with OsCIN1 primarily expressed in the caryopsis coat, OsCIN2 in embryo and endosperm, and OsCIN3 in embryo. Further RNA in situ hybridization analysis revealed that a strong signal of OsCIN2 mRNA was detected in the vascular parenchyma surrounding the xylem of the chalazal vein and the aleurone layer, whereas OsCIN3 transcript was strongly detected in the vascular parenchyma surrounding the phloem of the chalazal vein, cross-cells, the aleurone layer and the nuceUar tissue. These data indicate that the three cell-wall invertase genes play complementary/synergetic roles in assimilate unloading during the grain filling stage. In addition, the cell type-specific expression patterns of OsCIN3 in source leaf blades and anthers were also investigated, and its corresponding physiological roles were discussed.

Effects of Iron and Aluminum Oxides and Kaolinite on Adsorption and Activities of Invertase *1

Experiments were conducted to study the influences of synthetic bayerite, non crystalline aluminum oxide (N AlOH), goethite, non crystalline iron oxide (N FeOH) and kaolinite on the adsorption, activity, kinetics and thermal stability of invertase. Adsorption of invertase on iron, aluminum oxides fitted Langmuir equation. The amount of invertase held on the minerals followed the sequence kaolinite > goethite > N AlOH > bayerite > N FeOH. No correlation was found between enzyme adsorption and the specific surface area of minerals examined. The differences in the surface structure of minerals and the arrangement of enzymatic molecules on mineral surfaces led to the different capacities of minerals for enzyme adsorption. The adsorption of invertase on bayerite, N AlOH, goethite, N FeOH and kaolinite was differently affected by pH. The order for the activity of invertase adsorbed on minerals was N FeOH > N AlOH > bayerite > reak goethite > kaolinite. The inhibition effect of minerals on enzyme activity was kaolinite > crystalline oxides > non crystalline oxides. The pH optimum of iron oxide and aluminum oxide invertase complexes was similar to that of free enzyme (pH 4.0), whereas the pH optimum of kaolinite inv ertase complex was one pH unit higher than that of free enzyme. The affinity to substrate and the maximum reaction velocity as well as the thermal stability of combined invertase were lower than those of the free enzyme.

Research Progress in Plant Invertase

Invertase is a key enzyme in sucrose catabolism and crucial for plant assimilate distribution. With the development of molecularbiology, a lot of invertsae genes were cloned recently, and significant progress have been made in regulators on the expression of invertase genes.Thus, this article summarized theresearch progress of invertase in biological characteristics, molecular characteristics and expression regulation.

Vacuolar invertase genes SbVIN1 and SbVIN2 are differently associated with stem and grain traits in sorghum (Sorghum bicolor)

In higher plants, vacuolar invertases play essential roles in sugar metabolism, organ development, and sink strength. In sorghum(Sorghum bicolor), two vacuolar invertase genes,Sb VIN1(Sobic. 004 G004800) and Sb VIN2(Sobic. 006 G160700) have been reported, but their enzymatic properties and functional differences are largely unknown. We combined molecular, biochemical and genomic approaches to investigate their roles in sorghum stem and grain traits. Sb VIN1 and Sb VIN2 showed different expression levels in internodes,leaves, and panicles, indicating that their importance in each organ was different. In an in vitro sucrose hydrolysis assay, proteins of both genes heterologously expressed in Pichia pastoris displayed similar enzyme properties including the same optimum reaction p H(5)and similar Kmfor sucroe(49 mmol L-1 and 45 mmol L-1 for Sb VIN1 and Sb VIN2,respectively). The optimum reaction temperatures of Sb VIN1 and Sb VIN2 were 45 °C and65 °C, respectively. Sb VIN2 showed higher tolerance to high temperature than Sb VIN1. We characterized the sequence variation of these two vacuolar invertase genes in a panel of 216 diverse inbred lines of sweet and grain sorghum and performed gene-based association analysis. Sb VIN1 showed significant associations with stem traits including stem length,stem diameter, internode number, stem fresh weight, and Brix, as well as grain traits including hundred-grain weight and grain width. Significantly associated variation sites were mainly in 5′ upstream and intron regions. Sb VIN2 only associated with grain width and stem water-soluble carbohydrates(WSCs) content. We conclude that the vacuolar invertase genes Sb VIN1 and Sb VIN2 are differently associated with stem and grain traits in sorghum.

