禾谷类作物胚乳的淀粉合成

禾谷类作物胚乳拥有独特的淀粉合成途径,需要多种特异性同工酶的参与,这些酶在禾谷类其他组织或非禾谷类作物中是不存在的。近来明确了单个淀粉同工酶的功能,这有助于我们更深入地了解禾谷类淀粉直链、支链的合成与分布。在对禾谷类淀粉合成进行遗传分析的基础上,提出了脱分支酶作用模型。以水稻全基因组序列草图为背景,本文首次全面分析了禾谷类作物的淀粉合成。     

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。     

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。     

萌发中食松幼苗淀粉合酶同工酶与淀粉成分的关系

利用14C－ADPG标定法测定可溶性及与淀粉粒结合的淀粉合酶活性,采用过氯酸抽提、DMSO玻璃纤维纸层析、硫酸水解法定量测定各类淀粉成分,探讨了食松（PinusedulisEngelm）幼苗生长过程中淀粉合酶与淀粉成分间的关系。结果表明,在胚根出现以后,淀粉含量迅速增加,伴随着淀粉颗粒数目和质量的增加,两类淀粉合酶活性的增加以及淀粉合酶免疫印迹图谱的变化。支链淀粉是食松淀粉的主要成分,占总淀粉的84％。可溶性淀粉合酶峰值比淀粉粒结合的淀粉合酶活性峰值高1．3倍,与支链淀粉和直链淀粉的比例相对应。结果支持食松可溶性淀粉合酶是负责支链淀粉合成的主要酶的假说,同时表明淀粉粒结合的淀粉合酶在支链淀粉的合成中也有作用。     

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.     

合酶与合成酶

合酶与合成酶是生化教学中经常提到的,同时也是比较容易混淆的两个概念。例如,对于三羧酸循环中催化乙酰ＣｏＡ与草酰乙酸反应生成柠檬酸的酶,国内教科书就出现了命名的混乱,有的书称为柠檬酸合酶,更多的书称为柠檬酸合成酶。此外,不少教科书把脂肪酸合酶、ＨＭＧＣｏＡ合酶、ＡＬＡ合酶及前列腺内过氧化物合酶误译成脂肪酸合成酶、ＨＭＧＣｏＡ合成酶、ＡＬＡ合成酶及前列腺内过氧化物合成酶等。可见,合酶与合成酶误用存在一定的普遍性。这种状况不仅使青年教师茫然,更使学生无所适从,实在有必要澄清。出现这种错误的可能原因一是名词的错…     

小麦淀粉合酶基因Ⅲ片段的克隆及反义和RNA干扰载体的构建

采用RT-PCR方法从小麦品种豫教2号的发育籽粒中克隆出淀粉合酶Ⅲ基因(starch synthase Ⅲ,SSⅢ)部分cDNA片段(509bp) (GenBank No.EF466009),同源性比较结果显示,它与GenBank 上已报道的SSⅢ基因有高度同源性.以pWM101质粒为基础,构建了由35S启动子调控的SSⅢ基因的反义表达载体pWM101SSⅢA;另外,还以pFGC5941质粒为基础,构建了SSⅢ基因的RNAi干扰载体pFGC5941SSⅢsa,这些载体的构建为研究此基因的功能打下了很好的基础.     

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,而淀粉分酶支酶是其合成的关键酶。开展对该酶的深入研究不论是在基因理论研究领域还是在实现应用方面都具重要意义。     

Chain-length specificities of maize starch synthase I enzyme: studies of glucan affinity and catalytic properties

