Vacuolar invertases in sweet potato: molecular cloning, characterization, and analysis of gene expression

Two cDNAs (Ib beta fruct2 and Ib beta fruct3) encoding vacuolar invertases were cloned from sweet potato leaves, expressed in Pichia pastoris, and the recombinant proteins were purified by ammonium sulfate fractionation and chromatography on Ni-NTA agarose. The deduced amino acid sequences encoded by the cDNAs contained characteristic conserved elements of vacuolar invertases, including the sequence R[G/A/P]xxxGVS[E/D/M]K[S/T/A/R], located in the prepeptide region, Wxxx[M/I/V]LxWQ, located around the starting site of the mature protein, and an intact beta-fructosidase motif. The pH optimum, the substrate specificity, and the apparent K(m) values for sucrose exhibited by the recombinant proteins were similar to those of vacuolar invertases purified from sweet potato leaves and cell suspensions, thus confirming that the proteins encoded by Ib beta fruct2 and Ib beta fruct3 are vacuolar invertases. Moreover, northern analysis revealed that the expression of the two genes was differentially regulated. With the exception of mature leaves and sprouting storage roots, Ib beta fruct2 mRNA is widely expressed among the tissues of the sweet potato and is more abundant in young sink tissues. By contrast, Ib beta fruct3 mRNA was only detected in shoots and in young and mature leaves. It appears, therefore, that these two vacuolar invertases play different physiological roles during the development of the sweet potato plant.     

番木瓜酸性转化酶基因家族预测及生物信息学初步分析

酸性转化酶参与了番木瓜果实中蔗糖的韧皮部卸载,调控果实中糖类的积累。本文利用番木瓜基因组数据库信息,发现了番木瓜基因组中含有6个酸性转化酶基因,分别为细胞壁转化酶基因（CpCWINV1-3）和液泡转化酶基因（CpVINV1-3）。这些基因由3-7个外显子组成,其中CpCWINV1、CpVINV1和CpVINV2的N端均具有一个跨膜区域。蛋白质3D结构预测分析表明,CpCWINV1、CpVINV1和CpVINV2具有典型的N端β-螺旋桨模块（β-propeller module）和C端β-三明治模块（β-sandwich module）,而CpCWINV3和CpVINV3的桨叶I上,均没有保守区NDPNG/A;CpCWINV2和CpVINV3均缺失了β-三明治模块。推测番木瓜细胞壁转化酶CpCWINV1、液泡转化酶CpVINV1和CpVINV2能正确履行催化蔗糖分解的功能。本研究有助于我们进一步揭示酸性转化酶在番木瓜果实发育及糖类积累过程中的作用。     

Molecular cloning and functional identification of invertase isozymes from green bamboo Bambusa oldhamii

Three Bo beta fruct cDNAs encoding acid invertases were cloned from shoots of the green bamboo Bambusa oldhamii. On the basis of the amino acid sequences of their products and phylogenetic analyses, Bo beta fruct1 and Bo beta fruct2 were determined to encode cell wall invertases, whereas Bo beta fruct3encodes a vacuolar invertase. The recombinant proteins encoded by Bo beta fruct2 and Bo beta fruct3 were produced in Pichia pastoris and purified to near homogeneity using ammonium sulfate fractionation and immobilized metal affinity chromatography. The pH optima, pI values, and substrate specificities of the isolated enzymes were consistent with those of plant cell wall or vacuolar invertases. The growth-dependent expression of Bo beta fruct1 and Bo beta fruct2 in the base regions of shoots underscores their roles in sucrose unloading and providing substrates for shoot growth. Its high sucrose affinity suggests that the Bo beta fruct2-encoded enzyme is important for maintaining the sucrose gradient between source and sink organs, while the predominant expression of Bo beta fruct3 in regions of active cell differentiation and expansion suggests functions in osmoregulation and cell enlargement.     

