Temperature Stress at Grain Filling Stage Mediates Expression of Three Isoform Genes Encoding Starch Branching Enzymes in Rice Endosperm

An early-maturity indica rice variety Zhefu 49, whose grain quality and starch structure are sensitive to environmental temperature, was subjected to different temperatures （32℃ for high temperature and 22℃ for optimum temperature） at the grain filling stage in plant growth chambers, and the different expressions of three isoform genes （SBEI, SBEIII and SBE/V） encoding starch branching enzyme （SBE） in the endosperms were studied by the real-time fluorescence quantitative PCR （FQ-PCR） method. Effects of high temperature on the SBE expression in developing rice endosperrns were isoform-dependent. High temperature significantly down-regulated the expressions of SBEI and SBEIII, while up-regulated the expression of SBEIV. Compared with SBEIV and SBEIII, the expression of SBEI gene in Zhefu 49 rice endosperms was more sensitive to temperature variation at the grain filling stage. This study indicates that changes in weather/climate conditions especially temperature stress influence rice grain formation and its quality as evidenced by isoform expression.

Characterization and Expression Analysis of Starch Branching Enzymes in Sweet Potato

Spatial and temporal expression patterns of Sbel and Sbe2 that encode starch branching enzyme （SBE） Ⅰ and Ⅱ, respectively, in sweet potato （Ipomoea batatas L.） were analyzed. Expression of both genes in Escherichia coli indicate that both genes encoded active SBE. Analysis with real-time quantitative polymerase chain reaction technique indicates that IbSbel mRNA was expressed at very low levels in leaves but was the predominant isoform in tuberous root while the reverse case was found for lbSbe2. The expression pattern of IbSbel, closely resembles that of AGPase S, a gene coding for one of the subunits ofADP-glucose pyrophosphorylase, which is the key regulatory enzyme in the starch biosynthetic pathway. Western analysis detected at least two isoforms of SBE I in tuberous roots, those two isoforms showed adverse expression patterns with the development of the tuberous roots. Expression of the two IbSbe genes exhibited a diurnal rhythm during a 12-h cycle when fed a continuous solution of sucrose. Abscisic acid （ABA） was aother potent inducer of IbSbe expression, but bypassed the semidian oscillator.

Effects of Weak Light on Starch Accumulation and Starch Synthesis Enzyme Activities in Rice at the Grain Filling Stage

Dynamic changes of starch, amylose, sucrose contents and the activities of starch synthesis enzymes under shading treatments alter flowering were studied using two rice varieties IR72 （indica） and Nipponbare （japonica） as materials. Under shading treatments, the starch, amylose and sucrose contents decreased, while ADP-glucose pyrophosphorylase （ADPGPPase） activity only changed a little, soluble starch synthase activity and granule bound starch synthase activity decreased, soluble starch branching enzyme （SSBE, Q-enzyme） activity and granule bound starch branching enzyme （GBSBE, Q-enzyme） activity increased, and starch debranching enzyme （DBE, R-enzyme） activity varied with varieties. Correlation analyses showed that the changes of starch content were positively and significantly correlated with the changes of sucrose content in the weak light. Both ADPGPPase activity and SSBE activity were positively and significantly correlated with starch accumulation rate. It was implied that the decline of starch synthase activities was related to the decrease of starch content and the increase of the activity of starch branching enzyme played an important role in the decrease of the ratio of amylose to the total starch under the weak light.

Origin and evolution of the main starch biosynthetic enzymes

Starch,a semi-crystalline energy storage form primarily found in plant plastids plays a crucial role in various food or no-food applications.Despite the starch biosynthetic pathway’s main enzymes have been characterized,their origin and evolution remained a subject of debate.In this study,we conducted the comprehensive phylogenetic and structural analysis of three types of starch biosynthetic enzymes:starch synthase(SS),starch branching enzyme(SBE)and isoamylase-type debranching enzyme(ISA)from 51,151 annotated genomes.Our findings provide valuable insights into the possible scenario for the origin and evolution of the starch biosynthetic pathway.Initially,the ancestor of SBE can be traced back to an unidentified bacterium that existed before the formation of the last eukaryotic common ancestor(LECA)via horizontal gene transfer(HGT).This transfer event likely provided the eukaryote ancestor with the ability to synthesize glycogen.Furthermore,during the emergence of Archaeplastida,one clade of SS was transferred from Deltaproteobacteria by HGT,while ISA and the other clade of SS originated from Chlamydiae through endosymbiosis gene transfer(EGT).Both these transfer events collectively contributed to the establishment of the original starch biosynthetic pathway.Subsequently,after the divergence of Viridiplantae from Rhodophyta,all three enzymes underwent multiple duplications and N-terminus extension domain modifications,resulting in the formation of functionally specialized isoforms and ultimately leading to the complete starch biosynthetic pathway.By shedding light on the evolutionary origins of key enzymes involved in the starch biosynthetic pathway,this study provides important insights into the evolutionary events of plants.

cDNA Cloning and Sequence Analysis of Rice Sbe1 and Sbe3 Genes

Two starch-branching enzyme (SBE) in rice, is known to be a key enzyme in amylopectin biosynthesis. The cDNA of two SBE(starch-branching enzyme) genes Sbe1 and Sbc3 encoding SBE Ⅰ and SBE Ⅲ (two major isoforms in rice) were cloned by an improved RT-PCR technique, from a template cDNA library derived from the total mRNAs extracted from the immature seeds of a japonica rice Wuyunjing 7. DNA sequence analysis showed that the size of the cloned Sbe1 and Sbe3 cDNAs were 2490 and 2481 bp long, respectively, including their entire coding sequences. Comparison analysis indicated that the nucleotide sequence of Sbe3 was the same as that of sbc3 (Genbank Accession No. D16201) as reported previously. There were only four base-pairs difference, which resulted in changes of two deduced amino acids between the cloned Sbel cDNA and the reported sbe1 (Genbank Accession No. D11082). The cloned Sbe1 and Sbe3 cDNAs make it possible to improve rice starch quality through genetic engineering

Comparison of Five Endogenous Reference Genes for Specific PCR Detection and Quantification of Rice

Endogenous reference genes (ERGs) provide vital information regarding genetically modified organisms (GMOs). The successful detection of ERGs can identity GMOs and the source of genes, verify stability and reliability of the detection system, and calculate the level of genetically modified (GM) ingredients in mixtures. The reported ERGs in rice include sucrose-phosphate synthase (SPS), phospholipase D (PLD), RBE4 and rice root-specific GOS9 genes. Based on the characteristics of ERGs, a new ERG gene, phosphoenolpyruvate carboxylase (PEPC), was selected, and further compared with the four existing genes. A total of 18 rice varieties and 29 non-rice crops were used to verify the interspecies specificity, intraspecies consistency, sensitivity, stability and reliability of these five ERGs using qualitative and quantitative PCR. Qualitative detection indicated that SPS and PEPC displayed sufficient specificity, and the detection sensitivity was 0.05% and 0.005%, respectively. Although the specificity of both RBE4 and GOS9 were adequate, the amplicons were small and easily confused with primer dimers. Non-specific amplification of the PLD gene was present in maize and potato. Real-time quantitative PCR detection indicated that PLD, SPS and PEPC displayed good specificity, with R2 of the standard curve greater than 0.98, while the amplification efficiency ranged between 90% and 110%. Both the detection sensitivities of PLD and PEPC were five copies and that of SPS was ten copies. RBE4 showed typical amplification in maize, beet and Arabidopsis, while GOS9 was found in maize, tobacco and oats. PEPC exhibited excellent detection sensitivity and species specificity, which made it a potentially useful application in GM-rice supervision and administration. Additionally, SPS and PLD are also suitable for GM-rice detection. This study effectively established a foundation for GMO detection, which not only provides vital technical support for GMO identification, but also is of great significance for enhancing the comparability of detection results, and the standardization of ERG testing in GM-rice.

山药块茎膨大期淀粉积累及淀粉合成相关基因表达分析

为探讨山药块茎膨大期淀粉的积累机制,以大和长芋山药为试验材料,测定了块茎膨大期各类淀粉含量、淀粉合成关键酶活性以及淀粉合成相关基因的表达水平。结果表明,山药块茎膨大期总淀粉、直链淀粉及支链淀粉的积累呈先增加后降低的趋势,种植后150 d支链淀粉占总淀粉含量的88.35%,总淀粉含量的增加主要是支链淀粉的积累;ADP-葡萄糖焦磷酸化酶(AGPase)、淀粉分支酶(SBE)与支链淀粉的积累呈极显著正相关,是直接参与支链淀粉积累的重要功能酶。AGPase、SSⅡ、SSⅢ、SBEⅡ基因的表达量与支链淀粉含量呈极显著正相关,是支链淀粉积累的关键因素。SBE对淀粉积累表现为直接正效应,AGPase和束缚态淀粉合成酶(GBSS)在直链淀粉积累中体现为直接正效应。这些研究对揭示山药块茎淀粉合成的分子机制具有重要意义。

芋淀粉分支酶(SBE)基因的鉴定、生物信息学及表达分析

淀粉分支酶(starch branching enzyme,SBE)在支链淀粉生物合成中发挥关键作用,直接影响淀粉的含量和结构。芋(Colocasia esculenta)是一种主要的块茎类作物,在世界上热带和亚热带地区广泛栽培。目前,SBE在芋中的研究很少,对SBE基因在芋中的数量、分子结构特征和表达模式还不清楚。本研究首次对芋SBE基因进行了全面分析,鉴定了3个SBE基因(CeSBE1、CeSBE2和CeSBE3)。CeSBE1、CeSBE2和CeSBE3蛋白氨基酸数量分别为828、845和598,分子质量分别为92956.71、95625.13、69169.16 Da,等电点分别为5.22、5.41和7.36。系统进化分析显示3个CeSBE蛋白分别在3个不同的亚群。基因结构分析显示,CeSBE1、CeSBE2和CeSBE3外显子数量分别为16、22、10;保守结构域分析表明,CeSBE1和CeSBE2均具有alpha-amylase_C和alpha-amylase结构域及7个motif,而CeSBE3具有alpha-amylase和CBM_48结构域及3个motif。CeSBE基因启动子区域顺式作用元件分析表明,共预测到55个顺式作用元件,其中29个具有功能注释,涉及光响应、激素响应、植物生长发育及环境压力等相关元件。在不同组织中,3个CeSBE基因均能在所有组织中表达,其中CeSBE2在球茎和叶片显著表达(P<0.05);在球茎不同发育阶段中,CeSBE2在所有的发育阶段均有较高的表达量,呈现先升高后降低的表达趋势,在球茎发育120 d的表达量达到峰值。球茎不同发育阶段总淀粉和支链淀粉含量增加与CeSBE2表达量趋势一致,说明CeSBE2可能是芋支链淀粉生物合成的关键基因。本研究结果可为芋的产量、品质和营养性状的遗传改良提供基础。

土壤水分胁迫对马铃薯块茎淀粉合成关键酶基因表达及酶活性的影响

为探讨马铃薯块茎淀粉合成对土壤水分胁迫的响应机制,以马铃薯品种‘青薯9号’和‘闽薯1号’为材料,采用盆栽试验,设置3个水分胁迫处理:正常供水(保持土壤田间持水量的75%)、中度水分胁迫(保持土壤田间持水量的50%)、重度水分胁迫(保持土壤田间持水量的25%),研究了土壤水分胁迫对马铃薯块茎淀粉合成关键酶基因表达及酶活性的影响。结果表明:在块茎形成过程中,土壤水分胁迫显著提高了马铃薯块茎AGPase、GBSSI、SBEI、SBEII、SSII、SSIII基因表达量,随土壤水分胁迫程度的提高,马铃薯块茎SBEI、SBEII、SSII基因表达量上调;土壤水分胁迫显著降低了马铃薯块茎AGPase、GBSS、SBE、SSS酶活性,其中GBSS、SBE酶活性下调;而AGPase、GBSSI、SSIII基因表达量及AGPase、SSS酶活性在马铃薯青薯9号、闽薯1号品种间表现出相反的上、下调趋势。

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.

灌浆结实期弱光对水稻籽粒淀粉积累及相关酶活性的影响

选用IR72(籼稻)和日本晴(粳稻)为材料,在开花后进行遮光处理,对弱光条件下籽粒淀粉和直链淀粉含量的动态变化及相关酶的活性进行了研究。在弱光条件下,两品种籽粒的淀粉含量减少,直链淀粉含量下降,蔗糖含量降低,ADPG焦磷酸化酶活性的变化较小,可溶性淀粉合成酶和颗粒结合型淀粉合成酶活性减弱,可溶性淀粉分支酶Q酶和颗粒结合型淀粉分支酶活性增强,淀粉去分支酶活性因品种而异,IR72表现为减弱,日本晴则为增强。相关分析表明,遮光下蔗糖输入量的减少量和淀粉合成量的下降量呈显著正相关;ADPG焦磷酸化酶和淀粉分支酶活性与淀粉积累速率呈显著正相关。分析指出,遮光下淀粉合成酶活性的降低与淀粉合成量的下降有关,淀粉分支酶活性的升高是直链淀粉占淀粉总量的比率减少的重要原因。

RNA干扰水稻SBE3基因的表达对籽粒淀粉合成及其关键酶活性的影响

为探讨RNA干扰水稻SBE3基因的表达对籽粒淀粉合成及其关键酶活性的影响,以水稻品种中花11及以其为受体的转基因株系为材料,分别检测水稻SBE3基因的表达、籽粒发育各时期淀粉合成关键酶活性变化及直链、支链淀粉相对含量。结果表明,导入的SBE3基因RNA干扰结构成功地降低了目的基因的表达,并使其酶活性在籽粒发育各时期均显著降低并提前3d达高峰期,且不同株系间具有差异。同时也使不同发育时期籽粒的ADPG-PPase和SSS及DBE活性均不同程度地显著降低,尤其以SSS和ADPG-PPase活性峰值降幅最大。此外,两个转基因水稻株系各时期籽粒直链淀粉含量均显著高于对照,而其成熟籽粒千粒重却显著降低,直链淀粉含量越高,千粒重越低。

用RNA干扰技术创造高直链淀粉马铃薯材料

【目的】创造块茎高直链淀粉含量的转基因马铃薯材料。【方法】采用RT-PCR技术分别克隆了马铃薯Sbe1基因CDS内300bp的片段SⅠ和Sbe2基因CDS内410bp的片段SⅡ,并将SⅠ和SⅡ顺序连接得到融合片段SⅢ;以载体pHANNIBAL和pART27为基础,构建具有SⅢ反向重复结构的植物表达载体pRNAiⅢ;采用农杆菌介导法转化马铃薯优良品种陇薯3号、甘农薯2号和大西洋。【结果】获得了融合片段SⅢ,构建了以Sbe1基因和Sbe2基因为靶标的RNA干扰载体pRNAiⅢ,通过农杆菌介导法获得了24个转基因株系,其中21个转基因株系试管薯的淀粉粒形态发生明显变化,表观直链淀粉含量介于59.31%～87.14%,比受体材料平均高出3.2倍。RT-PCR分析表明,在8个直链淀粉含量超过80%的转基因株系中检测不到Sbe1和Sbe2基因mRNA的积累。【结论】采用RNAi技术通过沉默内源Sbe1和Sbe2,可获得高直链淀粉含量的马铃薯材料。

应用RNA干扰技术降低玉米支链淀粉含量

为了调控玉米淀粉的生物合成过程,克隆了玉米淀粉分支酶(starchbranchingenzymes,SBE)基因,构建高效的siRNA表达体系,通过花粉管通道法将其导入玉米自交系。PCR扩增和Southern杂交结果证明,目的基因已被整合到基因组中,且能够遗传。Northern杂交分析表明,该目的基因在转基因植株中能正常转录并导致内源SBEmRNA含量下降。对转基因植株淀粉分支酶活性和淀粉含量测定结果表明,分支酶活性明显地低于对照,相差最多的低85%;总淀粉含量与对照之间基本没有差异,但直链淀粉的含量提高了约50%。

水稻分支酶基因Sbe1和Sbe3 cDNA的克隆及全序列分析

通过改良的 RT- PCR法合成了水稻未成熟种子胚乳 c DNA库 ,并以此为模板 ,用 PCR技术从粳稻品种武运粳 7号中克隆了分别编码淀粉分支酶 SBE 和 SBE 的 2个基因 Sbe1和 Sbe3。序列分析表明 ,克隆 Sbe1和 Sbe3基因的大小分别为 2 4 90 bp和 2 4 81bp,包含了基因完整的编码序列。 Sbe3基因与已发表基因全序列完全相同 ,同源性达 10 0 % ;Sbe1基因与已发表基因序列有 4个碱基不同 ,同源性为 99.84 % ,推导氨基酸序列的差异为

不同类型玉米籽粒淀粉积累、相关酶活及基因表达差异分析

为探究不同类型玉米淀粉形成机理,对普通玉米、甜玉米、糯玉米淀粉积累、相关酶活及基因表达进行测定,分析不同类型玉米淀粉积累、相关酶活及基因表达之间的差异及相互关系。结果表明,不同类型玉米总淀粉和直链淀粉百分含量为:普通玉米>糯玉米>甜玉米,支链淀粉百分含量为:糯玉米>普通玉米>甜玉米,灌浆期间总淀粉和直链淀粉含量3个玉米类型间差异显著;灌浆期间,普通玉米各淀粉合成相关酶活性最高,甜玉米淀粉合成相关酶活性最低,糯玉米则介于普通玉米和甜玉米之间,但其GBSS酶活性很小。灌浆期间3个类型玉米除GBSS酶活性差异不显著外,其他淀粉合成相关酶活性差异显著;普通玉米淀粉合成相关基因表达量总体均高于甜玉米和糯玉米,甜玉米和糯玉米相关突变基因仍存在表达。表明不同类型玉米淀粉含量和组成上差异明显;普通玉米淀粉的形成需要淀粉合成相关酶相互作用,淀粉合成相关酶活性的缺失会改变淀粉组成;不同类型玉米淀粉合成相关基因表达差异显著,但都存在转录活性。对普通玉米进行相关性分析同时发现,淀粉的合成不仅受到转录调控,还受到转录后调控,淀粉的合成是淀粉合成各酶之间相互协调的结果。

花粉管通道法将淀粉分支酶基因反义表达载体转入玉米自交系的研究

实验主要利用花粉管通道技术将淀粉分支酶基因的反义表达载体转入玉米自交系中,同时探讨了不同导入时间、不同DNA浓度、不同导入量等对其转化率的影响,并从中得出花粉管通道法的最佳转化条件。同时将转化的玉米植株进行PCR检测,初步证明外源目的基因已整合到玉米基因组中,并得到了转化植株的种子。

利用正义RNAi技术提高玉米直链淀粉含量效果的研究

为了研究利用正义RNAi技术提高玉米直链淀粉含量效果,用基因枪法将构建的sbeⅡb正义RNAi表达载体pBAC506和pBAC508(筛选标记基因分别为35S启动子及Adh1-intron1增强驱动的epsps和bar)导入玉米(Zeamays)自交系幼胚愈伤组织,经过筛选、分化和再生获得了44株转基因当代植株,经PCR扩增、PCR-Southern检测有30株为阳性。选择9株健壮的阳性植株进行基因组Southern-blotting分析,结果表明7株T0植株整合了目的基因,其中4株整合了1个转基因拷贝,2株整合了2个转基因拷贝,1株整合了3个转基因拷贝。这7株中有2株结实,直链淀粉含量分别为23.22%和24.60%,有一定的小幅提高。下一步要进行较大规模的基因枪转化,得到比较大的转基因群体,以对更多转基因后代进行鉴定,期望筛选出外源基因多拷贝插入和直链淀粉含量大幅提高的株系。

玉米淀粉分支酶sbeