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.     

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。     

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。     

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。     

百合鳞茎发育过程中淀粉合成相关酶基因的克隆及表达分析

该研究通过同源克隆技术克隆腺苷二磷酸葡萄糖焦磷酸化酶(AGPase)、颗粒结合淀粉合酶(GBSS)和可溶性淀粉合酶(SSS) 3类百合淀粉合成关键酶基因,分析这三类淀粉合成关键酶基因的表达变化,测定百合鳞茎膨大发育中淀粉含量变化。结果表明:(1) AGPase具有GlgC家族蛋白PLN02241蛋白结构特征及cl11394家族蛋白ADP_Glucose_PP与NTP_transferase结构域,获登录号KP751443; GBSS与SSS具有cl10013家族蛋白Glyco_transf_5,GT1_Glycogen_synthase_DULL1_like结构域,获登录号分别为KP751444、KP751445。(2)百合鳞茎形成与膨大发育过程中,淀粉含量呈现递增趋势,鳞茎盘开始分化茎杆时其淀粉含量最高,达到44.52%。鳞茎与叶片部位的三个淀粉合成相关酶基因表达量均逐渐增加;在鳞茎膨大后茎杆分化阶段,三个淀粉合成相关酶基因表达量达到最高,AGPase、GBSS、SSS在鳞片中的表达量分别为10.79,6.92和5.12,叶片中的表达量分别为6.79,5.22和4.41,鳞片中的表达量大幅度高于叶片;淀粉合成相关酶基因的表达量变化与淀粉含量、鳞茎的膨大发育成正相关。这为鳞茎的繁殖生产提供了可通过调节淀粉合成关键酶基因表达促进百合鳞茎膨大发育的思路。     

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．     

温郁金萜类合酶基因CwTPS4的克隆和表达特征

温郁金是著名的"浙八味"之一,药用价值高、应用广泛,萜类化合物是其主要的药用成分.萜类合酶是植物萜类化合物生物合成途径中的关键酶.依据温郁金根茎的转录组数据,采用反转录PCR获得了1个萜类合酶基因CwTPS4(GenBank登录号:MW774935),其开放阅读框长1515 bp,编码504个氨基酸,含有萜类合酶特有的结构域和保守序列RXR、DDXXD等;生物信息学分析表明CwTPS4编码的蛋白定位在细胞质中、无跨膜区域,为水溶性的稳定蛋白,属于萜类合酶的TPS-a亚家族成员.实时荧光定量分析表明,CwTPS4基因主要在生长旺盛的叶片中表达,其次在根茎膨大初期的根茎中表达量较高,而在成熟或衰老的组织中表达量较低.本研究从温郁金中克隆得到1个新的萜类合酶基因CwTPS4,经生物信息学分析表明CwTPS4可能参与了温郁金中倍半萜类化合物的合成,为下一步研究其在温郁金萜类物质合成中的功能奠定了一定的基础.     

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。     

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.     

蔗糖合酶在植物生长发育中的作用研究

蔗糖合酶（SuSy）是植物蔗糖代谢的关键酶之一,在植物各组织中普遍存在。SuSy参与了植物体中许多代谢过程,包括淀粉及纤维素的合成,以及碳源的分配等。该酶还可影响植物的抗逆性、种子发育和生物固氮能力,因此,利用SUS基因改良作物品质具有良好的应用前景。对SuSy的性质、基因表达模式及其在植物生长发育中的作用进行综述。     

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.     

A novel shrunken endosperm mutant of barley

Although mutations affecting several enzymes of the starch synthetic pathway in developing cereal endosperm have been isolated, none has a major effect on soluble starch synthase We report a new recessive shrunken endosperm mutant in barley ( Hordeum vulgare L. cv. Bomi-like), shx , which has 25% of normal starch content. We have assayed the activity of sucrose synthase (EC 2.4.1.13), ADP and UDP-glucose pyrophosphorylases (EC 2.7.7.27 and 2.7.7.9), branching enzyme (EC.2.4.1.18), and granule-bound and soluble starch synthase (EC 2.4.1.21) in shx. Sucrose synthase activity is reduced by 49% and UDP-glucose pyrrphosphorylase is 80% of the normal level. Branching enzyme and starch-bound starch synthase activities are normal, but ADP-glucose pyrophosphorylase activity is reduced by 72%. The soluble starch synthase that is primer-independent in the presence of sodium citrate shows 14% of normal activity in shx. whereas the primer-dependent form is unaffected. This lower starch synthase activity in shx cannot be explained by inhibition, substrate destruction or lack of primer. Although several starch-synthetic enzymes are affected, it is suggested that the primer independent from of soluble starch synthase may be the primary-site of the mutation in shx.     

Expression of a chimaeric granule-bound starch synthase-GUS gene in transgenic potato plants

Granule-bound starch synthase is the key enzyme in amylose synthesis. The regulation of this gene was investigated using a chimaeric gene consisting of a 0.8 kb 5 upstream sequence of the granule-bound starch synthase gene from potato and the -glucuronidase gene which was introduced into potato using an Agrobacterium tumefaciens binary vector system. The chimaeric gene was highly expressed in stolons and tubers, whereas the expression in leaves, stems or roots from greenhouse-grown plants was relatively low. However, leaves from in vitro grown plantlets exhibited an elevated GUS expression. The expression of the chimaeric gene was inducible in leaves by growth on relatively high concentrations of sucrose, fructose and glucose and was about 30- to 50-fold higher than in leaves from greenhouse-grown plants. The granule-bound starch synthase gene is expressed organ-specifically since stolons and tubers showed GUS activities 125- to 3350-fold higher than in leaves. The activities in these two organs are 3- to 25-fold higher than the expression of the CaMV-GUS gene. Histochemical analysis of different tissues showed that only certain regions of leaves and roots express high GUS activities. Stolons and tubers show high expression.     

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.