It is widely known that some of the starch synthases and starch-branching enzymes are trapped inside the starch granule matrix during the course of starch deposition in amyloplasts. The objective of this study was to use maize SSI to further our understanding of the protein domains involved in starch granule entrapment and identify the chain-length specificities of the enzyme. Using affinity gel electrophoresis, we measured the dissociation constants of maize SSI and its truncated forms using various glucans. The enzyme has a high degree of specificity in terms of its substrate-enzyme dissociation constant, but has a greatly elevated affinity for increasing chain lengths of alpha-1, 4 glucans. Deletion of the N-terminal arm of SSI did not affect the Kd value. Further small deletions of either N- or C-terminal domains resulted in a complete loss of any measurable affinity for its substrate, suggesting that the starch-affinity domain of SSI is not discrete from the catalytic domain. Greater affinity was displayed for the amylopectin fraction of starch as compared to amylose, whereas glycogen revealed the lowest affinity. However, when the outer chain lengths (OCL) of glycogen were extended using the phosphorylase enzyme, we found an increase in affinity for SSI between an average OCL of 7 and 14, and then an apparently exponential increase to an average OCL of 21. On the other hand, the catalytic ability of SSI was reduced several-fold using these glucans with extended chain lengths as substrates, and most of the label from [14C]ADPG was incorporated into shorter chains of dp < 10. We conclude that the rate of catalysis of SSI enzyme decreases with the OCL of its glucan substrate, and it has a very high affinity for the longer glucan chains of dp approximately 20, rendering the enzyme catalytically incapable at longer chain lengths. Based on the observations in this study, we propose that during amylopectin synthesis shorter A and B1 chains are extended by SSI up to a critical chain length that soon becomes unsuitable for catalysis by SSI and hence cannot be elongated further by this enzyme. Instead, SSI is likely to become entrapped as a relatively inactive protein within the starch granule. Further glucan extension for continuation of amylopectin synthesis must require a handover to other SS enzymes which can extend the glucan chains further or for branching by branching enzymes. If this is correct, this proposal provides a biochemical basis to explain how the specificities of various SS enzymes determine and set the limitations on the length of A, B, C chains in the starch granule.     

The phenotype of soluble starch synthase IV defective mutants of Arabidopsis thaliana suggests a novel function of elongation enzymes in the control of starch granule formation

All plants and green algae synthesize starch through the action of the same five classes of elongation enzymes: the starch synthases. Arabidopsis mutants defective for the synthesis of the soluble starch synthase IV (SSIV) type of elongation enzyme have now been characterized. The mutant plants displayed a severe growth defect but nonetheless accumulated near to normal levels of polysaccharide storage. Detailed structural analysis has failed to yield any change in starch granule structure. However, the number of granules per plastid has dramatically decreased leading to a large increase in their size. These results, which distinguish the SSIV mutants from all other mutants reported to date, suggest a specific function of this enzyme class in the control of granule numbers. We speculate therefore that SSIV could be selectively involved in the priming of starch granule formation.     

Identification of multiple isoforms of soluble and granule-bound starch synthase in developing wheat endosperm

We have investigated the nature and locations of isoforms of starch synthase in the developing endosperm of wheat (Triticum aestivum L.). There are three distinct granule-bound isoforms of 60 kDa (the Waxy gene product), 77 kDa and 100–105 kDa. One of these isoforms, the 77-kDa protein, is also present in the soluble fraction of the endosperm but it contributes only a small proportion of the total soluble activity. Most of the soluble activity is contributed by isoforms which are apparently not also granule-bound. The 60-kDa and 77kDa isoforms of wheat are antigenically related to isoforms of very similar size in the developing pea embryo, but the other isoforms in the endosperm appear to have no counterparts in the pea embryo. The significance of these results in terms of the diversity of isoforms of starch synthase and their locations is discussed.Abbreviations DEAE diethylaminoethyl - GBSS granule-bound starch synthase - NT nullisomictetrasomic We are grateful to the late John Hawker (University of Adelaide, Australia) and to John Snape (John Innes Centre, UK) for useful discussions during the course of this work, to John Snape and Catherine Chinoy (John Innes Centre, UK) for the gift of the NT lines and to Richard Batt (University of Adelaide, Australia) for technical assistance.     

Starch synthase activity and heat shock protein in relation to thermal tolerance of developing wheat grains

Wheat (Triticum aestivum L.) cvs. HD 2285 (relatively tolerant) and WH 542 (susceptible) were exposed to ambient and elevated temperature (3–4 °C higher) in open top chambers during post anthesis period. The grain yield components were determined at the time of maturity. In order to elucidate the basis of differential tolerance of these cultivars, the excised developing grains (20 d after anthesis) of ambient grown plants were incubated at 15, 25, 35 and 45 °C for 2 h and then analysed for the activities of soluble starch synthase (SSS), granule bound starch synthase (GBSS), kinetic parameters of SSS and content of heat shock protein (HSP 100). The elevated temperature during grain development significantly decreased grain growth in WH 542 whereas no such decrease was observed in HD 2285. High temperature tolerance of HD 2285 was found to be associated with higher catalytic efficiency (V_max/K_m) of SSS at elevated temperature and higher content of HSP 100.     

