小麦籽粒胚乳淀粉合成酶基因表达及酶活性分析

为研究小麦籽粒淀粉合成酶基因表达与酶活性的特征,选用4个淀粉含量差异较大的普通六倍体小麦新春24、E28（高淀粉含量组）和宁春16、安农9912（低淀粉含量组）为试验材料,采用实时荧光定量RT-PCR ,对灌浆期籽粒淀粉合成酶相关基因的表达进行研究,并对其表达量与相应酶活性的相关性做了分析。结果表明,束缚态淀粉合成酶基因(GBSS)、可溶性淀粉合成酶基因(SSS)、淀粉分支酶基因( SBE)、淀粉去分支酶（DBE）基因均呈单峰曲线变化。利用实时荧光定量PCR技术检测9个基因在不同淀粉含量的4个供试品种花后不同时期的相对表达量,结果表明,花后6d这些基因开始表达,在灌浆的中期（花后12~18d不等）有表达的小高峰,但在不同时期,2个高淀粉含量的品种中各种酶活性及其酶基因的相对表达量均比2个低淀粉含量的品种相对较高;这些基因表达谱与酶活性相关分析显示, 除GBSS外其他几种淀粉合成酶基因均与相应酶活性呈显著或极显著正相关,而且GBSS酶活性到达峰值时间稍迟于DBE、SSS、SBE等酶,说明DBE、SSS、SBE基因可能主要通过转录水平来控制籽粒淀粉的合成,而GBSS基因可能主要通过转录后水平来控制籽粒淀粉的合成。     

不同氮、钾配比对杂交水稻灌浆期直链淀粉积累的影响

选用4个不同系列的杂交水稻组合,通过施用不同量氮、钾底肥处理,对水稻开花后子粒淀粉积累、淀粉合成关键酶活性及其之间的关系进行了研究。分析不同氮、钾处理及基因型因素对直链淀粉含量的影响以及淀粉合成酶（GBSS）与直链淀粉积累的关系。结果显示:品种基因型和氮肥施用量是决定直链淀粉含量的主要因素,其他因素对子粒直链淀粉的含量影响也达到显著水平;不同因素对GBSS的影响与直链淀粉相似;GBSS活性与直链淀粉含量在灌浆期间显著正相关。不同品种最高直链淀粉含量的氮钾处理也不同,在本试验条件下各水稻品种直链淀粉含量的最佳氮钾组合为:B优827为N180K135;红优2009为N180K135; C优2009为N180K180;冈优725为N90K180。各品种中B优827直链淀粉含量最高。     

垄沟集雨覆盖对旱作马铃薯块茎淀粉合成关键酶活性、基因表达及淀粉累积的影响

为探讨垄沟集雨覆盖栽培对旱作马铃薯块茎形成过程中淀粉合成关键酶活性、基因表达及淀粉累积的影响,以马铃薯栽培品种青薯9号为材料,设置全膜双垄和地膜垄作覆盖栽培模式,以露地平播为对照,测定马铃薯块茎形成过程中淀粉合成关键酶活性、基因表达及淀粉累积指标。结果表明,从块茎发育全过程来看,全膜双垄和地膜垄作覆盖栽培马铃薯可溶性淀粉合成酶(SSS)活性分别较露地平播显著提高77.70%、22.63%,可溶性淀粉合成酶SSIII基因表达量分别较露地平播显著提高32.26%、119.35%,同时,ADP-葡萄糖焦磷酸化酶(AGPP)、颗粒结合型淀粉合成酶(GBSS)活性和ADP-葡萄糖焦磷酸化酶AGPase、可溶性淀粉合成酶SSII、颗粒结合型淀粉合成酶GBSSI、淀粉分支酶SBEI、淀粉分支酶SBEII基因表达量均明显降低,而淀粉分支酶(SBE)活性无显著变化;全膜双垄覆盖栽培马铃薯总淀粉含量、直链淀粉含量及直/支链淀粉比较露地平播分别提高5.04%、17.57%和27.81%,达到显著水平,而地膜垄作明显降低了马铃薯总淀粉含量、直链淀粉含量及直/支链淀粉比;SSS活性与GBSSI基因表达量呈显著负相关,与直链淀粉含量及直/支链淀粉比呈显著正相关,其他淀粉合成关键酶活性均与基因表达量、淀粉累积指标无明显相关性。本研究结果对筛选青海东部旱区马铃薯适宜的淀粉优质生产措施具有重要意义。     

