萌发后食松幼苗淀粉粒结合与可溶性淀粉合酶的研究

裸子植物食松 (PinusedulisEngelm .)幼苗在萌发后具有淀粉积累的独特特征 ,而淀粉合酶在裸子植物中尚未有研究。这项研究对在室温下暗中萌发的食松幼苗的淀粉合酶进行了提取和研究 ,结果显示 :5 8kD和 91kD的淀粉粒结合蛋白均为豌豆淀粉合酶Ⅱ抗体所识别。5 8kD淀粉粒结合蛋白被纯化出来 ,其N端序列与其他几种被子植物淀粉粒结合的淀粉合酶Ⅰ的N端序列有很高的相似性 ,并与豌豆、大麦、马铃薯淀粉粒结合淀粉合酶Ⅰ有免疫相似性 ,被鉴定为淀粉粒结合的淀粉合酶Ⅰ。从DEAE_Sepharose柱上洗脱出来的可溶性淀粉合酶表现出两个淀粉合酶活性峰 ,表明至少有两类可溶性淀粉合酶同工酶。对可溶性淀粉合酶的前体亲和特性进行了研究。以兔肝糖原为前体时 ,可溶性淀粉合酶的亲和性最高。裸子植物淀粉合酶的酶学特性表现与已研究的被子植物相似。     

萌发后食松幼苗淀粉粒结合与可溶性淀粉合酶的研究

裸子植物食松(Pinus edulis Engelm.)幼苗在萌发后具有淀粉积累的独特特征,而淀粉合酶在裸子植物中尚未有研究.这项研究对在室温下暗中萌发的食松幼苗的淀粉合酶进行了提取和研究,结果显示: 58 kD和91 kD的淀粉粒结合蛋白均为豌豆淀粉合酶Ⅱ抗体所识别. 58 kD淀粉粒结合蛋白被纯化出来, 其N端序列与其他几种被子植物淀粉粒结合的淀粉合酶Ⅰ的N端序列有很高的相似性,并与豌豆、大麦、马铃薯淀粉粒结合淀粉合酶Ⅰ有免疫相似性,被鉴定为淀粉粒结合的淀粉合酶Ⅰ.从DEAE-Sepharose 柱上洗脱出来的可溶性淀粉合酶表现出两个淀粉合酶活性峰,表明至少有两类可溶性淀粉合酶同工酶.对可溶性淀粉合酶的前体亲和特性进行了研究.以兔肝糖原为前体时, 可溶性淀粉合酶的亲和性最高.裸子植物淀粉合酶的酶学特性表现与已研究的被子植物相似.     

水稻胚乳中淀粉合成相关酶活性的变化对支链淀粉精细结构的影响

分析了水稻（Oryza sativa L．）籽粒发育过程淀粉生物合成途径中的关键酶——ADP-葡萄糖焦磷酸化酶、可溶性淀粉合酶、淀粉分支酶以及淀粉脱支酶活性变化,同时研究了淀粉结构形成动态．与野生型晚粳9522相比,转基因晚粳9522中直链淀粉的合成被显著抑制,而总淀粉含量和籽粒终重量没有改变．淀粉生物合成途径中关键酶活性表达时间不一致,存在明显的时段特征,这与淀粉积累动态密切相关．可溶性淀粉合酶活性表达最早,其在灌浆前期驱动淀粉合成起始;而淀粉粒结合态淀粉合酶在胚乳发育的中期活性最大．两水稻实验材料间,除淀粉脱支酶活性变化有所不同外,ADP-葡萄糖焦磷酸化酶和淀粉分支酶活性的变化没有明显差异．并且,支链淀粉的分支模式在水稻籽粒发育过程中变化较大,且与品种有关．以上结果揭示,支链淀粉的合成要先于直链淀粉,并且在控制支链淀粉各分支的形成过程中有不同的酶在起特异的作用．     