Partition coefficient prediction of Baker's yeast invertase in aqueous two phase systems using hybrid group method data handling neural network

A hybrid GMDH neural network model has been developed in order to predict the partition coefficients of invertase from Baker's yeast. ATPS experiments were carried out changing the molar average mass of PEG(1500–6000 Da), p H(4.0–7.0), percentage of PEG(10.0–20.0 w/w), percentage of MgSO_4(8.0–16.0 w/w), percentage of the cell homogenate(10.0–20.0 w/w) and the percentage of MnSO_4(0–5.0 w/w) added as cosolute. The network evaluation was carried out comparing the partition coefficients obtained from the hybrid GMDH neural network with the experimental data using different statistical metrics. The hybrid GMDH neural network model showed better fitting(AARD = 32.752%) as well as good generalization capacity of the partition coefficients of the ATPS than the original GMDH network approach and a BPANN model. Therefore hybrid GMDH neural network model appears as a powerful tool for predicting partition coefficients during downstream processing of biomolecules.

Expression of Invertase Genes in Virus-free Sugarcane

Virus-free sugarcane seedlings have improved biomass and sucrose content compared with ordinary seedlings, and sucrose invertases are key enzymes regulating sugarcane growth and sucrose accumulation. In this study, the differences in the expression levels of 3 invertase genes, CWI, SAI and NI, between virus- free and ordinary sugarcane seedlings were analyzed. Compared with ordinary sugarcane plants, the expression of CWI was mainly up-regulated in immature leaves and stems at elongation stage and leaves and immature internodes at maturation stage, and especially, greatly up-regulated in immature interuedes at maturation stage of virus-free plants. The expression of SAI and NI were mainly up-regnlated in leaves and immature internedes of virus-free plants at maturation stage, which might be beneficial to sugar accumulation and rapid utilization of monosaccharide in the stalks of virus-free plants. It is further indicated that virus-free treatment could significantly improve the expression of sucrose invertases at late growth period, and might facilitate the increase of plant biomass.

Genome-wide analysis of the invertase genes in strawberry(Fragaria×ananassa)

Sugar is an important material basis in fruit development,and strawberry fruit flavour and sweetness largely depend on the sugar content and variety.Invertases(INVs)play an important role in the regulation of sugar accumulation because they irreversibly catalyse the hydrolysis of sucrose into the corresponding nucleoside diphosphate-glucose,glucose or fructose in fruit.In this work,we provided a comprehensive analysis of the INV gene family in octoploid strawberry(Fragaria×ananassa),including the gene structure,chromosomal locations,conserved domains,and gene evolution and expression profiles during strawberry fruit development.Our study revealed that polyploid events resulted in the abundant amplification(almost three-or four-fold)of the INV gene in the F.×ananassa genome,and these amplified INV genes showed dominant expression in strawberry fruit.More than half of the FaINVs transcripts with low expression had incomplete coding sequences by alternative splicing.Previous studies have shown that cell wall invertases(CWINV)are involved in the regulation of phloem unloading and sink strength establishment.The expression of FaCWINV1 was markedly upregulated during fruit development and strongly expressed in ripe fruit.Moreover,a significant correlation was observed between the total sugar content and the FaCWINV1 expression level.These findings suggest that FaCWINV1 may be involved in sugar accumulation in strawberry fruit.Taken together,the results of our study will be beneficial for further research into the functions of INVs in the regulation of fruit ripening.

