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。     

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。     

秸秆还田对旱作马铃薯块茎淀粉合成关键酶活性及基因表达的影响

为探讨秸秆还田下旱作马铃薯块茎形成过程中淀粉合成关键酶活性及基因表达特性,以马铃薯栽培品种"青薯9号"为材料,以露地栽培为对照,设置秸秆还田处理,研究了马铃薯块茎形成过程中淀粉合成关键酶活性、基因表达、淀粉糊化及累积指标。结果表明:秸秆还田显著提高了旱作马铃薯SSS酶活性,降低了AGPP、GBSS酶活性,而对SBE酶活性没有显著影响;显著提高了SSⅡ、SSⅢ基因表达量,降低了AGPase、GBSSⅠ、SBEⅠ、SBEⅡ基因表达量;显著增加了淀粉崩解值,减少了淀粉各阶段粘度、回生值,而对淀粉糊化温度没有显著影响;显著增加了直链淀粉含量及直/支链淀粉比,减少了总淀粉含量;GBSS酶活性与AGPase、SBEⅠ基因表达量呈显著正相关,与直链淀粉含量、直/支链淀粉比呈显著负相关;SBE酶活性与SSⅡ基因表达量、峰值粘度、低谷粘度、最终粘度、总淀粉含量呈显著正相关,与崩解值、糊化温度呈显著负相关;AGPase基因表达量与直链淀粉含量呈显著负相关;GBSSⅠ基因表达量与最终粘度、回生值呈显著正相关,与糊化温度呈显著负相关;淀粉糊化与累积无显著相关性。     

不同类型土壤上种植的小麦籽粒中与淀粉合成相关的酶活性变化

测定池栽条件下灰潮土、水稻土、砂姜黑土上种植的强筋小麦‘郑麦9023’籽粒灌浆过程中腺苷二磷酸葡萄糖焦磷酸化酶（AGPP）、尿苷二磷酸葡萄糖焦磷酸化酶（UGPP）、可溶性淀粉合成酶（SSS）、淀粉粒结合淀粉合成酶（GBSS）、淀粉分支酶（SBE）5个与淀粉合成有关的酶活性变化的结果表明,不同类型土壤上种植的小麦籽粒中AGPP、UGPP、SSS、GBSS、SBE活性均呈单峰曲线变化,花后18d,AGPP、UGPP、SSS和SBE活性达到峰值,而GBSS则在花后24d达到峰值。AGPP、SSS、SBE活性峰值表现为灰潮土〉水稻土〉砂姜黑土,UGPP峰值表现为灰潮土〉砂姜黑土〉水稻土,GBSS峰值则表现为水稻土〉灰潮土〉砂姜黑土。     

秸秆还田对旱作马铃薯块茎淀粉合成关键酶活性及基因表达的影响

为探讨秸秆还田下旱作马铃薯块茎形成过程中淀粉合成关键酶活性及基因表达特性,以马铃薯栽培品种"青薯9号"为材料,以露地栽培为对照,设置秸秆还田处理,研究了马铃薯块茎形成过程中淀粉合成关键酶活性、基因表达、淀粉糊化及累积指标。结果表明:秸秆还田显著提高了旱作马铃薯SSS酶活性,降低了AGPP、GBSS酶活性,而对SBE酶活性没有显著影响;显著提高了SSⅡ、SSⅢ基因表达量,降低了AGPase、GBSSⅠ、SBEⅠ、SBEⅡ基因表达量;显著增加了淀粉崩解值,减少了淀粉各阶段粘度、回生值,而对淀粉糊化温度没有显著影响;显著增加了直链淀粉含量及直/支链淀粉比,减少了总淀粉含量; GBSS酶活性与AGPase、SBEⅠ基因表达量呈显著正相关,与直链淀粉含量、直/支链淀粉比呈显著负相关; SBE酶活性与SSⅡ基因表达量、峰值粘度、低谷粘度、最终粘度、总淀粉含量呈显著正相关,与崩解值、糊化温度呈显著负相关; AGPase基因表达量与直链淀粉含量呈显著负相关;GBSSⅠ基因表达量与最终粘度、回生值呈显著正相关,与糊化温度呈显著负相关;淀粉糊化与累积无显著相关性。     