淀粉合酶的酶学与分子生物学研究进展

淀粉合酶作为淀粉合成的关键酶之一,一直是淀粉研究的重要内容。这些研究多集中在对其同工型的研究,淀粉合酶的两类主要同工型分别为淀粉粒结合的淀粉合酶和可溶性淀粉合酶,这两类同工型的作用极为复杂。本文介绍了淀粉合酶同工型的酶学和分子生物学近年来的研究进展,同时也讨论了这些同工型的分类、相互关系及其在淀粉合成过程中的生理功能等内容。     

植物支链淀粉生物合成研究进展

植物支链淀粉占贮存淀粉的70%～80%,是决定植物果实或种子品质的关键成分.对植物支链淀粉生物合成途径及其代谢酶基因的研究,可大大推动支链淀粉结构的改造和在食品工业上的应用.该文介绍了植物支链淀粉的结构组成,详细阐述了参与支链淀粉生物合成的三类酶,即淀粉分支酶(starch branchingenzyme,SBE)、可溶性淀粉合酶(soluble starch synthase,SSS)和淀粉脱支酶(starch debranching enzyme,SDBE)的编码基因、酶学特性及其在支链淀粉合成中的作用,并就植物支链淀粉的合成模型加以探讨.同时提出了该研究领域尚待解决的问题,对其应用前景作了展望.     

淀粉合酶的酶学与分子生物学研究进展

淀粉合酶作为淀粉合成的关键酶之一,一直是淀粉研究的重要内容,这些研究多集中在对其同工型的研究,淀粉合酶的两类主要同工型分别为淀粉粒结合的淀粉合酶和可溶性淀粉合酶,这两类同工型的作用极为复杂,本文介绍了淀粉合酶同工型的酶学和分子生物学近年来的研究进展,同时也讨论了这些同工型的分类,相互关系及其在淀粉合成过程中的生理功能等内容。     

花后高温对不同耐热性小麦品种籽粒淀粉形成的影响

以耐热性不同的2个小麦品种济麦20和鲁麦21为材料,分别于花后5～9d（T1）和15～19d（T2）进行高温处理,研究了小麦花后不同阶段高温对籽粒淀粉积累、淀粉粒分布及相关酶活性的影响。结果表明,花后高温显著降低籽粒淀粉积累量;显著降低籽粒淀粉及支链淀粉含量,但提高直链淀粉含量、直／支链淀粉比例。处理间比较,他处理对籽粒淀粉积累的影响程度较T1处理大。品种间比较,高温对济麦20的影响程度较鲁麦21大。高温使A型淀粉粒的体积、数量和表面积比例显著增加,B型淀粉粒的体积、数量和表面积比例显著降低。T1处理后,两品种籽粒蔗糖含量、蔗糖合酶（SS）和腺苷二磷酸葡萄糖焦磷酸化酶（AGPP）、可溶性淀粉合酶（SSS）、束缚态淀粉合酶（GBSS）和淀粉分支酶（SBE）活性均略高于对照;但济麦20、鲁麦21上述指标分别于花后15、20d开始低于对照。他处理后,两品种籽粒蔗糖含量、SS、AGPP、SSS、GBSS和SBE活性显著低于对照,济麦20上述指标的降幅较鲁麦21大。与其它淀粉合成相关酶相比,高温对籽粒GBSS活性的影响程度较小。两品种处理间籽粒蔗糖含量、SS、AGPP、SSS、GBSS及SBE活性的变化趋势,与淀粉积累量的变化趋势基本一致。说明灌浆期高温使籽粒淀粉积累量降低,一方面是由于籽粒蔗糖供应较低引起糖源不足;另一方面则是由于灌浆中后期淀粉合成相关酶活性下降使淀粉合成受抑所致。     