水稻不同Wx等位基因对籽粒直链淀粉积累特性的影响

为了解水稻Wx等位变异对籽粒直链淀粉积累特性的影响,本研究选用携带有5种不同Wx等位基因的水稻单片段代换系(Single-segment substitution line,SSSL)为材料,在同一遗传背景下对水稻籽粒灌浆过程中表观直链淀粉含量(Apparent amylose content,AAC)的动态变化及其与颗粒结合性淀粉合成酶(Granule-bound starch synthase,GBSS)酶活的关系进行了研究。结果表明,不同Wx等位基因水稻材料籽粒灌浆过程中的AAC差异在灌浆早期(花后6d)已较明显,随着籽粒发育,非糯水稻花后6~11d是直链淀粉的快速增长期。籽粒灌浆过程中,不同Wx等位基因间籽粒AAC存在显著性差异,并且在直链淀粉积累特性上存在明显不同。较高和高AAC品系(携Wxg2和Wxg3基因)直链淀粉积累速率快,在灌浆初期(花后11d)便达到与籽粒成熟期一致的水平,之后变化不大;低、中等AAC材料(携Wxt和Wxg1基因)直链淀粉积累速率相对中等,灌浆初期(花后11d)AAC并不高,其直链淀粉的积累是个渐近的过程,直至灌浆后期(花后26d)才达到最大含量;糯稻(携wx基因)则在整个籽粒发育过程中AAC变化不大,始终保持着极低的含量水平。在整个灌浆期间,籽粒GBSS酶活与AAC均呈正相关,其中灌浆早期(花后6d)相关度最高,随后相关度逐渐降低,但始终维持在较高的水平(r0.820)。本研究结果可为进一步深入研究Wx座位等位变异机制奠定基础。     

施钾对夏玉米子粒发育过程中糖代谢相关酶活性的影响

选用高淀粉玉米品种费玉3号及低淀粉玉米品种豫玉22为材料,研究了钾肥不同用量对玉米开花后子粒淀粉积累、淀粉合成关键酶活性的影响;分析不同处理对直、支链淀粉含量的影响以及淀粉合成关键酶活性与直、支链淀粉积累的关系。结果表明,施K2O 225kg/hm2的处理显著比不施钾肥处理提高了两品种子粒中腺苷二磷酸葡萄糖焦磷酸化酶（ADPG-PPase）的活性与尿苷二磷酸葡萄糖焦磷酸化酶 （UDPG-PPase）,显著提高了费玉3号子粒中可溶性淀粉合成酶（SSS） 的活性;施钾降低了豫玉22子粒SSS、束缚态淀粉合成酶（GBSS）的活性。施K2O 225kg/hm2提高了两品种穗位叶蔗糖含量和蔗糖合成能力,促进了子粒淀粉的合成;显著提高子粒直链、支链及总淀粉含量及积累速率,收获时总淀粉含量分别提高13.6%和8.2%,施钾更容易提高费玉3号子粒淀粉含量。中、高量钾提高了高淀粉玉米费玉3号子粒可溶性糖含量,有利于淀粉的合成;而对低淀粉玉米豫玉22子粒可溶性糖含量没有显著影响。除可溶性糖含量外,品种与施钾的交互作用对以上各指标的影响均达到显著性水平。     