RNA干涉对马铃薯内源酸性转化酶活性的影响

酸性转化酶是植物体内降解蔗糖为还原糖（葡萄糖、果糖）的关键酶,而还原糖在马铃薯（Solanum tuberosum L.）低温贮藏中的快速积累是影响油炸加工品质的重要因素。为了实现对马铃薯块茎低温糖化的品质改良,本研究利用所克隆的酸性转化酶基因构建了RNA干涉载体,并转化马铃薯品种N2。经PCR、Northern鉴定,酸性转化酶基因的干涉片段已经成功导入马铃薯植株中。对干涉转基因株系的试管苗和试管薯的酸性转化酶活性进行测定,结果显示,植株的酸性转化酶的活性平均下降69.8%（Ni-1除外）,最大降幅为78%（Ni-4）,而试管薯的酶活性最大降幅为68%。与反义RNA的表现较好的转基因株系进行比较,RNA干涉对酶活的调节作用与之相当。研究结果表明,RNA干涉对马铃薯内源酸性转化酶活性的调节作用显著,这种转录表达的调控技术对马铃薯低温糖化改良具有良好的应用前景。     

Invertase inhibitors from sweet potato (Ipomoea batatas): purification and biochemical characterization

Two proteinaceous invertase inhibitors, designated ITI-L and ITI-R, were purified to electrophoretic homogeneity. ITI-L was purified from acetone powder of sweet potato leaves through sequential steps entailing buffer extraction, acid treatment, DEAE-Sephacel ion-exchange chromatography, and Sephacryl S-100 gel filtration. ITI-R was purified from sweet potato tuberous roots by sequentially applying buffer extraction, Con A-Sepharose affinity chromatography, DEAE-Sephacel ion-exchange chromatography, Sephacryl S-200, and Superose 12 gel filtration. The optimal pHs for interaction between ITI-L and ITI-R and acid invertase from sweet potato leaves were 5.5 and 5.0, respectively. The molecular masses of ITI-L and ITI-R were 10 and 22 kDa, respectively, as estimated by both gel filtration and SDS-PAGE. Both inhibitors were thermostable (90% of the activity remained after incubation at 100 degrees C for 20 min), and Western blotting showed them to be immunologically related.     

钾素对食用型甘薯糖代谢相关酶活性的影响

为了探讨钾素提高甘薯块根可溶性糖含量的生理基础。选用典型的食用型甘薯品种北京553,设置不同施钾处理,于2009～2010年2个生长季在山东农业大学农学试验站进行试验。采用甘薯块根膨大过程中定期取样的方法,测定块根可溶性糖和淀粉含量及相关酶活性、功能叶蔗糖含量及相关酶活性。结果表明,与对照比较,施用钾肥能显著提高块根产量、可溶性糖及各糖组分含量,其中K2O用量为24 g/m2处理增幅最大,为最适用量。进一步研究发现,适宜供钾处理显著提高了功能叶磷酸蔗糖合成酶活性和蔗糖含量,生育期内平均增幅分别为10.31%和34.13%,同时提高了块根中蔗糖合成酶、不溶性酸性转化酶的活性,生育期内平均增幅为16.47%和3.66%,在提高源端光合产物供应的同时促进蔗糖在库端的卸载,促进块根中淀粉和可溶性糖的积累;适宜供钾处理还提高了块根中-和-淀粉酶的活性,生育期内平均增幅分别为26.06%和14.64%,促进淀粉向可溶性糖转化。此外,适宜供钾处理还显著提高了生长前期和后期块根中可溶性酸性转化酶活性、以及生长后期块根中蔗糖-蔗糖果糖基转移酶活性,促进了葡萄糖、果糖和果聚糖在块根中的积累。在甘薯收获期,块根可溶性糖和淀粉含量分别提高了13.52%和3.02%。即钾肥能够增加块根中蔗糖的供应量、促进块根对蔗糖的吸收、促进淀粉水解,是其提高块根可溶性糖含量的生理原因。     

Changes in invertase activities and reducing sugar content in sweetpotato stored at different temperatures