Contribution of sucrose synthase, ADP-glucose pyrophosphorylase and starch synthase to starch synthesis in developing pea seeds

Using genetic variability existing amongst nine pea genotypes (Pisum sativum L.), the biochemical basis of sink strength in developing pea seeds was investigated. Sink strength was considered to be reflected by the rate of starch synthesis (RSS) in the embryo, and sink activity in the seed was reflected by the relative rate of starch synthesis (RRSS). These rates were compared to the activities of three enzymes of the starch biosynthetic pathway [sucrose synthase (Sus), ADP-glucose pyrophosphorylase and starch synthase] at three developmental stages during seed filling (25, 50 and 75% of the dry seed weight). Complete sets of data collected during seed filling for the nine genotypes showed that, for all enzyme activities (expressed on a protein basis), only Sus in the embryo and seed coat was linearly and significantly correlated to RRSS. The contribution of the three enzyme activities to the variability in RSS and RRSS was evaluated by multiple regression analysis for the first two developmental stages. Only Sus activity in the embryo could explain, at least in part, the significant variability observed for both the RSS and the RRSS at each developmental stage. We conclude that Sus activity is a reliable marker of sink activity in developing pea seeds.     

Two isoforms of the GBSSI class of granule-bound starch synthase are differentially expressed in the pea plant (Pisum sativum L.)

In addition to the GBSSI isoform of starch synthase described previously, the pea plant contains a second, granule-bound isoform, GBSSIb. GBSSI is abundant in pea embryos and Rhizobium root nodules, is present at low levels in pods and is absent from leaves. Mutations at the lam locus eliminate GBSSI from all of these organs. GBSSIb is present in pods, leaves and nodules and is unaffected by mutations at the lam locus. GBSSI and GBSSIb are very similar in molecular mass, primary sequence, activity and antigenic properties. GBSSIb, like GBSSI, can synthesize amylose in the presence of malto-oligosaccharides in isolated starch granules. However, its role in vivo is unclear. The lam mutation eliminates amylose from the starch of embryos but does not affect the relatively small amounts of amylose-like material in the starch of pods, leaves and nodules. The significance of these results for understanding of the regulation of amylose synthesis is discussed.     

Enhancing the expression of starch synthase class IV results in increased levels of both transitory and long-term storage starch

Starch is an important renewable raw material with an increasing number of applications. Several attempts have been made to obtain plants that produce modified versions of starch or higher starch yield. Most of the approaches designed to increase the levels of starch have focused on the increment of the amount of ADP-glucose or ATP available for starch biosynthesis. In this work, we show that the overexpression of starch synthase class IV (SSIV) increases the levels of starch accumulated in the leaves of Arabidopsis by 30%-40%. In addition, SSIV-overexpressing lines display a higher rate of growth. The increase in starch content as a consequence of enhanced SSIV expression is also observed in long-term storage starch organs such as potato tubers. Overexpression of SSIV in potato leads to increased tuber starch content on a dry weight basis and to increased yield of starch production in terms of tons of starch/hectare. These results identify SSIV as one of the regulatory steps involved in the control of the amount of starch accumulated in plastids.     

The purification and characterisation of the two forms of soluble starch synthase from developing pea embryos

Soluble starch synthase was purified 10000-fold from developing embryos of pea (Pisum sativum L.). The activity was resolved into two forms which together account for most if not all of the soluble starchsynthase activity in the embryo. The two isoforms differ in their molecular weights but are similar in many other respects. Their kinetic properties are similar, neither isoform is active in the absence of primer, and both are unstable at high temperatures, the activity being abolished by a 20-min incubation at 45° C. Both isoforms are recognised by antibodies raised to the granule-bound starch synthase of pea. Isoform II, which has the same molecular weight (77 kDa) as the granulebound enzyme, is recognised more strongly than Isoform I.     

小麦淀粉粒束缚淀粉合成酶基因多态性的分子鉴定

运用6％的SDS-PAGE对14个小麦品种成熟籽粒Wx蛋白的多态性进行了鉴定,结果表明,14个小麦品种根据其Wx蛋白的缺失情况可分为6种组合类型。另外,根据Wx-A1、Wx-D1和Wx-D1这3个位点基因序列和变异情况分别设计了PCR引物,扩增结果表明：Wx-A1位点突变材料扩增产物为327bp,正常材料中扩增不到该特异带;在Wx-B1位点扩增出187bp目标带,突变材料没有该扩增产物;在Wx-D1位点扩增出约700bp目标带,突变材料没有该特异带。与前人的研究结果相比,Wx-B1引物在3个位点的扩增产物长度更短,差异更大,在2％琼脂糖胶上即可清楚分开,缩短了鉴定时间,提高了效率,为大规模筛选优质面条小麦品种提供了可能。