小麦腺苷二磷酸葡萄糖焦磷酸化酶同工酶基因型与酶活性及淀粉含量的关系

采用非变性聚丙烯酰胺凝胶电泳鉴定了我国60个代表性小麦品种的腺苷二磷酸葡萄糖焦磷酸化酶(AGP)同工酶基因型, 并测定了AGP活性及总淀粉含量, 以明确小麦籽粒AGP同工酶基因型组成及其与AGP活性和淀粉含量的关系。结果表明, AGP有AGPa、AGPb、AGPc和AGPd 4个等位基因位点, 其出现频率分别为96.7%、80.0%、86.7%和16.7%; 共检测到5种基因型, 其中基因型AGPabc出现频率最高, 为46.7%。不同基因型的品种间AGP活性和总淀粉含量差异显著(P<0.05)。其中具有基因型AGPabcd的品种酶活性及总淀粉含量最高, 表明小麦籽粒中不同AGP同工酶基因型对酶活性及淀粉含量有不同遗传效应。     

小麦籽粒淀粉分支酶同工酶结构组成及时空表达

本研究的目的是阐明小麦支链淀粉合成的酶学机制。以8个小麦品种的籽粒为材料, 采用非变性聚丙烯酰胺凝胶电泳(Native-PAGE)和SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定SBE同工酶类型、时空表达谱及亚基组成, 分析SBE同工酶空间分布特点和器官表达特异性。共检测到4种SBE同工酶, 其中B和SBEIIa分布在胚乳和叶片中, 而A和D_i专一定位于胚乳中。在小麦籽粒灌浆过程中, D_i和SBEIIa首先表达, 而后是B, A最后表达;至灌浆末期, B和SBEIIa停止表达。SBE同工酶都是单亚基酶, 均由一条86~92 kD的多肽链组成。SBE同工酶的空间分布具有器官特异性, 并在籽粒发育进程中顺序表达。D_i、B和SBEIIa是占主导地位的SBE同工酶, 可能是决定SBE总酶活性的主效应酶, 在籽粒和叶片支链淀粉合成中起关键作用。     

玉米淀粉分支酶SBEⅠ基因RNAi干扰载体的构建

淀粉分支酶(SBE)催化葡萄糖以α-(1,6)糖苷键连接,形成分支结构。SBE有3种同工酶形式：SBEⅠ、SBEⅡa和SBEⅡb,目前的研究主要集中在SBEⅡb上,而对于SBEⅠ在淀粉合成的作用报道极少。为了明确SBEⅠ在玉米淀粉合成中的作用,扩增了玉米SBEⅠ基因的337 bp片段,以pMD19-T为中间载体,pCambia3301为目标载体,构建玉米SBEⅠ基因的RNAi载体,命名为pC3301-SBEⅠ。通过限制性内切酶酶切鉴定,表明pC3301-SBEⅠ载体构建正确,可用于农杆菌介导的玉米自交系转化工作。     

小麦籽粒淀粉分支酶同种型SBE Ⅱb的亚细胞定位及遗传多样性

以70个有代表性的小麦品种,采用SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)方法检测淀粉粒结合SBE Ⅱb的品种间差异;并对SDS-PAGE凝胶中SBE Ⅱb进行回收、复性和酶活性测定.结果表明,淀粉粒结合蛋白(SGP)中分子量为85 kD的SGP-2是与淀粉粒结合的SBE Ⅱb,具有淀粉分支酶活性;SBE Ⅱb从淀粉粒释放并去除SDS后活性立即恢复,其活性高峰出现在花后21 d左右;SBE Ⅱb在小麦籽粒发育早期开始表达,不同发育时期表达量有差异,但其表达图谱在品种间没有差异,即编码SBE Ⅱb的基因位点不具有品种的遗传多态性.     