Cured sweetpotato roots were stored at different temperatures (4.5, 15.6, and 24 degrees C) for 7 weeks and assayed for invertase activities and reducing sugar levels during two separate years. Invertase activities and reducing sugar concentration significantly increased in the roots kept at low temperature. Of the three types of invertases assayed, acid invertase specific activity was the highest. Acid invertase was the most influential in determining reducing sugar levels in stored sweetpotato. Cultivar differences were found in invertase specific activities and reducing sugar concentration. Reducing sugar content was highly correlated to acid and total invertase activity, regardless of cultivar.     

patatin启动子调控烟草液泡转化酶抑制子在马铃薯抗低温糖化中的作用

为了降低马铃薯(Solanum tuberosum )块茎低温贮藏下还原糖的积累，实验构建了马铃薯块茎特异性启动子（CIPP）调控的烟草液泡转化酶抑制子Nt-VIF基因表达载体pBICNI，并转化马铃薯植株。PCR、Northern杂交和Southern杂交分析结果显示，CIPP调控的Nt-VIF基因全长cDNA成功地导入鄂马铃薯3号（E-3）植株。14个转基因株系块茎分别贮藏在4℃和20℃条件下，贮藏1个月后进行还原糖含量和液泡酸性转化酶（VI）活性测定。结果表明，在20℃条件下转基因株系块茎还原糖（RS）含量与对照相比差异不明显，在4℃条件下RS含量则显著下降，与对照相比下降幅度从34%（株系B-13）至76.8%（株系B-1），说明Nt-VIF cDNA在马铃薯中的表达，成功地抑制了液泡酸性转化酶的活性，导致还原糖含量降低。进一步分析表明，转基因块茎低温贮藏其液泡转化酶活性与还原糖含量呈显著的正直线相关（VI = 0.3084RS + 0.0673）。实验获得的B-1、B-2、B-6、B-9、B-14等5个转基因株系，块茎低温贮藏后能直接满足炸片加工对还原糖含量的要求。     

不同乳酸菌发酵甘薯渣产物组分分析

为提高甘薯淀粉加工副产物的高值化利用水平,本试验以植物乳杆菌(Lp)、干酪乳杆菌(Lc)、保加利亚乳杆菌(Lb)、戊糖片球菌(Pp)、嗜热链球菌(St)、商业植物乳杆菌(SZ)6种乳酸菌分别对甘薯渣进行发酵,系统分析不同发酵产物的pH值、总酸含量及营养功能成分,并采用灰色理论加权关联度对甘薯渣发酵产物进行综合营养评价,筛选适宜发酵甘薯渣的乳酸菌菌种。结果表明,6种乳酸菌的发酵能力存在显著差异,其中Lb组pH值最低(3.15),Lc组总酸含量最高(27.90);同时,不同乳酸菌对发酵产物中营养功能成分的影响存在显著差异,其中Lb组乳酸含量最高(11.60 mg·mL^-1),SZ组乙酸含量最高(66.99μg·mL^-1),Lb组可溶性膳食纤维含量最高(0.74 g·100 mL^-1),St组总酚含量最高(146.87μg GAE·mL^-1)。与未发酵样品相比,Lc组游离氨基酸总量提高了3.71倍;所有发酵产物中的Mg、Ca、Fe、Zn、Se等矿物质元素含量均显著提高。进一步通过灰色理论加权关联度分析发现...     

Purification and biochemical characterization of insoluble acid invertase (INAC-INV) from pea seedlings

Invertase (EC 3.2.1.26) catalyzes the hydrolysis of sucrose into D-glucose and D-fructose. Insoluble acid invertase (INAC-INV) was purified from pea (Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation, ion exchange chromatography, absorption chromatography, reactive green-19 affinity chromatography, and gel filtration. The purified INAC-INV had a pH optimum of 4.0 and a temperature optimum of 45 °C. The effects of various concentrations of Tris-HCl, HgCl(2), and CuSO(4) on the activities of the purified invertase were examined. INAC-INV was not affected by Tris-HCl and HgCl(2). INAC-INV activity was inhibited by 6.2 mM CuSO(4) up to 50%. The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis. The K(m) and V(max) values of INAC-INV were determined to be 4.41 mM and 8.41 U (mg protein)(-1) min(-1), respectively. INAC-INV is a true member of the β-fructofuranosidases, which can react with sucrose and raffinose as substrates. SDS-PAGE and immunoblotting were used to determine the molecular mass of INAC-INV to be 69 kDa. The isoelectric point of INAC-INV was estimated to be about pH 8.0. Taken together, INAC-INV is a pea seedling invertase with a stable and optimum activity at lower acid pH and at higher temperature than other invertases.     

甘薯加工过程酶促褐变及控制研究

甘薯加工过程中极易产生酶促褐变。应用分光光度法，研究了甘薯中多酚氧化酶（PPO）的特性及褐变的控制方法。结果表明，甘薯皮部的PPO活性和褐变强度是心部的2倍左右，在pH值为4.5时其活性较低。在甘薯淀粉生产过程中，结合去皮工艺，分别选择添加0.01%～0.05%柠檬酸或0.005%～0.01%抗坏血酸或0.05%～0.1%亚硫酸钠，均可有效控制PPO产生的褐变，提高甘薯淀粉的质量。     

Quantity and potential biological activity of caffeic acid in sweet potato [Ipomoea batatas (L.) Lam.] storage root periderm

The caffeic acid content of storage root periderm and cortex tissues of genetically diverse sweet potato [Ipomoea batatas (L.) Lam.] cultivars and breeding clones was quantified by high-performance liquid chromatography. Periderm caffeic acid content of the clones ranged from 0.008 to 7.97 mg/g dry weight, whereas the highest cortex content was 0.047 mg/g. Clones varied greatly in periderm caffeic acid content in all experiments, but there were also differences between experiments in content averaged for all clones. This indicates that periderm caffeic acid content is subject to genetic and environmental influences. Caffeic acid inhibited the growth of four sweet potato pathogenic fungi and germination of proso millet seeds in bioassays. Inhibitory activity in the bioassays suggests that high periderm caffeic acid levels contribute to the storage root defense chemistry of some sweet potato genotypes.     

外源脱落酸增强甘薯幼苗耐盐性的作用

【目的】本文系统研究了不同浓度NaCl胁迫及外源脱落酸(ABA)对NaCl胁迫下甘薯幼苗生根及一些生理生化特性的影响,探讨了外源ABA对盐胁迫下甘薯幼苗的缓解效应,为增强盐碱地甘薯耐盐性、 提高产量提供理论依据。【方法】以甘薯种植品种徐薯25为实验材料,在装有石英砂的具孔塑料盆中放入培养室自然光照/昼夜温度[(261)/(171)℃]中培养,并进行不同浓度NaCl处理以及对NaCl 300 mmol/L胁迫甘薯幼苗叶片喷施ABA溶液,连续处理7 d后,测定生根数,使用CIRAS-1型便携式光合仪测定光合作用指标、 植物效率分析仪测定叶绿素荧光参数、 采用比色法测定丙二醛、 脯氨酸、 可溶性糖含量和超氧化物歧化酶活性、 用原子吸收分光光度计测定Na+、 K+、 Ca2+含量,利用SPSS13.0和Excel软件对数据进行处理分析。【结果】低浓度 NaCl胁迫(50 mmol/L)对甘薯幼苗影响较小; 随着盐度的增加,甘薯生根不断减少,相对电导率、 丙二醛(MDA)、 脯氨酸和可溶性糖含量持续增加,甘薯叶片超氧化物歧化酶(SOD)活性呈先增加后降低趋势; 叶片净光合速率(Pn)、 蒸腾速率(Tr)、 气孔导度(Gs)、 光系统Ⅱ(PSⅡ)最大光化学效率(Fv/Fm)、 捕获的激子将电子传递到电子传递链中超过QA的其他电子受体的概率(0)、 用于电子传递的量子产额(E0)逐渐降低,放氧复合体活性(Vk)和用于热耗散的量子比率(D0)不断增加; 叶片中Na+含量增加,K+、 Ca2+和K+/Na+水平降低。高浓度(300 mmol/L)NaCl胁迫下,甘薯幼苗的正常生理代谢受到显著抑制。适当增加外源ABA浓度,能够显著缓解NaCl胁迫造成的伤害作用,以ABA浓度为70 mol/L的缓解效果最好。【结论】外源ABA可显著促进盐胁迫下甘薯幼苗生根,维持细胞膜的稳定性,降低膜脂过氧化程度,调节植物细胞的渗透和离子动态平衡,使甘薯幼苗叶片维持较高的Fv/Fm、 0、 E0和较低的Vk、 D0,缓解PSⅡ光抑制的程度,改善植物的光合作用,提高植物的耐盐性。因此,喷施70 mol/L ABA是缓解NaCl胁迫效应,提高甘薯幼苗耐盐性的一种有效方法。     

甘薯抗疮痂病多胺与吲哚乙酸代谢的研究

研究不同抗性甘薯品种受疮痂病菌侵染前后多胺和吲哚乙酸代谢变化结果表明 ,感染疮痂病菌后抗病与感病品种甘薯叶片中吲哚乙酸含量和多胺氧化酶活性降低 ,多胺总量及腐胺含量增加 ,腐胺 / (精胺 亚精胺 )比值升高 ,感病品种变化幅度高于抗病品种。疮痂病菌侵入后感病品种甘薯叶片中绿原酸含量下降 ,吲哚乙酸氧化酶和过氧化物酶活性升高 ,而抗病品种则相反。     

甘薯横向水平复式移栽机栽苗装置设计及试验

针对目前市场上缺少甘薯横向水平移栽机的问题，该研究根据横向水平栽插法移栽甘薯苗的农艺要求，设计了甘薯横向水平复式移栽机的栽苗装置。首先，通过理论分析栽苗装置栽插运动，确定影响甘薯苗移栽质量的主要因素为机器前进速度、链条速度、入土深度及栽苗爪高度等。再基于EDEM-RecurDyn耦合仿真建立栽苗装置-柔性甘薯苗-薯垄耦合作用模型，模拟栽苗作业过程，确定栽苗爪运功轨迹为短摆线，栽苗爪高度为50 mm，确定甘薯苗最终位姿和甘薯垄形态符合甘薯水平栽植农艺要求。最后采用Box-Behnken试验设计方法，以机器前进速度、链条前进速度和入土深度为试验因素，以栽植深度合格率、栽植株距合格率、移栽效率为评价指标，进行了响应面试验，构建优化模型。试验确定移栽机最优工作参数组合为：机器前进速度0.4 m/s，链条前进速度0.2 m/s，入土深度46 mm。该参数组合下栽植深度合格率为92%，栽植株距合格率为92%，移栽效率为263株/min。研究结果可为甘薯横向水平移栽机设计及优化提供参考。     

紫甘薯对硒的吸收和累积特征

以紫甘薯为试验材料,采用盆栽试验的方法研究了基施硒酸钠[Se(VI)]和亚硒酸钠[Se(IV)]条件下,紫甘薯对外源硒的吸收累积规律,并比较了施用两种不同价态硒的紫甘薯富硒效果。结果表明:两种硒源均可显著提高紫甘薯各器官含硒量,且紫甘薯含硒量均随施硒量的增加而增大。当土壤施硒量为Se 8 mg/kg时,施用硒酸钠、亚硒酸钠收获期薯块的硒含量(干基)分别达到6.69、0.88 mg/kg。紫甘薯生育期40 d时各器官硒含量叶茎薯块,130 d时硒含量叶薯块茎。当硒酸钠施用量为Se 4 mg/kg时,紫甘薯薯块中的硒累积量最高达923.81μg/株,硒在紫甘薯块根中的分配率可达67%~70%,硒酸钠处理下,紫甘薯对硒的吸收利用率远远高于亚硒酸钠处理。综合紫甘薯含硒量和施硒量对生长的影响结果分析,施用硒酸钠和亚硒酸钠均能增加紫甘薯薯块的硒含量,紫甘薯对硒酸钠敏感性高于亚硒酸钠,生产过程中应充分考虑施用硒酸钠对作物造成的毒害。     

58个不同品种甘薯茎叶营养与功能成分的研究

为了明确不同品种甘薯茎叶的营养与功能成分组成,本试验系统比较了58个不同品种甘薯茎叶中的粗蛋白、粗脂肪、膳食纤维、维生素、多酚类物质、黄酮类物质含量及抗氧化活性。结果表明,甘薯茎叶富含粗蛋白(9.35~38.45 g·100 g^-1 DW)、粗脂肪(1.36~12.30 g·100 g^-1 DW)、总膳食纤维(35.30~45.0 g·100 g^-1 DW)、维生素C(1.47~131.64 mg·100 g^-1 DW)、β-胡萝卜素(6.75~59.35 mg·100 g^-1 DW)及维生素E(0.39~23.30 mg·100 g^-1 DW);湛薯01-2品种甘薯茎叶中总酚含量(21.39 g CAE·100 g^-1 DW)及抗氧化活性(40.28 g TE·100 g^-1 DW)最高。抗氧化活性与营养成分之间的相关性分析结果表明,甘薯茎叶中主要的抗氧化活性物质为多酚类物质(R²=0.748)。本研究为甘薯茎叶的合理开发利用提供了一定的指导。     

Direct absorption of acylated anthocyanin in purple-fleshed sweet potato into rats

Absorption of acylated anthocyanins in purple-fleshed sweet potato (Ipomoea batatas cv. Ayamurasaki) in rats was studied to obtain evidence that the acylated anthocyanins themselves could exert a physiological function in vivo. Peonidin 3-caffeoylsophoroside-5-glucoside (Pn 3-Caf*sop-5-glc) in purple-fleshed sweet potato was directly absorbed into rat and present as an intact acylated form in plasma. After oral administration of the purple-fleshed sweet potato anthocyanin (PSA) concentrate containing 38.9 micromol of Pn 3-Caf*sop-5-glc/kg of body weight, Pn 3-Caf*sop-5-glc was detected in the plasma, and the C(max) value and t(max) were estimated as 50.0 +/- 6.8 nmol/Lof plasma and 30 min, respectively. Furthermore, the plasma antioxidant capacity was significantly elevated from 58.0 +/- 12.0 to 89.2 +/- 6.8 micromol of Trolox equivalent/L of plasma 30 min after the administration of the PSA concentrate.     

不同1-甲基环丙烯熏蒸处理对甘薯贮藏保鲜及抗氧化能力的影响

甘薯在常温环境下放置极易出现失重、腐烂、发芽等问题,较难贮藏。为明确不同1-甲基环丙烯(1-MCP)熏蒸处理对甘薯贮藏保鲜及抗氧化能力的影响,并筛选出适宜甘薯常温贮藏的保鲜技术,本试验以烟薯25号为试验材料,研究不同熏蒸浓度(0.5、1.0、2.0、4.0、8.0 μL·L^-1)、不同熏蒸时间(6、12、24、36、48 h)及其交互作用对甘薯贮藏保鲜效果的影响,并通过模糊评价方法对不同1-MCP处理组在不同贮藏时间(0、7、14、22、30、60、90、120 d)的动态时间点上进行综合评判与分析。结果表明,在相同熏蒸时间条件下,高浓度1-MCP处理显著抑制甘薯发芽率、发芽指数和丙二醛(MDA)含量上升,抗氧化酶活性随着1-MCP处理浓度的增加呈降低趋势。相同熏蒸浓度条件下,12 h熏蒸处理与其他处理组相比贮藏保鲜效果更好。双因素方差分析结果显示,熏蒸浓度、熏蒸时间及其交互作用对甘薯贮藏期不同理化指标的影响随贮藏时间的延长逐渐减小,其中熏蒸浓度对甘薯发芽率、发芽指数、失重率、MDA含量及过氧化氢酶(CAT)、坏血酸过氧化酶(APX)活性的影响较大,熏蒸时间对甘薯超氧化物歧化酶(SOD)活性影响更大,熏蒸浓度与熏蒸时间的交互作用主要影响甘薯的呼吸强度。模糊综合评价模型表明, 2 μL·L^-1 1-MCP密闭熏蒸12 h最接近标准物元的最优状态,贮藏保鲜效果最好。本研究结果可为延长甘薯货架期与贮藏期提供技术参考。