Genome-Wide Identification and Expression Profiling Suggest that Invertase Genes Function in Silique Development and the Response to Sclerotinia sclerotiorum in Brassica napus

Invertase(INV),a key enzyme in sucrose metabolism,irreversibly catalyzes the hydrolysis of sucrose to glucose and fructose,thus playing important roles in plant growth,development,and biotic and abiotic stress responses.In this study,we identified 27 members of the BnaINV family in Brassica napus.We constructed a phylogenetic tree of the family and predicted the gene structures,conserved motifs,cis-acting elements in promoters,physicochemical properties of encoded proteins,and chromosomal distribution of the BnaINVs.We also analyzed the expression of the BnaINVs in different tissues and developmental stages in the B.napus cultivar Zhongshuang 11 using qRT-PCR.In addition,we analyzed RNA-sequencing data to explore the expression patterns of the BnaINVs in four cultivars with different harvest indices and in plants inoculated with the pathogenic fungus Sclerotinia sclerotiorum.We used WGCNA(weighted coexpression network analysis)to uncover BnaINV regulatory networks.Finally,we explored the expression patterns of several BnaINV genes in cultivars with long(Zhongshuang 4)and short(Ningyou 12)siliques.Our results suggest that BnaINVs play important roles in the growth and development of rapeseed siliques and the defense response against pathogens.Our findings could facilitate the breeding of high-yielding B.napus cultivars with strong disease resistance.

Using a Membrane Reactor for Sucrose Hydrolysis： The Effect of Reactor＇s Design on Invertase Stability

Invertase hydrolyses sucrose, produces inverted sugar syrup, which is used, mainly, as a food composition in industries. To carry out the hydrolysis properly, the invertase should be stable in the soluble form through a considerable reaction period and recovered afterwards. The chosen reactor was a CSTR-type （continuous stirred tank reactor-type） coupled with an UFM （ultrafiltration-membrane）, the so-called MR （membrane reactor）. The varied parameters were： sucrose concentration （10-300 mM）, temperature （5-65 ℃）, reaction volume （14 mL and 65 mL）, stirring （100-500 rpm）, volumetric feeding rate （2.2-12 mL/h） and UFM MWCO （molecular weight cut off） （10, 20, 30, 50 and 100 kDa）. The invertase kinetic constants （KM = 23.5 mM; Vmax = 2,758 μmolgluJmin-mgprot; Ea = 9.1 kcal/mol） and the temperature deactivation energy （Ead= 20 kcal/mol） were calculated. Moreover, the invertase was unstable as the MR capacity diminished and the agitation increased up to 500 rpm most likely due to the damaging effect of shearing forces （present inside the MR） on the invertase molecule. Finally, both the MWCO and the chemical nature of the UFM affected the invertase stability along the hydrolysis. The enzyme stability increased as the UFM cut-off decreased, the highest value being observed with the 10 kDa-UFM.

Pollen Germination and Pollen Tube Elongation of Tomato （Lycopersicum esculentum L.） Regulated by Cell Wall Invertase through Sucrose Hydrolysis

Sucrose is a sugar required for pollen germination and pollen tube elongation. It is little known on the regulation mechanism. As such, this research was conducted to reveal mechanism pathway of the sugar in regulating pollen germination and pollen tube elongation by cell wall invertase. The pollen grains, respectively originated from wild type and transgenic tomato plants, which had been silenced their inhibitor gene （INVINH1） of the cell wall invertase were used in this study. The pollen grains were cultured in media containing glucose, fi＇uctose or sucrose. Results of the study showed that percentage of the pollen germination derived from transgenic plants was much higher than that from wild type plants. Moreover, pollen tube elongation was longer in transgenic plants compared with wild type plants. Interestingly, these results were observed in medium containing sucrose only, but not in glucose and fructose. This result suggests that cell wall invertase activity regulates pollen germination and pollen tube elongation through sucrose hydrolysis.

Effects of copper pollution on the activity of soil invertase and urease in loquat orchards

Dealt with in this paper is the relationship between Cu contents of soil and activity parameters for soil invertase and urease from the Changtai loquat orchards in Fujian Province. The soil invertase activity and maximum reaction velocity (enzyme kinetic parameters: Vmax and Vmax/Km) have no remarkable negative correlation with Cu concentrations, but Cu concentrations have an obvious positive effect on invertase Km, indicating a reversible competitive restraint on the reaction between Cu and invertase. The soil urease activity and maximum reaction velocity (Vmax,Vmax/Km) have a remarkable negative correlation with Cu concentrations, and in this aspect, they can be used as ecological indices of the soil polluted by Cu. A slightly variable Km indicates the non-reversible competitive restraint between Cu and soil urease. Cu concentration= 2.00 mg·L-1 in soil is an indicative of the moderate Cu pollution level.