糯性和非糯性小麦灌浆期胚乳直/支链淀粉积累及其相关酶活性研究

选用3份糯性和2份非糯性小麦材料,通过田间试验在灌浆过程中分别检测了各材料的籽粒直链和支链淀粉积累量、淀粉积累速率及淀粉合成关键酶活性的动态变化过程,探讨籽粒淀粉累积与相关酶活性的关系.结果表明:(1)非糯小麦在花后7 d前均未检测到直链淀粉存在,而此时已经检测到支链淀粉含量,并且糯小麦仅含有支链淀粉,支链淀粉早于直链淀粉合成.(2)糯性和非糯性小麦灌浆期籽粒的直、支链淀粉积累速率均呈先增加后降低的趋势,且直、支链淀粉最终积累量取决于最大积累速率和平均积累速率的大小,而积累活跃期的调节作用较小;糯性和非糯性小麦在淀粉合成过程中的腺苷二磷酸葡萄糖焦磷酸化酶(AGPP)、可溶性淀粉合成酶(SSS)、颗粒结合型淀粉合成酶(GBSS)和淀粉分支酶(SEB)活性均呈单峰曲线变化,活性峰值基本上都出现在花后20～25 d左右.(3)直链淀粉积累速率与AGPP、SSS、GBSS和SBE活性变化显著或极显著正相关,而支链淀粉积累速率仅与SSS活性变化极显著正相关,总淀粉积累速率与AGPP和SSS活性变化显著或极显著正相关.     

氮素水平对冬小麦籽粒灌浆过程中淀粉合成关键酶活性的影响

小麦籽粒灌浆过程中,淀粉合成关键酶腺苷二磷酸葡萄糖焦磷酸化酶(ADPG-PPase)、可溶性淀粉合成酶(SSS)、淀粉分支酶(SBE)和束缚态淀粉合成酶(GBSS)均随着灌浆进程呈单峰曲线变化,峰值出现在花后25d;不同氮肥施用量对灌浆前期酶活性的影响较小,而在花后20d之后影响较大;随着氮肥施用量的增加,4种酶活性均呈增加趋势,但氮肥过量时酶活性下降,表明适当增加施氮量有利于淀粉合成关键酶活性的提高。     

植物支链淀粉合成的关键酶——淀粉分支酶

支链淀粉是植物淀粉的主要成分,而淀粉分支酶是其合成的关键酶。淀粉分支酶可分为两同形体家族,本文从酶学特性、染色体定位、基因及基因表达方面阐明了它们之间的联系和区别,并证实不同同形体在植物支链淀粉合成和结构决定上所起作用不同。开展对该酶的深入研究不论是在基础理论研究领域还是在现实应用方面都具重要意义。     

Relationship Between Isozymes of Starch Synthases and Starch Composition in Germination Pinyon Seedlings

The relation between starch synthases and starch composition in the germinating pinyon ( Pinus edulis Engelm) seedlings was studied. Using the method of 14C-glucose transferred from 14C-ADPG in the assay of starch synthases activity. Starch was extracted with 32% HC1O4, separated on glass fiber with DMSO, and assayed with the sulfuric acid-phenol method. After the emergence of radicle, starch content increased rapidly accompanied with the increase of starch grains in number and size, the increase of both soluble and granulebound starch synthase activity and the change of the pattern of Western-blot. Amylopectin was the major composition in pinyon starch, accounted for 84% of the total starch. The activity of soluble starch synthase was 1.3 times higher than that of the granule-bound starch synthase, corresponding to the ratio of amylopectin to amylose. This result supports the conventional theory that soluble starch synthase is the major enzyme responsive for the synthesis of amylopectin, and also supports that granule-bound starch synthase is functional in the synthesis of amylopectin.     

Soluble starch synthases and starch branching enzymes from developing seeds of sorghum

Soluble starch synthases and branching enzymes have been partially purified from developing sorghum seeds. Two major fractions and one minor fraction of starch synthase were eluted on DEAE-cellulose chromatography. The minor enzyme eluted first and was similar to the early eluting major synthase in citrate-stimulated activity, faster reaction rates with glycogen primers than amylopectin primers, and in K_m for ADP-glucose (0.05 and 0.08 mM, respectively). The starch synthase peak eluted last had no citrate-stimulated activity, was equally active with glycogen and amylopectin primers, and had the highest K_m for ADP-glucose (0.10 mM). Four fractions of branching enzymes were recovered from DEAE-cellulose chromatography. One fraction eluted in the buffer wash; the other three co-eluted with the three starch synthases. All four fractions could branch amylose or amylopectin, and stimulated α-glucan synthesis catalysed by phosphorylase. Electrophoretic separation and activity staining for starch synthase of crude extracts and DEAE-cellulose fractions demonstrated complex banding patterns. The colour of the bands after iodine staining indicated that branching enzyme and starch synthase co-migrated during electrophoresis.     

