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

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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.

Research Progress in Plant Invertase

Abstract 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.

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.

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.

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。

前蛋白转化酶枯草溶菌素9抑制剂与阿托伐他汀联用对介入治疗急性心肌梗死术后的影响

目的:分析急性心肌梗死(acute myocardial infarction,AMI)患者行经皮冠状动脉介入(percutaneous coronary intervention,PCI)术治疗后联用前蛋白转化酶枯草溶菌素9(proprotein convertase subtilisin kexin type 9,PCSK9)抑制剂与阿托伐他汀治疗对其心室功能、炎性因子、血脂水平及主要不良心血管事件(major adverse cardiovascular events,MACE)的影响。方法:选择2020年9月至2021年9月,本院收治的98例AMI并行PCI术治疗患者加以研究,随机数字表法分观察组及对照组,各49例,对照组予以阿托伐他汀治疗,观察组于对照组的基础之上联用PCSK9抑制剂治疗;比较两组治疗前与治疗3个月后LVEDD、LVESD及LVEF、hs-CRP、CK-MB、TNF-α、PCSK9、TG、载脂蛋白B(pdipop oteir B,apoB)、LDL-C的变化,比较用药60d中MACE事件以及不良反应的发生状况。结果:治疗后,两组LVEDD、LVESD、hs-CRP、CK-MB、TNF-α、PCSK9、TG、apoB、LDL-C水平较治疗前均显著降低(P<0.05),LVEF水平较治疗前显著升高(P<0.05),且观察组LVEDD、LVESD、hs-CRP、CK-MB、TNF-α、PCSK9、TG、apoB、LDL-C水平的降低值及LVEF水平的升高值均大于对照组(P<0.05)。两组Ⅱ级、Ⅲ级心力衰竭、靶血管血运重建、心源性死亡、再发心肌梗死的发生率,均差异无统计学意义(P>0.05),而观察组60 d中MACE事件总发生率显著低于对照组(P<0.05)。两组肌痛、心律失常、神经认知异常、恶心呕吐、新发糖尿病的发生率以及不良反应总发生率比较,差异无统计学意义(P>0.05)。结论:与阿托伐他汀单独治疗相比,PCSK9抑制剂与阿托伐他汀联用治疗AMI行PCI术后患者疗效更为显著,更有助于改善心室功能,降低炎性因子与血脂水平,减少MACE事件的发生状况,不会增加不良反应。

StvacINV1负调控马铃薯的耐旱性

植物液泡酸性转化酶不可逆的催化蔗糖转化为葡萄糖和果糖,在植物的生长和发育及其逆境适应中扮演重要的角色。马铃薯液泡转化酶基因StvacINV1参与调控薯块的糖化,但其是否参与对干旱胁迫的调控尚不清楚。本研究以马铃薯品种‘Atlantic’和‘Russet Burbank’的StvacINV1 RNA干扰表达的转基因株系及其野生型为材料,采用停止浇水进行自然干旱处理,研究StvacINV1对马铃薯植株耐旱性的调控机制。结果表明干旱胁迫引起马铃薯叶片StvacINV1的表达量及其液泡酸性转化酶活性显著降低;与野生型相比,干旱胁迫下StvacINV1干扰效率高的转基因株系植株不易失水萎蔫、离体叶片的失水率低,MDA含量低且叶片的相对水分含量高,由此表明StvacINV1干扰效率高的转基因株系的耐旱性高于野生型,即StvacINV1负调控马铃薯的耐旱性;进一步的分析表明,干旱胁迫下StvacINV1干扰效率高的转基因株系的气孔开度和气孔导度显著低于野生型,水分利用率显著高于野生型,因此推测StvacINV1可能通过介导气孔的关闭来调节马铃薯植株的耐旱性;干旱胁迫下StvacINV1干扰株系的蔗糖含量显著高于野生型,且外源高浓度蔗糖处理可诱导气孔的关闭,因此推测StvacINV1可能通过其底物蔗糖参与调控气孔的关闭;外源ABA诱导的气孔关闭过程中,StvacINV1干扰株系比野生型更敏感。综上所述,干旱胁迫下StvacINV1通过介导气孔的关闭负调控马铃薯植株的耐旱性,StvacINV1可能通过其所催化的底物蔗糖参与调控气孔的关闭,StvacINV1也参与ABA介导的气孔关闭。本研究为选育既耐糖化(块茎)又耐干旱的马铃薯品种提供理论依据。