Low Peptone Dose as Inductor of Alkaline Protease Promoter Used for Invertase Gene Expression in Yarrowia lipolytica

According literature, the induction of Yarrowia lipolytica alkaline protease promoter （PXPR2） is efficient in pH 〉 6.0 and with high peptone dose. To establish optimal pH and peptone concentration for induction of invertase gene （suc2 of Saccharomyces cerevisaie） under PXPR2 in new Y. lipolytica A-101 invertase positive （Suc＋） transformants their growth on Bioscreen C was analyzed. Minimal mineral medium with thiamine （MMT） and sucrose （1%）, adjusted to pH from 5.8 to 7.6 and supplemented by 0-0.1% of peptone was used. Biomass （OD）, maximal specific growth rate （μmax） and consumed sucrose were measured. Maximal yeasts growth, resulting from the optimal PXPR2 induction, was observed at pH 7.2 and with very low peptone doses （0.0025% and 0.01%）. For five clones （A-101 1356-5; A-101 B54-6; A-101 B57-4; A-101 A18 and W29 ura3-302） only 0.005% of peptone was needed. Amount of hydrolyzed sucrose varied from 24% to 83% and μmax from 0.06 to 0.28 hl. Suc^＋ clones differ in growth parameters, so the site of yeast cassette integration into genome influences expression level of suc2 under PXPR2. Designing large scale processes with Y. lipolytica Suc^＋ clones peptone concentration has to be 100 times smaller than recommended so far.

Production of Invertases by Anamorphic(Aspergillus nidulans)and Teleomorphic(Emericela nidulans)Fungi under Submerged Fermentation Using Rye Flour as Carbon Source

The production of invertases by anamorph (A. nidulans) and teleomorph (E. nidulans) was investigated. The best level of extracellular enzymatic production for anomorph was obtained in Khanna medium containing sucrose as carbon source, whereas for teleomorph the best production was archived using M5 medium containing inulin as carbon source. Despite this, rye flour was selected as carbon source. The extracellular enzyme production was higher for teleomorph than that observed for anomorph for all carbon sources used. The enzyme production was inhibited by the addition of fructose and glucose in the medium containing rye flour as carbon source. The best conditions to recover the higher enzymatic activity were temperature of 54℃ - 62℃ and pH of 4.8 5.6 for both enzymes determined by experimental design (CCRD). The stability of the temperatures at 40℃ and 50℃were similar for both enzymes. The invertases from the anomorph and teleomorph were activated by Mn2+, but the response of each one towards the presence of this cation was different with best activation observed for the anomorph enzyme (+80%). The extracellular enzymes were able to hydrolyze inulin, sucrose and raffinose. However, the affinity was higher for sucrose than inulin. In conclusion, the carbon source assimilation and the invertase production, as well as the enzymes properties, were different for the anomorph and teleomorph mycelia.

菠萝AcNINV家族全基因组分离及表达分析

【目的】鉴定出菠萝NINV家族全基因组成员,初步阐明NINV基因与蔗糖代谢的关系。【方法】采用生物信息学方法鉴定并分析菠萝NINV家族全基因组成员,通过实时荧光定量PCR分析其表达特性,利用HPLC对蔗糖含量进行测定。【结果】在菠萝中共鉴定出6个NINV基因,分布于5条不同染色体上,其二级结构主要由α-螺旋和无规则卷曲组成,该基因家族启动子区域光反应元件数量最多。AcNINV外显子数量介于4~6个之间,其中AcNINV3、6外显子的数量为6个,亚细胞定位预测发现这2个基因都分布于叶绿体,属于α亚族;AcNINV1、2、4、5外显子的数量为4个,亚细胞定位预测发现这4个基因都分布于质膜,属于β亚族。在果柄、果皮、果心中表达量最高的是AcNINV2基因,在果肉中表达量最高的是AcNINV6基因。蔗糖含量随着菠萝果实成熟呈先升高后降低的变化趋势,而AcNINV4基因在菠萝果实成熟过程中呈下调表达。【结论】AcNINV4可能是催化果实蔗糖降解的水解酶基因。