灌浆期高温与干旱胁迫对小麦籽粒淀粉合成关键酶活性及淀粉积累的影响

高温与干旱是小麦灌浆期的主要胁迫逆境, 影响小麦同化物的生成及其在籽粒中的积累。本文以郑麦366为试验材料, 采用盆栽和人工气候室模拟相结合的方式, 研究了灌浆期高温(HT)、干旱(DS)和高温干旱复合胁迫(HT+DS)对小麦籽粒淀粉合成关键酶活性、淀粉及其组分的影响。于花后10 d将长势均匀一致的盆栽小麦转移至人工气候室, 至小麦成熟。人工气候室设适温(昼25℃/夜15℃)和高温(昼32℃/夜22℃)两种温度模式, 每种温度模式下又设置正常水分(土壤相对含水量75%左右)和轻度干旱胁迫(土壤相对含水量50%左右)两个土壤水分处理, 以适温、正常水分处理为对照。与对照相比, HT、DS及HT+DS条件下籽粒可溶性淀粉合酶(SSS)和结合态淀粉合酶(GBSS)活性在胁迫初期显著升高, 之后迅速下降; 淀粉分支酶(SBE)、ADPG焦磷酸化酶(AGPase)和蔗糖合酶(SS)活性在小麦籽粒整个灌浆过程中均低于对照; 上述酶活性还受高温与干旱互作的显著影响。HT、DS及HT+DS逆境下, 小麦籽粒淀粉积累速率减小, 籽粒直、支链淀粉和总淀粉含量下降, 生育期缩短, 粒重和产量降低。其中, HT的影响大于DS, 复合胁迫的影响大于单一胁迫。相关分析表明, SSS和GBSS活性与籽粒直、支链淀粉和总淀粉含量在多数测定时期呈正相关(P<0.01), AGPase、SBE和SS活性在灌浆后期(花后22~26 d)与籽粒直、支链淀粉和总淀粉含量呈正相关(P<0.01)。表明高温干旱胁迫通过淀粉合成关键酶而影响籽粒淀粉的合成与积累, 最终影响小麦产量和品质。     

两种供水条件下两穗型小麦品种籽粒淀粉积累及相关酶活性的变化特征

在灌溉和旱作2种栽培条件下,研究了大穗型（山农710331和潍麦8号）和多穗型（济南17和鲁麦21）小麦籽粒淀粉积累及相关酶活性的变化特征。结果表明,淀粉积累速率（SAR）和蔗糖合成酶（SS）、ADPG焦磷酸化酶（AGPase）、淀粉合成酶（SSS和GBSS）和淀粉分支酶（SBE）等活性均存在明显的基因型差异。灌溉条件下大穗型品种籽粒淀粉合成相关酶的活性显著高于多穗型品种,旱作栽培条件下两穗型品种间差异变小。旱作栽培宜于增加灌浆前、中期AGPase、SS和SBE的活性,尤其对多穗型品种。大穗型品种在灌浆中后期比多穗型品种具有更强的淀粉合成能力,但对水分较为敏感。用 Richards方程模拟籽粒淀粉积累过程表明,大穗型品种籽粒淀粉积累时间长、速率高,是其淀粉积累量高的原因。     

反义Trxs基因导入对弱筋小麦豫麦18淀粉积累及淀粉合成关键酶表达的影响

以转反义硫氧还蛋白基因小麦TY18-99和TY18-100及其相应未转基因对照为材料,于2007-2009年通过盆栽试验系统研究了反义Trxs基因(anti-Trxs)导入对弱筋小麦豫麦18籽粒灌浆过程中淀粉积累、淀粉合成关键酶活性以及淀粉合成酶基因表达的影响。结果表明,反义Trxs基因对小麦籽粒淀粉积累有一定的正效应。2个转基因株系的总淀粉和支链淀粉的积累速率较对照显著提高;在整个籽粒形成过程中,反义Trxs基因导入显著提高了淀粉分支酶(SBE)活性。在籽粒灌浆中期和后期,反义Trxs基因导入显著提高了腺苷二磷酸葡萄糖焦磷酸化酶(AGPase)和可溶性淀粉合酶(SSS)活性,降低了颗粒束缚型淀粉合酶(GBSS)活性。实时荧光定量RT-PCR分析表明,反义Trxs基因促进了AGPase、SBE I和SSS (SSS I、SSS II和SSS III)基因的表达,抑制了GBSS I基因的表达。上述结果说明,反义Trxs基因导入后促进了AGPase、SBE I和SSS基因的转录,从而使籽粒灌浆期间的淀粉积累速率发生了改变,最终显著提高了总淀粉和支链淀粉的含量,降低了直链淀粉含量,进而提高了淀粉的支/直比,这可能是转基因小麦淀粉品质改善和产量提高的主要原因。     

