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．     

淀粉分支酶和去分支酶编码基因的功能

淀粉分支酶(SBE)和淀粉去分支酶(DBE)是直接参与淀粉生物合成的5类酶中的2类分支点形成支链淀粉,后者水解葡萄糖苷链中α(1-6)糖苷键.文章概述了已克隆的编码SBE和DBE同种型编码基因及其在体内外的表达特征,并提出DBE多聚体酶的结构和功能将成为淀粉粒起始形成研究中的起点,SBEⅠ和SBEⅡ表达调控是支链淀粉分子改良的途径的看法.     

玉米种子发育过程中直链淀粉积累规律的分析

淀粉是玉米种子的主要组成成分,它包括直链淀粉和支链淀粉。支链淀粉的合成需要淀粉合成酶、分支酶和脱支酶的共同作用,而直链淀粉的合成则是在颗粒结合型淀粉合成酶的作用下进行的。颗粒结合型淀粉合成酶基因的突变造成玉米种子的腊质（糯性）表型。与支链淀粉合成的分子机制的研究相比,目前对玉米种子中直链淀粉合成的分子机制了解相对较少。以野生型黄早4玉米自交系和突变体糯玉米为实验材料,研究了种子不同发育时期直链淀粉的积累规律。通过碘染色的方法,观察了玉米种子发育过程中淀粉积累的形态变化。定量分析表明,从授粉后10d至25d,黄早4种子中直链淀粉的含量逐渐增加,同时颗粒结合型淀粉合成酶（GBSS）的活性逐渐提高;而在糯玉米中,直链淀粉和GBSS活性均未检测到。进而,通过RT-PCR方法,从黄早4种子中分离出编码GBSSI的cDNA片段。在授粉后10d至25d的玉米胚乳中均可检测到GBSSI的表达,而在胚中直到授粉后25d才检测到该基因表达的微弱信号。在糯玉米种子中没有检测到该基因的表达。研究结果表明,在玉米种子发育过程中,GBSSI基因的表达通过控制GBSS的合成,最终控制直链淀粉的合成。研究工作为理解玉米种子中直链淀粉合成的分子机制提供了重要信息。     

糯玉米SW70和SW22是一类新的淀粉合成酶基因功能缺失多突变体

糯玉米是一类相对于粮食玉米的重要天然突变体，其淀粉合成的遗传机制复杂而又多样。以糯玉米自交系5形22、普通玉米自交系5003、SW22（♀）和5003（♂）的杂种定向选育的糯玉米自交系SW70（自交4代）为材料，研究了自交系授粉后1、2、4周时，SW22、SW70和5003籽粒中ADP-葡萄糖焦磷酸化酶（AGPase）、淀粉合酶（SS）、淀粉分支酶（SBE）和淀粉去分支酶（DBE）共4个家族21个成员编码基因表达水平的变化。结果表明：1）SW22中多个淀粉合成酶编码基因的表达动态与SW70及5003中有显著差异。差异主要存在于淀粉合酶SS和淀粉去分支酶DBE基因家族：2）SW22及SW70籽粒中有明显的阢转录产物累积，但累积水平均显著低于5003籽粒中：3）SW22及SW70中颗粒结合淀粉合成酶编码基因Gbssm和异淀粉酶型去分支酶编码基因Iso2完全不表达．意味着SW22胚乳中GbssIIb基因和Iso2基因的功能完全缺失。目前，有关玉米籽粒中GbSSIIb和异淀粉酶型DBEIso2同时突变或单个基因突变均未见报道。推测淀粉合成酶基因GbssIIb和异淀粉酶型DBEIso2功能缺失以及耽功能部分缺失．共同导致了SW22和SW70籽粒中直链淀粉合成部分缺陷。     

基因工程改良淀粉品质

淀粉对人类生活十分重要,它不仅是人们的能量和营养来源,而且还是重要的工业原材料。对于淀粉合成过程及淀粉的加工、使用一直是淀粉研究的重点内容。淀粉的合成在最后阶段涉及到３个关键性的酶是：ＡＤＰＧ焦磷酸化酶、淀粉合成酸以及淀粉分支酶。它们分别催化ＡＤＰ－葡萄糖的形成、葡聚糖链的延伸以及分支链的形成。另外淀粉去分支酶对淀粉最终结构的形成也起到重要作用。本文将介绍上述４个酶近年来的生物化学和分子生物学研究     

植物叶片暂时淀粉分解途径研究进展

植物叶片暂时淀粉主要分解途径包括如下过程:叶绿体中半结晶状淀粉粒在葡聚糖-水双激酶(GWD)和磷酸葡聚糖-水双激酶(PWD)作用下磷酸化,使淀粉粒结构松散;异淀粉酶(ISA3)作用于松散淀粉粒而释放出磷酸葡聚糖,再经磷酸葡聚糖磷酸酶(SEX4)水解去除磷酸而生成可溶性线性葡聚糖;葡聚糖在β-淀粉酶(BAM3)催化下水解生成麦芽糖后,再通过麦芽糖载体(MEX1)转运至细胞质.该文主要综述了以上转化过程中涉及的底物、生成物和催化酶类的研究进展情况,同时简述了植物叶片暂时淀粉分解的次要途径和抗逆性相关途径,并提出了该领域目前存在的问题和今后研究方向.     