用壳寡糖修饰的方法提高酵母转化酶的催化活性

为了对来自酵母细胞的天然转化酶分子进行改造,以使其适用于工业和医药领域,利用壳寡糖(COS)对酵母转化酶(YInv)的氨基进行修饰。结果发现:COS修饰后的糖链成功连接到转化酶肽链的氨基上,修饰酶(COS-Inv)的分子量比天然酶(YInv)的明显增大;COS-Inv的催化活性比YInv的提高200.3%。YInv和COS-Inv分子中的多糖含量分别为2.75和6.34 mg/mg。COS-Inv的Km值(58.92 mmol/L)比YInv的Km值(125.45 mmol/L)低得多。YInv和COS-Inv的三维荧光光谱测定结果表明,COS-Inv的荧光强度比YInv的荧光强度低得多,说明COS-Inv与YInv两者的三维分子结构有非常大的差别。由此可见,利用壳寡糖修饰酵母转化酶以改造其分子结构是提高其催化活性的有效办法。

尕海湿地区沼泽草甸退化对土壤氮转化酶活性的影响

为探讨高寒湿地退化对土壤氮转化酶活性的影响,以青藏高原东缘尕海湿地未退化(ND)、轻度退化(LD)、中度退化(MD)和重度退化(HD)4种不同退化程度0~40 cm土层沼泽草甸为研究对象,研究不同退化与土层中土壤氮转化酶(蛋白酶、脲酶、硝酸还原酶和亚硝酸还原酶)活性的变化特征及其与土壤理化性质之间的关系。结果表明:1)随沼泽草甸退化程度加剧,土壤含水量、全氮、铵态氮和微生物生物量氮含量均显著降低,土壤温度与硝态氮含量却显著增加。2)随退化程度加剧,各土层土壤脲酶活性增加、蛋白酶活性降低,且仅在20~40 cm土层存在显著差异;硝酸还原酶活性增加、亚硝酸还原酶活性降低,在0~20 cm土层存在显著差异。3)各退化程度中,土壤脲酶、蛋白酶、亚硝酸还原酶活性均随土层深度的增加而显著下降,硝酸还原酶活性仅在HD显著下降。4)退化程度和土层对4种土壤氮转化酶活性均存在显著影响,且对土壤硝酸酶和亚硝酸还原酶活性存在显著交互作用。5)冗余分析表明土壤含水量对土壤氮转化酶活性变化的贡献率高达67.1%,其是驱动尕海沼泽草甸退化演替过程中土壤氮转化酶活性变化的主导因素。研究结果可为高寒湿地生态系统退化中的土壤酶活性变化规律提供理论依据。

葡萄蔗糖转化酶基因家族的生物信息学及响应逆境胁迫分析

蔗糖转化酶(invertase,INV)在植物生长发育和抵御胁迫中发挥着重要作用。研究从葡萄基因组数据库中鉴定出19个蔗糖转化酶基因,对基因结构和编码蛋白质的理化性质进行生物信息学分析,并利用qRT-PCR技术分析基因在不同激素和非生物胁迫条件下的表达特征,为进一步探索葡萄INV基因家族参与葡萄逆境响应提供了一定的理论依据。结果表明,(1)该基因家族编码蛋白的氨基酸长度在150~766 aa之间,理论等电点介于4.43~9.1之间,亚细胞定位预测发现其主要在细胞质中表达,此外液泡和细胞壁也存在部分基因表达;(2)共线性结果显示VvCINV与其他5个物种复制频率较高;(3)保守基序分析表明VvCwINV包含了所有的保守基序,且Glyco_32和Glyco_hydro_100是VvINV基因主要结构域;(4)组织特异性表达分析发现多数基因在葡萄生长发育进程中都有表达;(5)qRT-PCR分析结果显示,VvINV基因家族在叶片中对激素处理和非生物胁迫的响应出现上调,VvCINV1在50 mg/L GA_(3)和10%PEG处理后上调表达极显著,VvCINV4在盐胁迫、ABA和低温处理后也上调表达显著。这些研究结果表明,VvINVs可能调控葡萄生长发育过程中激素和对非生物胁迫的响应。