黑膜覆盖对旱地马铃薯块茎淀粉积累和关键酶活性的影响

为探明宁南半干旱雨养农业区马铃薯块茎中淀粉和关键酶活性对黑色地覆盖膜的响应机理,在2016年和2017年以宁南山区马铃薯主栽品种青薯9号为材料,通过两年大田试验,研究了黑膜覆盖(BF)、白膜覆盖(WF)和不覆膜(NF)对马铃薯块茎中淀粉及其组分积累、淀粉合成关键酶活性的影响。结果表明,与WF和NF相比,BF的直链淀粉、支链淀粉、总淀粉含量分别增加11.37%~35.68%、8.56%~27.05%、8.33%~27.60%,淀粉积累速率提高13.80%~37.90%;BF、WF、NF条件下块茎ADPG焦磷酸化酶(AGPase)、UDPG焦磷酸化酶(UGPase)、淀粉分支酶(SBE)、可溶性淀粉合酶(SSS)、束缚态淀粉合酶(GBSS)活性均呈单峰曲线变化,BF的淀粉合成关键酶活性明显高于WF和NF。与WF和NF相比,BF的AGPase活性分别增加12.81%和40.24%;UGPase活性分别增加15.34%和36.52%、SBE活性分别增加16.64%和44.17%、SSS活性分别增加13.69%和34.76%、GBSS活性分别增加15.75%和45.44%。BF产量较WF和NF分别提高14.54%和57.23%。相关分析表明,马铃薯块茎中直链淀粉、支链淀粉和总淀粉含量与AGPase、UGPase、SBE、SSS、GBSS活性在多数测定时期呈正相关(P<0.01)。因此,在干旱半干旱地区采用黑色地膜覆盖栽培能够通过影响淀粉合成关键酶,进而提高马铃薯块茎中的淀粉及其组分含量,最终影响马铃薯的产量。     

种植密度对不同株型玉米蔗糖代谢和淀粉合成相关酶活性的影响

试验选取平展型玉米"长玉13"、半紧凑型玉米"东单60"和紧凑型玉米"郑单958"为材料,分别在2600株·667m-2、3200株·667m-2、3800株·667m-23种密度下种植,探讨种植密度对不同株型玉米灌浆过程中功能叶蔗糖代谢和籽粒淀粉合成相关酶活性的影响。结果表明,种植密度对功能叶片蔗糖含量、磷酸蔗糖合成酶和蔗糖合成酶活性影响较大,高密度不利于功能叶片中蔗糖的合成,低密度有利于蔗糖积累;而籽粒中的淀粉合成相关酶——ADPG焦磷酸化酶和UDPG焦磷酸化酶活性受种植密度影响较小;平展型"长玉13"和紧凑型"郑单958"是源端限制型品种,而半紧凑型"东单60"是库端限制型品种;种植密度对不同株型玉米品种的影响程度有差异,对平展型"长玉13"影响最大,而对紧凑型"郑单958"影响最小。     

花后阴雨对小麦籽粒淀粉合成和干物质积累的影响

针对长江中下游小麦开花期常遇连阴雨导致减产的现象,研究阴雨寡照对小麦籽粒淀粉合成和干物质积累的影响,旨在为该地区小麦抗逆稳产栽培提供理论依据。选用长江中下游小麦主栽品种‘扬麦18’(受渍迟钝型)和‘皖麦52’(受渍敏感型)为试验材料,在小麦开花后设置7 d、11 d和15 d的渍水遮阴处理,研究渍水遮阴对小麦籽粒发育过程中淀粉合成相关酶活性及淀粉、干物质积累的影响。结果表明,渍水遮阴处理后,小麦籽粒中腺苷二磷酸葡萄糖焦磷酸化酶(AGPase)、可溶性淀粉合成酶(SSS)和结合态淀粉合成酶(GBSS)活性在灌浆前期(花后10～15d)与对照差异不显著,随着灌浆进程的推进,渍水遮阴处理与对照之间差异增大。灌浆中期(花后20 d)小麦籽粒中AGPase和SSS活性达到峰值时,渍水遮阴处理11 d、15 d的‘扬麦18’和‘皖麦52’籽粒中AGPase活性分别较对照下降1%、10%和11%、24%,SSS活性则下降5%、11%和9%、32%,且渍水遮阴处理11 d和15 d的小麦籽粒中SSS和GBSS活性在灌浆后期显著低于对照。用Logistic方程分别拟合籽粒淀粉和干物质的积累,花后渍水遮阴处理缩短了籽粒灌浆缓增期,降低了小麦籽粒灌浆的平均速率、淀粉积累的最大速率及平均速率,减少了籽粒淀粉和干物质的积累量。同时,渍水遮阴处理降低了小麦穗粒数和千粒重,使产量显著下降。随着渍水遮阴处理时间的延长,小麦籽粒中淀粉合成相关酶活性、干物质积累量及产量的下降幅度越大。迟钝型品种‘扬麦18’各指标的下降幅度均小于敏感型品种‘皖麦52’。小麦开花后渍水遮阴处理降低了籽粒中AGPase、SSS和GBSS活性,不利于籽粒淀粉合成及干物质的积累,导致产量下降显著。     