淀粉含量不同的玉米自交系籽粒淀粉积累及其关键酶活性

以2个高淀粉和2个低淀粉玉米自交系为材料,分析了玉米籽粒淀粉的动态积累规律,同时对高低淀粉玉米籽粒灌浆过程中淀粉生物合成关键酶活性的动态变化及其与淀粉积累动态的相关性进行讨论分析。研究结果表明:灌浆过程中4个自交系淀粉含量变化趋势均呈sigmoid型曲线。灌浆过程中ADPG-PPase(腺苷二磷酸葡萄糖焦磷酸化酶)、SSS(可溶性淀粉合成酶)、GBSS(颗粒结合淀粉合成酶)活性均呈单峰曲线变化,峰值都出现在20～30DAP(授粉后天数)。2个高淀粉自交系的Q酶(淀粉分支酶)活性也呈单峰曲线变化,峰值也出现在20DAP,而2个低淀粉自交系的Q酶活性则呈双峰曲线变化,2个峰值分别出现在15～20DAP和30～35DAP。4个自交系籽粒淀粉的积累速率与各自交系ADPG-PPase、SSS和GBSS的活性变化呈极显著正相关。各自交系关键酶活性之间,ADPG-PPase、SSS和GBSS三者间活性变化呈极显著正相关,这3种酶活性变化与Q酶活性变化也呈不同程度的正相关。     

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.     

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。     

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。     

鲜食糯玉米采后糖代谢相关酶活性变化

鲜食糯玉米采后腺苷二磷酸葡萄糖焦磷酸化酶（ADPGPPase）、尿苷二磷酸葡萄糖焦磷酸化酶（UDPGPPase）、束缚态淀粉合成酶（GBSS）、淀粉脱分支酶（DBE）等活性均呈现单峰曲线变化,第1～2天出现峰值然后略有下降;可溶性淀粉合成酶（SSS）活性呈较大幅度上升趋势,活性远高于采收初期;淀粉分支酶（SBE）在第3天出现极高峰值。与20℃贮藏温度相比,采后0℃低温贮藏可增强UDPGPPase活性,促进蔗糖降解;降低SSS和GBSS活性,延缓淀粉合成进程;抑制SBE活性,增强DBE活性,促进直链淀粉生成。     

Introduction of Wx transgene into rice wx mutants leads to both high- and low-amylose rice

The Waxy (Wx) gene encodes a granule-bound starch synthase (GBSS) that plays a key role in the amylose synthesis of rice and other plant species. Two functional Wx alleles of rice exist: Wx(a), which produces a large amount of amylose, and Wx(b), which produces a smaller amount of amylose because of the mutation at the 5' splice site of intron 1. Wx(b) is largely distributed in Japonica cultivars, and high amylose cultivars do not exist in Japonica cultivars. We introduced the cloned Wx(a) cDNA into null-mutant Japonica rice (wx). The amylose contents of these transgenic plants were 6-11% higher than that of the original cultivar, Labelle, which carries the Wx(a) allele, although the levels of the Wx protein in the transgenic rice were equal to those of cv. Labelle. We also observed a gene-dosage effect of the Wx(a) transgene on Wx protein expression, but a smaller dosage effect was observed in amylose production with over 40% of amylose content in transgenic rice. Moreover, one transgenic line carrying eleven copies of the transgene showed low levels of Wx expression and amylose in the endosperm. This suggested that the integration of excessive copies of the transgene might lead to gene silencing.     

Molecular aspect of good eating quality formation in Japonica rice

The composition of amylopectin is the determinant of rice eating quality under certain threshold of protein content and the ratio of amylose and amylopectin. In molecular biology level, the fine structure of amylopectin is determined by relative activities of starch branching enzyme (SBE), granule-bound starch synthase (GBSS), and soluble starch synthase (SSS) in rice grain under the same ADP-Glucose level. But the underlying mechanism of eating quality in molecular biology level remains unclear. This paper reports the differences on major parameters such as SNP and insertion-deletion sites, RNA expressions, and enzyme activities associated with eating quality of japonica varieties. Eight japonica rice varieties with significant differences in various eating quality parameters such as palatability and protein content were used in this experiment. Association analysis between nucleotide polymorphism and eating quality showed that S12 and S13 loci in SBE1, S55 in SSS1, S58 in SSS2A were significantly associated with apparent amylose content, alkali digestion value, setback viscosity, consistency viscosity, pasting temperature, which explained most of the variation in apparent amylose content, setback viscosity, and consistency viscosity; and explained almost all variations in alkali digestion value and pasting temperature. Thirty-five SNPs and insertion-deletions from SBE1, SBE3, GBSS1, SSS1, and SSS2A differentiated high or intermediate palatability rice varieties from low palatability rice varieties. Correlation analysis between enzyme activities and eating quality properties revealed that SBE25 and SSS15/W15 were positively correlated with palatability, whereas GBSS10 and GBSS15 were negatively correlated. Gene expressions showed that SBE1 and SBE3 expressions in high palatability varieties tended to be higher than middle and low palatability varieties. Collectively, SBE1, SBE3, SSS1, and SSS2A, especially SBE1 and SBE3 could improve eating quality, but GBSS1 decreased eating quality. The results indicated the possibility of developing high palatability cultivars through modification of key genes related to japonica rice eating quality formation in starch biosynthesis.     

Gene expression of the biosynthetic enzymes and biosynthesis of starch during Rice Grain development

Amylose and amylopectin are determinants of the physicochemical properties for starch and grain quality in rice. Their biosynthesis is catalyzed by the interplay of ADP-glucose pyrophosphorylase (AGPase), granule-bound starch synthase (GBSS), soluble starch synthase (SSS), a starch branching enzyme (SBE), and a starch debranching enzyme (SDE). In this study, the genes for these enzymes were highly expressed 7 to 28 days after flowering during grain development, and their expression closely matched increases in both starch content and grain weight Among all the tested cultivars, amylose contents in the rice grains remained essentially constant throughout their development The AGPase gene was highly expressed in the high-yield cultivars of both glutinous and non-glutinous rice. The SSS gene was actively expressed when mature GBSS mRNA decreased. Genes responsible for amylopectin biosynthesis were simultaneously expressed in the late stage of grain development. We have now demonstrated that the expression patterns of starch biosynthetic genes differ between glutinous and non-glutinous rice, and between Tongil (a Japonica/ Indica hybrid) and Japonica types.     

水稻Wx基因表达调控的研究进展

水稻Wx基因编码颗粒结合淀粉合成酶(GBSS),是控制直链淀粉合成的主效基因。文中主要从转录水平和转录后水平介绍水稻Wx基因表达调控的研究进展,同时介绍转基因、遗传背景以及环境温度对Wx基因表达的影响,并提出Wx基因表达调控研究中一些期待解决的问题。     

Temperature induced changes in the starch components and biosynthetic enzymes of two rice varieties

The effects of temperature on starch and amylose accumulation, fine structure of amylopectin and activities of some enzymes related to starch synthesis in developing rice endosperms was examined. Two early indica rice varieties were used, differing in amylose concentration (AC, %), namely Jia 935 (low AC) and Jia 353 (high AC). The results showed that the effects of high temperature on AC and amylopectin fine structure were variety-dependent. High temperature caused a reduction in amylose concentration and an increase in the short chain (CL<22) proportion of amylopectin for Jia 935; while opposite was true for Jia 353. High temperature also reduced and increased the activity of granule-bound starch synthase (GBSS) in Jia 935 and in Jia 353, respectively. This suggests that a change in the ratio of amylose/starch due to temperature was attributable to a change in GBSS activity. Moreover, obvious differences between the two rice varieties were detected in the activities of sucrose synthase (SuSy), ADP-glucose pyrophosphorylase (ADPG-Ppase), soluble starch synthase (SSS), starch branching enzyme (SBE), starch de-branching enzyme (SDBE) and starch phosphorylase (SPase) to high temperature. Accumulation rate of amylose was significantly and positively correlated with GBSS for Jia 935, but not for Jia 353. Amylose accumulation was also significantly and positively correlated with the activities of SDBE, SBE, ADPG-Ppase and SuSy for both varieties. The results suggest that the ratio of amylose to starch in rice endosperm is not only related to GBSS, but also affected by the activities of SDBE, SBE, ADPG-Ppase and SuSy.     

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