Starch biosynthesis: mechanism for the elongation of starch chains

Starch granules from eight diverse plant sources all had active starch synthases and branching enzymes inside the granules. The enzymes synthesized both amylose and amylopectin from ADPGlc. Pulsing of the granules with ADP-[14C]Glc gave synthesis of starch that on reduction and glucoamylase hydrolysis gave 14C-labeled D-glucitol. The pulsed label could be chased by nonlabeled ADPGlc to give a significant decrease of 14C-label in D-glucitol. Evidence further indicated that the synthase forms a high-energy covalent complex with D-glucose and the growing starch chain, and that the D-glucopyranosyl group is added to the reducing end of the growing starch chain by a two-site insertion mechanism.     

作物淀粉生物合成与转基因修饰研究进展

淀粉是高等植物中碳水化合物的主要贮藏形式 ,也是粮食作物产品的最主要成分。淀粉虽然都由直链淀粉和枝链淀粉组成 ,但在不同作物中两者的比例和枝链淀粉结构的存在很大差异。现已明确 ,直链淀粉是在颗粒结合淀粉合成酶 (granule boundstarchsynthase,GBSS)催化下合成的 ,而枝链淀粉是四种酶共同作用的结果 ,它们分别是腺嘌呤 -葡萄糖焦磷酸化酶 (ADP glucosepyrophosphorylase ,AGP) ,可溶性淀粉合成酶 (solublestarchsynthase ,SSS) ,淀粉分枝酶 (starchbranchingenzyme ,SBE)和脱分枝酶 (starchdebranchingenzyme ,DBE)。一方面 ,在不同作物中 ,这些酶本身存在多种形式 ,如在玉米胚乳中 ,AGP有大亚基和小亚基之分 ,SBE又可分BE1,BEIIa ,BEIIb 3种 ,SSS也可分为SSI和SSIII(或SSIIa)两种 ,而DBE也有异淀粉酶 (isoamylase)和限制性糊精酶 (pullu lanase)两种。另一方面 ,控制特定酶的基因 ,在不同作物甚至在同一种作物的不同品种中也可能存在不同的复等位基因 ,如籼稻和粳稻的GBSS分别由蜡质基因Wxa 和Wxb 控制 ,两者编码的GBSS活性差异显著。此外 ,环境条件也可通过影响基因的转录使酶的含量或催化性能发生变化。迄今 ,国内外已获得多种马铃薯和水稻的转基因材料 ,对淀粉合成进行修饰 ,试图培育优质品     

Starches from A to C. Chlamydomonas reinhardtii as a model microbial system to investigate the biosynthesis of the plant amylopectin crystal.

Wide-angle powder x-ray diffraction analysis was carried out on starch extracted from wild-type and mutant Chlamydomonas reinhardtii cells. Strains containing no defective starch synthases as well as mutants carrying a disrupted granule-bound starch synthase structural gene displayed the A type of diffraction pattern with a high degree of crystallinity. Mutants carrying a defect for the major soluble starch synthase (SSS), SSS II, were characterized by a switch to the B type of diffraction pattern with very low crystallinity. Mutant strains carrying SSS I as the only glucan elongation enzyme regained some of their crystallinity but switched to the C type of diffraction pattern. Differential scanning calorimetry analysis correlated tightly with the x-ray diffraction results. Together with the electron microscopy analyses, these results establish C. reinhardtii as a microbial model system displaying all aspects of cereal starch synthesis and structure. We further show that SSS II is the major enzyme involved in the synthesis of crystalline structures in starch and demonstrate that SSS I alone builds a new type of amylopectin structure.     