开花期外施表油菜素内酯(epi-BR)对小麦籽粒淀粉积累及其关键酶活性的影响

在大田条件下，以豫麦49 (Tritictun aestivurn cv. Yumai 49)为试材，研究了开花期外施0、0.1、1.0、10.0 µg/L表油菜素内酯(epi-BR)对小麦籽粒灌浆过程中腺苷二磷酸葡萄糖焦磷酸化酶(ADPGPPase)、可溶性淀粉合成酶(SSS)、颗粒束缚型淀粉合成酶(GBSS)和淀粉分支酶(SBE)活性，以及淀粉积累速率的影响，同时对收获籽粒的淀粉成分和淀粉特性进行了测定。结果表明，开花期外施epi-BR提高了小麦籽粒中ADPGPPase、SSS和SBE活性，epi-BR 0.1和1.0 µg/L 处理可降低GBSS活性，10.0 µg/L处理可提高 GBSS活性，从而改变小麦籽粒灌浆期间的淀粉积累速率，改善小麦籽粒淀粉成分与特性，提高淀粉品质。在本试验条件下，以epi-BR 1.0 µg/L处理对籽粒淀粉的调控效果最好，使小麦籽粒中总淀粉含量、淀粉的支/直比、高峰黏度值和崩解值分别提高8.88% (α＜0.01 ）、34% (α＜0.01）、32.4 BU (α＜0.05）和13.7 BU (α＜0.05)。     

小麦籽粒淀粉分支酶同种型SBE IIb 的亚细胞定位及遗传多样性

以70个有代表性的小麦品种,采用SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)方法检测淀粉粒结合SBE IIb的品种间差异;并对SDS-PAGE凝胶中SBE IIb进行回收、复性和酶活性测定。结果表明,淀粉粒结合蛋白(SGP)中分子量为85 kD的SGP-2是与淀粉粒结合的SBE IIb,具有淀粉分支酶活性;SBE IIb从淀粉粒释放并去除SDS后活性立即恢复,其活性高峰出现在花后21 d左右;SBE IIb在小麦籽粒发育早期开始表达,不同发育时期表达量有差异,但其表达图谱在品种间没有差异,即编码SBE IIb的基因位点不具有品种的遗传多态性。     

水稻淀粉合成相关基因对稻米品质效应的研究

颗粒淀粉合成酶(GBSS)、淀粉分支酶1(SBE1)和淀粉分支酶3(SBE3)是淀粉合成过程中的3个关键酶,这3个酶主要由耽、Sbel、She3 3个基因控制.设计这3个基因位点的分子标记检测了183个水稻品种的基因型,分析了3个基因对稻米理化品质和RVA谱特征的效应.结果表明:3个分子标记均能很好的区分3个位点上等位基因的籼梗来源,根据3个位点的基因型可将183个品种分为8种类型;单个基因遗传效应分析表明,不同基因型品种间淀粉的理化特性(AC、GC、RVA)存在显著或极显著差异,3个基因的联合效应在不同基因型组合间也存在显著的差异.Wx基因对大多数品质性状效应显著,Sbe3效应次之,Sbel效应较小;Sbel和Sbe3基因在不同的Wx等位基因背景下,对稻米的品质的理化特征的效应不同.3基因间存在互作效应,Wx与Sbe3的互作效应较大.     

木薯淀粉分支酶SBEⅠ反义基因遗传转化木薯的研究

旨在获得转淀粉分支酶反义SBEⅠ基因的‘华南木薯8号’转基因植株,为利用转基因技术改良木薯淀粉品质打下基础。在建立了木薯从胚状体子叶到完整植株的再生体系的基础上,用块根特异表达启动子Sporamin驱动的木薯淀粉分支酶SBEⅠ反义基因,通过农杆菌介导法对‘华南木薯8号’进行遗传转化。共接种‘华南木薯8号’子叶517块,获得7株生长良好的转化再生植株,转化再生频率达到1.35%。经PCR检测,其中5株转化再生植株扩增出目的条带,初步证实木薯淀粉分支酶SBEⅠ反义基因已整合进了‘华南木薯8号’基因组中。通过农杆菌介导法可以将淀粉分支酶SBEⅠ反义基因导入到‘华南木薯8号’基因组中,获得了5株转基因植株。     

An approach to DNA polymorphism screening in <Emphasis Type="Italic">SBEIIa</Emphasis> homeologous genes of polyploid wheat (<Emphasis Type="Italic">Triticum</Emphasis> L.)

The non-transgenic manipulation of starch properties in common wheat (Triticum aestivum L.) generally implies combining mutant alleles of the particular gene copies in all three subgenomes (A, B and D). The redundancy of the hexaploid wheat chromosome set substantially complicates the identification of recessive mutations and breeding. Nevertheless, naturally occurring or induced genetic polymorphism has already been successfully exploited for the production of waxy (GBSSI-deficient) and elevated amylose (SSIIa-deficient) wheats. However, in order to achieve the amylose content above 50% of wheat endosperm starch, it may be necessary to inactivate the starch branching enzyme (SBEIIa) isoforms, as the RNAi repression results and gene expression data strongly suggest. The identification of null SBEIIa alleles and their combination in a single genotype is therefore a promising approach to the production of non-transgenic high-amylose wheat; however, wheat SBEIIa polymorphism has not been characterized as of yet. In order to develop an approach to SBEIIa mutation screening, we sequenced the SBEIIa central region (exons 9–12) from the three subgenomes of common wheat cv. Chinese Spring and the A genome of diploid einkorn T. monococcum. The genome-specific primers were developed that amplify the exons downstream from intron 11 selectively from each homeologous gene. Using a single-stranded DNA conformation polymorphism (SSCP) approach, we screened 60 wheat cultivars, landraces, and rare species for naturally occurring SNPs in exons 12, 13 and 14 of the three SBEIIa homeologs. In total, 13 SNPs were discovered in the A and B wheat genomes. Two of these SNPs affect the amino acid sequences of SBEIIa isoforms and may change the enzyme functional properties. The presence of restriction site polymorphism at SNP positions enables their easy genotyping with CAPS assays. Our results indicate that the mining for naturally occurring sequence polymorphism in starch biosynthesis genes of wheat can be successfully performed at the DNA level, providing the starting point for a search for SBEIIa mutants at a larger scale.     

秸秆还田配施化学氮肥对冬小麦氮效率和产量的影响

2006—2007年生长季, 通过田间定位试验, 研究了秸秆还田配施化学氮肥对冬小麦产量干物质积累、氮效率和产量的影响。结果表明, 与单施氮肥相比, 秸秆还田配施氮肥的冬小麦全生育期干物质积累总量较高, 干物质积累量及其占全生育期干物质积累量的百分比抽穗前阶段较低, 而抽穗到成熟阶段则较高。秸秆还田配施纯氮90、180、270和360 kg hm^-2比单施同量氮肥分别增产7.1%、8.4%、11.1%和10.2%, 其中秸秆还田配施纯氮270 kg hm^-2的产量最高, 显著高于其他各处理。秸秆还田配施氮肥比单施氮肥提高冬小麦的氮效率, 氮肥表观利用率、氮素生产效率和氮肥农学效率分别提高3.3%~9.2%、0.7~4.0和2.7~7.3 kg kg^-1。     