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(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.     

稻米品质的分子水平研究进展

随着分子生物学和各项技术的发展,稻米品质的研究取得了一些进展。从与稻米品质中的直链淀粉含量、胶稠度、垩白、糊化温度及碾磨品质有关的基因或QTL,ADP葡聚糖焦磷酸化酶、淀粉合成酶、分支酶及脱支酶在淀粉合成中的作用以及胚乳淀粉粒的形成、形态与结构等方面作了综述 。     

复合调控Wx与SBE3基因对水稻籽粒直链淀粉含量的影响

颗粒淀粉合成酶（GBSS）和淀粉分支酶3（SBE3）是淀粉合成过程中的两个关键酶,这两个酶主要由耽和SBE3两个基因分别控制,它们的表达量直接影响直链淀粉和支链淀粉的含量比例。为了探讨水稻淀粉关键酶基因耽过量与SBE3干涉复合表达对直链淀粉含量的影响,构建了Wx过量表达与SBE3干涉结合的多基因表达载体,并通过农杆菌介导的方法将其导入日本晴水稻中。经过PCR检测分析获得了65株转基因阳性植株,半定量RT—PCR检测表明转基因株系中Wx基因表达量明显增加,而SBE3基因表达量显著减少。转基因株系籽粒透明度明显降低,直链淀粉含量比野生型的平均高45％,但是千粒重变化不大,与野生型相当。遗传分析表明这些转基因株系多数可稳定遗传。     

Effects of the activities of key enzymes involved in starch biosynthesis on the fine structure of amylopectin in developing rice (Oryza sativa L.) endosperms

The dynamic changes of the activities of enzymes involving in starch biosynthesis, including ADP-glucose pyrophosphorylase (AGPase), soluble starch synthases (SSS), starch branching enzyme (SBE) and starch debranching enzymes (DBE) were studied, and changes of fine structure of amy- lopectin were characterized by isoamylase treatment during rice grain development, using trans anti-waxy gene rice plants. The relationships between the activities of those key enzymes were also analyzed. The amylose synthesis was significantly inhibited in transgenic Wanjing 9522, but the total starch content and final grain weight were less affected as compared with those of non-transgenic Wanjing 9522 rice cultivar. Analyses on the changes of activities of enzymes involving in starch bio- synthesis showed that different enzyme activities were expressed differently during rice endosperm development. Soluble starch synthase is relatively highly expressed in earlier stage of endosperm de- velopment, whilst maximal expression of granule-bound starch synthase (GBSS) occurred in mid-stage of endosperm development. No obvious differences in changes of the activities of AGPase and SBE between two rice cultivars investigated, except the DBEs. Distribution patterns of branches of amy- lopectin changed continually during the development of rice grains and varied between two rice culti- vars. It was suggested that amylopectin synthesis be prior to the synthesis of amylose and different enzymes have different roles in controlling syntheses of branches of amylopectin.     

Effect of high temperature on fine structure of amylopectin in rice endosperm by reducing the activity of the starch branching enzyme

Rice (Oryza sativa L.) grain quality is affected by the environmental temperature it experiences. To investigate the physiological molecular mechanisms of the effect of high temperatures on rice grain, a non-waxy indica rice was grown under two temperature conditions, (29/35 degrees C) and (22/28 degrees C), during the ripening stage in two phytotrons. The activities and gene expression of key enzymes for the biosynthesis of amylose and amylopectin were examined. The activity and expression levels of soluble endosperm starch synthase I were higher at 29/35 degrees C than that at 22/28 degrees C. In contrast, the activities and expression levels of the rice branching enzyme1, the branching enzyme3 and the granule bound starch synthase of the endosperm were lower at 29/35 degrees C than those at 22/28 degrees C. These results suggest that the decreased activity of starch branching enzyme reduces the branching frequency of the branches of amylopectin, which results in the increased amount of long chains of amylopectin of endosperm in rice grain at high temperature.     

Coordinated Regulation of the Genes Participating in Starch Biosynthesis by the Rice Floury-2 Locus

The recessive floury-2 (flo-2) locus of rice (Oryza sativa L.), which is located on chromosome 4, causes a strong reduction in expression of the gene encoding an isoform of branching enzyme RBE1 in immature seeds 10 d after flowering. Mapping of the RBE1 gene demonstrated the localization on rice chromosome 6, suggesting that the wild-type Floury-2 (Flo-2) gene regulates RBE1 gene expression in trans. However, reduced expression of the genes encoding some other starch-synthesizing enzymes, including another isoform of branching enzyme RBE3 and granule-bound starch synthase, was also found in the flo-2 seeds. In spite of the low level of RBE1 gene expression in the immature seeds of the flo-2 mutants, the RBE1 gene was equally expressed in the leaves of the wild type and flo-2 mutants. Thus, these results imply that the Flo-2 gene may co-regulate expression of some of the genes participating in starch synthesis possibly in a developing seed-specific manner.     