番茄液泡蔗糖转化酶抑制蛋白克隆、生物信息学及表达分析

液泡转化酶抑制蛋白(vacuolar invertase inhibitor, VIF)在调节植物蔗糖代谢,调控生长发育及响应逆境等方面发挥重要作用。本试验从番茄Alisa中克隆到VIF基因(SlVIF)和cDNA序列。结果显示,SlVIF基因与cDNA序列全长均为528 bp,不含有内含子,共编码175个氨基酸。SlVIF蛋白分子量为19.92 ku,理论等电点为5.53,含有一个信号肽序列,保守结构域为PMEI。系统进化关系表明,茄科物种的VIF亲缘关系最近,且基序具有一致性。组织特异性表达结果显示,在不同的番茄材料中,SlVIF的表达模式存在差异,在Heinz番茄中,其在根部有高水平的表达;在LA1589番茄中,其在果实发育的各阶段,尤其是在成熟果实及根部有高水平的表达。说明SlVIF可能通过介导蔗糖代谢影响果实成熟并调控根系生长。SlVIF受到水分胁迫的诱导,响应非生物胁迫过程。

双启动子过表达蔗糖转化酶Suc2酿酒酵母菌株的构建

为了提高蔗糖转化酶Suc2在酿酒酵母中的表达水平,本研究利用串联双启动子联合启动Suc2基因表达,构建高效菊糖基乙醇的生产菌株。首先构建含有串联磷酸甘油酸激酶组成型强启动子PGK1p的Suc2重组表达载体pYC230-DP-mSuc2,电击转化至酿酒酵母BY4741中,成功构建重组菌BY-DP。经实时荧光定量PCR、高效气相色谱和摇瓶培养验证,证明了在含15%菊糖的培养基中发酵48 h后,重组菌BY-DP具有更高的Suc2基因转录水平、酶活以及乙醇产量,与含单个PGK1p启动子的对照重组菌BY-mS相比,分别提高了30.0%、18.6%和25.3%。实验通过构建串联双PGK1p启动子,有效强化了Suc2的表达,提高了重组菌发酵菊糖生产乙醇能力。

甘薯CIN基因家族鉴定及影响块根膨大关键CIN基因的挖掘

细胞质转化酶(CIN)催化蔗糖不可逆地水解为葡萄糖和果糖,在模式植物根系生长发育中发挥着重要作用。目前尚未对甘薯CIN基因家族进行研究,更不清楚CIN在甘薯块根膨大中的具体作用。本研究系统鉴定了甘薯CIN基因家族的种类和数量并分析其蛋白质理化性质、染色体定位、系统发育、基因结构和保守基序、启动子顺式元件,同时,分析甘薯CIN家族基因在不同组织部位进行的表达特异性分析及其在不同类型根系中的表达水平和酶活性,初步筛选出在甘薯块根膨大中发挥重要作用的关键候选CIN基因。结果如下:(1)从甘薯(Ipomoeabatatas)基因组中共鉴定出12个IbCIN基因(IbCIN1-12),分布于8条染色体上,编码蛋白质的氨基酸数量范围为417~825 aa,分子量范围为46.60~93.75 kDa,等电点范围为4.83~7.17。(2)系统发育分析发现IbCIN可分为3个亚组,其中α1亚组1个,α2亚组3个,β亚组8个。α1、α2亚组成员之间在保守基序和基因结构上差异较小、相对保守,而β亚组成员之间则差异较大,表明β亚组的IbCIN基因可能在功能上更为多样,可以参与更多的生物过程。此外,IbCIN1、IbCIN7、IbCIN10、IbCIN12与拟南芥和木薯根系发育相关CIN基因的亲缘关系较近,推测其可能与非块根类型根系发育密切相关。(3)IbCINs在不同组织部位(幼叶、成熟叶、茎、花、60 d块根)和不同类型根系(白色纤维根、红色纤维根、柴根和60 d块根)中的表达水平分析表明,IbCIN4、IbCIN8和IbCIN11在块根中的表达水平最高,同时块根中的CIN酶活性也显著高于非块根类型根系,推测这3个基因可能在甘薯块根膨大中发挥着重要作用。(4)生物信息学分析进一步揭示IbCIN4、IbCIN8和IbCIN11可能通过调控蔗糖转运和代谢、光周期反应、赤霉素信号转导等途径和机制来共同促进甘薯块根的发育。本研究可为后续通过转基因技术等手段深入研究甘薯CIN基因的功能奠定基础。