施氮对高淀粉玉米和普通玉米子粒可溶性糖和淀粉积累的影响

采用田间试验、植株生理生化分析和电镜观察等方法,研究了不同氮肥用量对高淀粉玉米吉单535和普通玉米军单8号子粒糖分与淀粉形成的影响。结果表明,在施氮量N 0～200 kg/hm2之间,两品种玉米子粒淀粉含量随着施氮量的增加而增加,过量施氮则淀粉含量降低。灌浆期吉单535子粒蔗糖、果糖、葡萄糖含量高于军单8号,适量施氮使子粒灌浆期保持较高的蔗糖、果糖和葡萄糖含量,有利于淀粉合成。高淀粉玉米在成熟期淀粉粒充满了细胞,大小均匀,排列有序;普通玉米的淀粉粒则没有充满细胞,大小不一,排列混乱。适宜施氮下淀粉粒大小较为均匀,淀粉粒之间挤压轻微,而过量施氮下淀粉粒相互挤压成多面体状。     

有机无机肥料及抽穗期气温升高对水稻籽粒淀粉合成相关酶活性及淀粉品质形成的影响

为探讨不同肥料处理下抽穗期高温胁迫对水稻籽粒淀粉酶活性及淀粉品质形成的影响,本试验以优质食味水稻南粳9108为材料,设置施用有机肥(OF)和常规化肥(CF)处理,于抽穗期进行常温(NT)、+2℃(较常温增加2℃,MT)和+5℃(较常温增加5℃,HT)处理,对籽粒淀粉合成特性进行研究。结果表明,抽穗期温度升高降低了蔗糖合成酶(SS)、淀粉合成酶(SSS)和淀粉分支酶(SBE)的活性,提高了蔗糖磷酸合成酶(SPS)和焦磷酸化酶(AGP)的活性。蔗糖含量、淀粉平均粒径、热焓值与峰值温度均表现为HT>MT>NT;淀粉含量、直链淀粉含量与黏度值则随着温度升高而下降。在肥料处理方面,各淀粉相关酶活性均表现为OF>CF,且在OF处理下有较好的淀粉品质。综上所述,温度升高通过抑制淀粉合成,加速了形成淀粉原料的积累,进而导致籽粒中蔗糖含量升高;有机肥处理能促进蔗糖合成并提高淀粉合成相关酶活性。从气候变暖应对措施方面,可选择有机肥替代化肥调控淀粉相关酶活性,进一步改善淀粉品质。本研究结果为减少高温对水稻的危害与提高淀粉品质提供了技术参考。     

Association of Starch Granule Proteins with Starch Ghosts and Remnants Revealed by Confocal Laser Scanning Microscopy

Starch suspensions (0.25%) were gelatinized to 70 and 100°C, and starch ghosts (defined as gelatinized starch granule envelopes after the majority of internal starch polymers have been released) and remnants were collected by centrifugation and washed with water. Protein was revealed in isolated gelatinized normal starch ghosts using confocal laser scanning microscopy and a protein‐specific dye that fluoresces only after reaction with primary amines in protein. This technique eliminates background interference from residual dye. Observation of fluorescent‐labeled protein in the starch ghosts at different optical depths of field revealed that protein was concentrated in the envelopes of swollen, gelatinized potato, maize, and wheat starch ghosts. Only traces of protein were found in gelatinized starch granule remnants of waxy maize and amylose‐free potato starches after they were heated to 100°C, indicating that the proteins observed in gelatinized normal maize starch were largely granule‐bound starch synthase (GBSS). Moreover, fragility of the gelatinized waxy and amylose‐free starch granule remnants might be caused in part by the lack of GBSS. Gel electrophoresis of proteins in starch ghosts confirmed that GBSS in potato and maize was tightly associated with the starch ghosts. The study provides a structural explanation for a role of granule‐associated proteins in maintaining the integrity of starch ghosts and remnant structures, and their consequent effect on paste rheology.     

Functional Characteristics of Bread Wheat (Triticum aestivum L.) Near‐Isogenic Lines Differing at the Waxy (Wx) Locus

The Waxy (Wx) gene in hexaploid wheat (Triticum aestivum L.) encodes granule‐bound starch synthase (GBSS1), which is involved in the synthesis of amylose, a mostly linear glucan polymer that makes up ∼25% of wheat starch. A null mutation of the Wx gene in each of the three genomes is associated with starch almost entirely consisting of the branched glucan polymer amylopectin (waxy starch), with corresponding changes in functionality. However, the rheological behavior of partially waxy starch remains unclear. The objective of this study was to characterize flour and baking quality in 16 near‐isogenic lines, null at the Wx locus on zero, one, two, or all three genomes, grown in four different environments. Across allelic groups, significant variations in amylose concentrations, flour paste viscosity, loaf structure and texture, dough stability, and proximate variables were observed. Because waxy wheat starch has greater water absorbance and resistance to retrogradation than normal starch, its inclusion in flour blends has been suggested as a means of improving the texture and appearance of bakery products and noodles. The results indicate that wheat encoding <3 functional homeologs of GBSS1 produces starch that has potential in the production of certain food items, such as Asian noodles. However, further research is necessary to determine the optimal amylose‐to‐amylopectin ratio to improve baking quality.     

Structural Changes of Debranched Corn Starch by Aqueous Heating and Stirring

Aqueous dispersions (2 mg/mL) of debranched corn starches of different amylose contents (waxy, normal, and high‐amylose) were subjected to extensive autoclaving and boiling‐stirring, and then the changes in starch chain profile were examined using medium‐pressure, aqueous, size‐exclusion column chromatography. As autoclaving time increased from 15 to 60 min, weight‐average chain length (CL_w) of waxy, normal, and high‐amylose corn starches determined using pullulan standards decreased from 46 to 41.2, from 122.1 to 96.3, and from 207.3 to 151.8, respectively. Number‐average chain length (CL_n) measured by the Nelson‐Somogyi method also decreased from 23.0 to 18.4, from 26.4 to 21.8, and from 66.5 to 41.5, respectively, indicating that thermal degradation of starch chains occurred. The CL_w/CL_n ratio for normal corn starch was higher than that for waxy corn starch, indicating an increase in polydispersity of the amylose fraction. Thermal degradation was also observed when the debranched starch was subjected to the boiling‐stirring treatment (0–96 hr). During 96 hr, the CL_w and relative proportion of B≥2 chains of amylopectin released by debranching waxy corn starch increased, whereas those of B1 chains decreased. This change may indicate physical aggregation of B1 chains. But branches from normal and high‐amylose corn starches showed increases in CL_w and the proportion of both B1 and B≥2 chains, along with substantial decreases in those of amylose chains. Therefore, thermal degradation of amylose was greater than that of amylopectin.     

Molecular Size Distribution and Amylase Resistance of Maize Starch Nanoparticles Prepared by Acid Hydrolysis

The molecular size distribution of maize starch nanoparticles (SNP) prepared by acid hydrolysis (3.16M H₂SO₄) and their amylase‐resistant counterparts, before and after debranching, was investigated. The weight average molecular weight (M_w) and linear chain length distribution were determined by high‐performance size‐exclusion chromatography (HPSEC) and high‐performance anion‐exchange chromatography (HPAEC), respectively. The objective was to understand the role of amylose involvement in the formation of SNP showing different crystalline structures (A‐ and B‐types). The HPSEC profiles of SNP before debranching from waxy, normal, and high‐amylose maize starches showed broad monomodal peaks. Debranched SNP from waxy maize eluted in a single narrow peak, whereas those from nonwaxy starches showed a multimodal distribution. Similar trends were also observed for the chain length distribution patterns, for which the longest detectable chains (degree of polymerization [DP] 31) in waxy maize were significantly lower than those of nonwaxy maize starches (DP 55–59). This indicated the potential amylose involvement in the SNP structure of normal and high‐amylose starches. Further evidence of amylose involvement was ascribed to the resistance of SNP toward amylolysis (Hylon VII > Hylon V > normal > waxy). The amylase‐resistant residues of SNP from high‐amylose maize starches were composed of both low M_w linear and branched chains.     