云南野生稻籽粒淀粉合成关键酶活性测定

为研究云南3种野生稻直链淀粉合成机制并利用其直链淀粉含量较低的优良品质,以云南3种野生稻和4种当地栽培稻为材料,研究野生稻灌浆期籽粒4种淀粉合成关键酶(包括ADPG焦磷酸化酶、可溶性淀粉合成、颗粒凝结型淀粉合成酶、淀粉分支酶)活性变化。结果表明,野生稻中4种淀粉合成酶的变化趋势与栽培稻相似,但活性有较大差别。颗粒凝结型淀粉合成酶的活性与直链淀粉含量呈正相关,说明在野生稻中同样是由颗粒凝结型淀粉合成酶控制直链淀粉的合成。同时发现在同一灌浆期,同种材料中可溶性淀粉合成酶和淀粉分支酶的活性变化呈相反趋势,推测这两种酶之间可能在淀粉合成过程中存在某种反馈调节机制。     

Starch branching enzyme IIb in wheat is expressed at low levels in the endosperm compared to other cereals and encoded at a non-syntenic locus

Studies of maize starch branching enzyme mutants suggest that the amylose extender high amylose starch phenotype is a consequence of the lack of expression of the predominant starch branching enzyme II isoform expressed in the endosperm, SBEIIb. However, in wheat, the ratio of SBEIIb and SBEIIa expression are inversely related to the expression levels observed in maize and rice. Analysis of RNA at 15 days post anthesis suggests that there are about 4-fold more RNA for SBE IIa than for SBE IIb. The genes for SBE IIa and SBE IIb from wheat are distinguished in the size of the first three exons, allowing isoform-specific antibodies to be produced. These antibodies were used to demonstrate that in the soluble fraction, the amount of SBE IIa protein is two to three fold higher than SBIIb, whereas in the starch granule, there is two to three fold more SBE IIb protein amount than SBE IIa. In a further difference to maize and rice, the genes for SBE IIa and SBE IIb are both located on the long arm of chromosome 2 in wheat, in a position not expected from rice–maize–wheat synteny.     

Starch biosynthesis in cereal endosperm

Stored starch generally consists of two d-glucose homopolymers, the linear polymer amylose and a highly branched glucan amylopectin that connects linear chains. Amylopectin structurally contributes to the crystalline organization of the starch granule in cereals. In the endosperm, amylopectin biosynthesis requires the proper execution of a coordinated series of enzymatic reactions involving ADP glucose pyrophosphorylase (AGPase), soluble starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE), whereas amylose is synthesized by AGPase and granule-bound starch synthase (GBSS). It is highly possible that plastidial starch phosphorylase (Pho1) plays an important role in the formation of primers for starch biosynthesis in the endosperm. Recent advances in our understanding of the functions of individual enzyme isoforms have provided new insights into how linear polymer chains and branch linkages are synthesized in cereals. In particular, genetic analyses of a suite of mutants have formed the basis of a new model outlining the role of various enzyme isoforms in cereal starch production. In our current review, we summarize the recent research findings related to starch biosynthesis in cereal endosperm, with a particular focus on rice.     

Starch branching enzymes from immature rice seeds.

Four forms of branching enzyme, termed RBE1, RBE2 (a mixture of RBE2A and RBE2B), RBE3, and RBE4, were apparently separated by DEAE-cellulose column chromatography of soluble extract from immature rice seeds, and each of these four forms was further purified by gel-filtration. RBE1, RBE2A, and RBE2B were the predominant forms of the enzyme. The molecular size, amino-terminal amino acid sequence, and immunoreactivity with anti-maize branching enzyme-I (BE-I) antibody were identical among these three forms, except that the molecular mass of RBE2A was almost 3 kDa higher than those of RBE1 and RBE2B. These results indicate that RBE1, RBE2A, and RBE2B are the same (termed rice BE-I). The cDNA clones coding for rice BE-I have been identified from a rice seed library in lambda gt11, using the maize BE-I cDNA as a probe. The nucleotide sequence indicates that rice BE-I is initially synthesized as an 820-residue precursor protein, including a putative 64- or 66-residue transit peptide at the amino terminus. The rice mature BE-I contains 756 (or 754) amino acids with a calculated molecular mass of 86,734 (or 86,502) Da, and shares a high degree of sequence identity (86%) with the maize protein. The consensus sequences of the four regions that form the catalytic sites of amylolytic enzymes are conserved in the central region of the rice BE-I sequence. Thus, rice BE-I as well as the maize protein belongs to a family of amylolytic enzymes.