细胞壁转化酶调控番茄响应灰霉菌侵染的转录组分析

由死体营养型病菌(necrotrophic pathogen)灰葡萄孢菌(Botrytis cinerea)侵染引起的灰霉病是番茄生产上的常见病害,可导致番茄大面积减产甚至绝收。蔗糖分解代谢在植物抗病中发挥着重要作用,不仅可为植物防御反应提供碳骨架和能量,还可通过信号途径调控抗病基因的表达。研究表明,分解蔗糖的细胞壁转化酶(cell wall invertase,CWIN)可增强植物对死体营养型病害的抗性。然而,目前尚无CWIN对番茄灰霉病抗性影响的相关研究。本研究以CWIN活性上调的转基因番茄(RNAi/R)及野生型番茄(WT/W)为材料,对其叶片进行灰霉菌(Bc)离体接种,研究CWIN对番茄灰霉病抗性的影响,同时对接种后12、60 h的叶片进行取样,通过转录组测序初步阐明CWIN调控番茄灰霉病抗性的分子机制。结果表明:(1)提高番茄CWIN活性可增强番茄叶片对灰霉病菌的抗性。(2)KEGG注释表明,接种12 h的DEGs(W-Bc-12 h-vs-R-Bc-12 h)共获得5个显著性富集通路,包括次生代谢物生物合成(biosynthesis of secondary metabolites)、代谢途径(metabolic pathways)、DNA复制(DNA replication)、淀粉和蔗糖代谢(starch and sucrose metabolism)以及甾族化合物生物合成(steroid biosynthesis);接种60 h的DEGs(W-Bc-60 h-vs-R-Bc-60 h)则未发现显著性富集通路,表明接种早期是CWIN调控番茄抗病性的关键时期。(3)植物-病原菌互作图分析表明,参与超敏反应和防御相关基因诱导的富含亮氨酸重复(LRR)受体类丝氨酸/苏氨酸蛋白激酶基因FLS2和热激蛋白基因HSP90在接种后的RNAi叶片中上调,其可能是CWIN增强番茄抗病性机制中的重要基因。(4)植物激素信号通路和MapMan作图分析表明,接种后RNAi番茄的抗病激素茉莉酸(JA)和乙烯(ET)信号途径上调,同时水杨酸(SA)信号途径减弱,表明其可能共同作用提高RNAi番茄的抗病性。此外,接种后RNAi番茄叶片的生长促进激素生长素(IAA)和细胞分裂素(CTK)信号途径增强,而衰老促进激素脱落酸(ABA)信号途径减弱,这样可以抑制病菌侵染期间细胞的死亡,从而阻止死体营养型病菌灰霉病菌从死亡寄主细胞上获得必要的养分用于侵染。此外,MapMan作图还揭示接种后RNAi番茄叶片在细胞壁增厚、蛋白水解、活性氧(氧化还原状态和过氧化物酶)、次生代谢物方面也得到极大的加强,这些途径均有助于提高番茄的抗病性。综上,提高CWIN活性可增强番茄灰霉病抗性,转录组分析不仅验证已有的CWIN抗病分子机制,如细胞壁加厚、活性氧积累和超敏反应(HR)、抗病激素积累(SA和JA/ET)、病程相关蛋白(如HSP和PR)和次生代谢物的合成(如植物毒素和多酚等)等,而且还挖掘出一些新的CWIN抗病机制,包括生长促进激素(IAA和CTK)和衰老促进激素(ABA)信号途径和蛋白水解途径。研究结果可为下一步利用基因工程、分子育种等现代生物技术手段提高番茄灰霉病抗性提供理论指导。