Production of Bihon-type Noodles from Maize Starch Differing in Amylose Content

Maize starches extracted from selected maize cultivars with 0.2–60.8% amylose contents were used to produce bihon-type noodles. Starch dough using a pregelatinized starch binder was prepared and extruded through a laboratory-scale extruder simulating the traditional process of making bihon in the Philippines. The normal maize starches with amylose content of ≈28% were successfully used for bihon-type noodle production, but waxy maize starches with 0.2–3.8% amylose content and high-amylose maize starches with 40.0–60.8% amylose content failed to produce bihon-type noodles. Viscoamylograph profile parameters and swelling volume are significantly correlated to amylose content of maize starch samples evaluated. These physicochemical properties may be used to indicate that the starch samples at normal amylose levels may be used for bihon-type noodles. Starch noodles produced in the laboratory were not significantly different in terms of either cooking quality or textural properties from two commercially produced maize noodle samples, except for adhesiveness. The laboratory process and fabricated extruder can be used to produce bihon-type noodles.     

Effect of green tea catechins on the postprandial glycemic response to starches differing in amylose content

The effect of tea polyphenols (TPLs), specifically tea catechins, on the postprandial glycemic response to cooked starches differing in amylose contents was investigated. The in vivo test using a mouse model showed a moderate reduction of the postprandial glycemic response to co-cooked normal (containing 27.8% amylose) or waxy corn starch with 10% TPLs (dry weight of starch), while an augmented glycemic response with a delayed blood glucose peak was observed when high amylose corn starch (HAC, containing 79.4% amylose) was used as the starch component. Enzyme kinetics results demonstrated that TPLs noncompetitively inhibit the digestion of waxy or normal corn starch, while the digestion rate of HAC starch was increased in the presence of TPLs, which supports the observed postprandial glycemic responses. Further studies using X-ray powder diffraction showed that the diffraction intensity (area under the diffraction curves) of normal and HAC starch was increased by 45% and 74%, respectively, whereas no change was observed for waxy corn starch. Consistently, dynamic laser light scattering studies using a solution of pure amylose showed an increased hydrodynamic radius of amylose molecules from ～54 nm to ～112 nm in the presence of TPLs. These experimental results indicate that there might exist an interaction between TPLs and amylose, which facilitates the association of amylose molecules to form a special nonordered structure that can produce a high and sustained postprandial glycemic response. Thus, a combination of tea polyphenols and specific starches could be used to manipulate postprandial glycemic response for glycemic control and optimal health.     

冻结-高压湿热解冻对多种淀粉中支链淀粉断裂的影响

冻结和高压湿热解冻是含淀粉面团在食品加工过程的必要工艺,在此过程中淀粉球破裂、部分支链淀粉断裂成为直链淀粉,给面团和食品的物理性状控制带来许多不可预见的结果。该文通过研究加热时间、加热温度、冷冻时间和解冻时间对糊化后的小麦、甘薯、马铃薯和玉米淀粉中直链淀粉含量的影响,间接反映支链淀粉断裂情况,结合冻结解冻前后淀粉分子量分布、链长分布、光学和电子显微镜图谱提出了冻结-解冻过程不同淀粉中支链淀粉可能断裂方式。结果表明,4种淀粉中的直链淀粉含量先升高后下降,直链淀粉含量在4种淀粉的中达到峰值的时间分别为48,48,48,72 h。光学显微照片观察表明,冻融处理会导致更多凝胶化淀粉球的破裂。对于小麦支链淀粉,冻融解冻过程支链淀粉中侧链长度为5、6、7个葡萄糖残基的侧链对应3种可能的断裂方式:2+2+1、2+2+2及2+2+2+1;对于甘薯支链淀粉,支链淀粉中侧链长度为10、11和13个葡萄糖残基的侧链对应3种可能的断裂方式:3+3+4、2+2+3+4及2+2+2+3+4;对于马铃薯支链淀粉,支链淀粉中侧链长度为5和6个葡萄糖残基的侧链对应3种可能的断裂方式:2+3、2+4、3+3;而玉米支链淀粉中,支链淀粉中侧链长度为7、8、9个葡萄糖残基的侧链对应3种可能的断裂方式:2+5,3+5,和3+3+3(其中1表示1个葡萄糖;2表示含2个葡萄糖的麦芽糖、3表示含3个葡萄糖的麦芽多糖、4表示含4个葡萄糖的麦芽多糖和5表示含5个葡萄糖的麦芽多糖)。该论文结果为培育具有冻融稳定性的淀粉种子提供一种全新的思路,即通过基因方法控制植物减少容易断裂淀粉侧链的合成。