不同类型玉米发育籽粒中淀粉合成及相关酶活性比较

以普通玉米、爆裂玉米、甜玉米和糯玉米为试材,分析和比较不同类型的玉米品种之间籽粒发育过程中淀粉合成及相关酶活性的变化。结果表明,淀粉合成速率和蔗糖合成酶(SS)、可溶性淀粉合成酶(SSS)、束缚态淀粉合成酶(GBSS)、淀粉分支酶(SBE)、去分支酶(DBE)活性都呈单峰曲线变化。30～40 DAP,普通玉米的SS活性显著高于其他3种类型;类型间平均和最大SSS活性水平的顺序为普通玉米>糯玉米>爆裂玉米>甜玉米;30～40 DAP,普通玉米GBSS活性最高,糯玉米GBSS活性最低;20～40 DAP,糯玉米SBE活性最高;甜玉米的DBE活性很低,并且在40 DAP完全丧失。淀粉合成速率与SS、SSS、GBSS和SBE活性相关程度比较高,与腺苷二磷酸葡萄糖焦磷酸化酶(AGP酶)和DBE活性相关不显著。推测AGP酶虽然为淀粉合成提供直接前体ADPG,但可能SS活性过低致使其限速作用比AGP酶的还强,AGP酶潜在的限速作用无法表现,SS成为玉米籽粒淀粉合成的限速因子。GBSS对直链淀粉积累起重要的促进作用;SSS和SBE对支链淀粉积累起重要的促进作用。     

Soluble starch synthase I: a major determinant for the synthesis of amylopectin in Arabidopsis thaliana leaves

A minimum of four soluble starch synthase families have been documented in all starch-storing green plants. These activities are involved in amylopectin synthesis and are extremely well conserved throughout the plant kingdom. Mutants or transgenic plants defective for SSII and SSIII isoforms have been previously shown to have a large and specific impact on the synthesis of amylopectin while the function of the SSI type of enzymes has remained elusive. We report here that Arabidopsis mutants, lacking a plastidial starch synthase isoform belonging to the SSI family, display a major and novel type of structural alteration within their amylopectin. Comparative analysis of beta-limit dextrins for both wild type and mutant amylopectins suggests a specific and crucial function of SSI during the synthesis of transient starch in Arabidopsis leaves. Considering our own characterization of SSI activity and the previously described kinetic properties of maize SSI, our results suggest that the function of SSI is mainly involved in the synthesis of small outer chains during amylopectin cluster synthesis.     

A combined reduction in activity of starch synthases II and III of potato has novel effects on the starch of tubers

A chimeric antisense construct has been used to generate transgenic potatoes ( Solanum tuberosum L.) in which activities of both of the main starch synthases responsible for amylopectin synthesis in the tuber (SSII and SSIII) are reduced. The properties of starch from tubers of these plants have been compared with those of starches from transgenic plants in which activity of either SSII or SSIII has been reduced. Starches from the three types of transgenic plant are qualitatively different from each other and from the starch of control plants with unaltered starch synthase activities, with respect to granule morphology, the branch lengths of amylopectin, and the gelatinisation behaviour analysed by viscometry. The effects of reducing SSII and SSIII together cannot be predicted from consideration of the effects of reducing these two isoforms individually. These results indicate that different isoforms of starch synthase make distinct contributions to the synthesis of amylopectin, and that they act in a synergistic manner, rather than independently, during amylopectin synthesis.     

Specificity of starch synthase isoforms from potato.

In higher plants several isoforms of starch synthase contribute to the extension of glucan chains in the synthesis of starch. Different isoforms are responsible for the synthesis of essentially linear amylose chains and branched, amylopectin chains. The activity of granule-bound starch synthase I from potato has been compared with that of starch synthase II from potato following expression of both isoforms in Escherichia coli. Significant differences in their activities are apparent which may be important in determining their specificities in vivo. These differences include affinities for ADPglucose and glucan substrates, activation by amylopectin, response to citrate, thermosensitivity and the processivity of glucan chain extension. To define regions of the isoforms determining these characteristic traits, chimeric proteins have been produced by expression in E. coli. These experiments reveal that the C-terminal region of granule-bound starch synthase I confers most of the specific properties of this isoform, except its processive elongation of glucan chains. This region of granule-bound starch synthase I is distinct from the C-terminal region of other starch synthases. The specific properties it confers may be important in defining the specificity of granule-bound starch synthase I in producing amylose in vivo.