绿肥还田对土壤有机碳组分及碳转化酶活性的影响

【目的】探究绿肥还田对旱作麦田土壤有机碳组分和碳转化酶活性的影响,为土壤质量提升及“碳中和”目标的实现提供数据支撑。【方法】在甘肃陇东旱塬典型黑垆土上开展毛苕子(Vicia villosa Roth)-冬小麦(Triticum aestivum L.)和饲用油菜(Brassica napus L.)-冬小麦5年轮作系统绿肥还田试验,分析毛苕子、饲用油菜覆盖和翻压还田后冬小麦不同生育时期4个土层(0—5、5—10、10—20、20—25 cm)土壤有机碳(SOC)、易氧化有机碳(EOC)、微生物量碳(MBC)含量和β-1,4-葡萄糖苷酶(βG)、纤维二糖水解酶(CBH)、β-木糖苷酶(βX)、几何平均酶(GMEA)活性的变化特征。【结果】绿肥还田方式对土壤有机碳组分含量及CBH和βX活性有显著影响。与覆盖还田相比,毛苕子和饲用油菜翻压还田能够使0—25 cm土层土壤SOC、EOC和MBC含量提高12.9%、12.1%、53.8%,βG和CBH活性提高了3.2%、10.2%,且对20—25 cm土层的影响最显著。冬小麦不同生育时期的土壤活性有机碳含量及土壤酶活性有显著差异,其中土壤EOC和MBC含量分别在冬小麦成熟期和返青期达到最高,βG、CBH、βX和GMEA活性在冬小麦孕穗期达到最高。βG在不同覆盖方式下变化最显著,活性最高,是绿肥还田后参与土壤碳转化过程的主要酶。不同土层土壤碳组分含量和碳转化酶活性存在显著差异,均随着土层深度的增加而降低。绿肥类型也显著影响土壤碳组分及酶活性,其中饲用油菜还田土壤SOC、MBC含量和βG、CBH、βX、GMEA活性是毛苕子还田的1.08、1.21、1.15、1.23、1.19、1.18倍。结构方程模型表明,绿肥还田方式通过调控其累积分解速率影响SOC的积累,通过影响土壤pH、SOC的累积及绿肥累积分解速率影响GMEA活性。土壤有机碳累积受绿肥还田量影响的效应大于还田方式,而碳转化酶活性反之。【结论】黄土高原夏闲期种植饲用油菜并翻压还田能提高0—25 cm土层中有机碳组分含量和碳转化酶活性,是黄土高原夏闲期资源高效利用的有效措施。

刺梨果实转化酶提取方法比较及酶学特性分析

刺梨(Rosa roxburghii Tratt.)果实转化酶(蔗糖酶)通过调控蔗糖分解影响果实的生长发育和品质形成。提取方法能够影响植物转化酶的提取效率和活性。以“贵农7号”刺梨花后45 d的果实为材料,比较了液氮研磨样品和提取液冰上研磨样品下6种提取转化酶方法的效果,探究了可溶性转化酶的酶学特性。结果表明,液氮研磨法对刺梨果实转化酶活性影响较大,其中可溶性酸性转化酶(soluble acid invertase,SAI)活性丧失严重,而细胞壁酸性转化酶(cell wall-bound invertase,CWI)和中碱性转化酶(neutral or alkaline invertaes,N/A-INV)均无活性。采用提取液冰上研磨样品,用Hepes-NaOH法提取刺梨果实转化酶的效果最好,SAI、CWI和N/A-INV的活性最高,分别为3.16、2.09和2.09μmol/(g·h),磷酸钠法次之,且Hepes-NaOH法提取的可溶性转化酶(SAI和N/A-INV)的比活性最高。酶学特性分析表明,SAI的最适温度为60℃,最适pH值为4.5~5.0,Km值为87.15 mmol/L;N/A-INV的最适温度为45~50℃,最适pH值为8.5,K m值为3.46 mmol/L。研究结果为刺梨果实转化酶的提取和转化酶生物学功能的深入研究奠定了基础。