Development of near-isogenic lines of wheat carrying different null Wx alleles and their starch properties

The granule-bound starch synthase (GBSS I) encoded by the Wxgenes, is involved in amylose synthesis. For analyses of mechanisms of amylose synthesis and associated starch properties in hexaploid wheat, eight possible genotypes having different combinations of the three null alleles at the Wx loci with a common genetic background are a prerequisite. A near-isogenic population of doubled haploid (DH) lines was produced from Chinese Spring × waxy Chinese Spring F₁ plants using the wheat × maize method. The Wx protein phenotypes of the DH progeny were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and found that the null alleles at each of the three Wx loci segregated in a Mendelian fashion. A field trial demonstrated no differences between the eight types for ear emergence time, plant height and grain yield traits. Amylose content in the endosperm starch was highest in the wild type while lowest in the waxy type having no Wx proteins. Comparison between single null types and double null types indicated that the amylose synthesis capacity of Wx-A1a allele is the lowest. Pasting properties of starch are the highest in the waxy type, followed by the double null types. Consequently, both peak viscosity and breakdown were negatively correlated with amylose content. The chain-length distribution analysis of amylopectin structure revealed no clear difference among the eight types,suggesting that the reduced GBSS I activity due to introgression of the null Wx alleles does not affect either the chain length or the degree of branching of amylopectin. This revised version was published online in July 2006 with corrections to the Cover Date.     

杂交水稻恢复系桂99的抽穗期基因及其效应分析

以基因型明确的抽穗期主基因近等基因系EG0~EG7、ER~LR、T65系列为测验系（TLs）,在江西南昌（28^o 36’ N）夏季自然高温长日（14 h/d）和人工遮光短日（10 h/d）,以及海南三亚（18^o 14’ N）旱季自然低温短日（11.6 h/d）处理条件下,对籼型杂交水稻恢复系桂99的抽穗期基因及其感温性和基因位点间的互作效应进行了分析。结果表明,桂99在E₁、E₂和E₃位点分别带有感光迟熟等位基因E₁、E₂和E₃,在Se-1位点带有非感光等位基因Se-1^e,在Ef-1位点带有早熟基因Ef-1,由此推断其抽穗期基因型为E₁E₁E₂E₂E₃E₃Se-1^eSe-1^eEf-1Ef-1。迟熟基因E₁、E₃与早熟基因Ef-1同时存在,E₁、E₃与Se-1^e基因位点间的互作使桂99农艺性状表现弱感光性。感光基因Se-1^u（或Se-1ⁿ）的存在能增强E位点感光迟熟基因的感光性,感光基因对“TLs×桂99”F₂植株抽穗的影响,是延长平均抽穗期,增加迟抽穗植株分布频率。分析了感温性对TLs抽穗期的影响,讨论了以桂99为恢复系配置的杂交水稻组合丰产性和广适性的遗传基础。     

Waxy proteins in diploid, tetraploid and hexaploid wheats

Electrophoretic analyses of waxy proteins, encoded by genes present at the Wx‐1 loci, present in several cultivars and accessions of hexaploid wheat, Triticum aestivum, have permitted the detection of null alleles at the Wx‐B1 and Wx‐D1 loci. Polymorphism at the Wx‐A1 and Wx‐B1 loci was also investigated in several accessions of tetraploid wheats, Triticum durum, Triticum dicoccoides and Triticum timopheevi, and in diploid species, Triticum urartu, Triticum boeoticum and Triticum monococcum. One null allele at the Wx‐A1 locus and three polymorphic alleles at Wx‐B1 locus were detected in T. durum; a new allele at one of the two waxy loci was identified in the tetraploid wheat T. timopheevi; no polymorphism was detected in diploid species. Polymerase chain reaction techniques made possible the detection of further polymorphism existing at the Wx‐1 loci and the reason for the lack of expression of the null genotypes to be investigated. The null forms detected at each locus have been used to produce complete sets of partial and total waxy lines in durum and bread wheat.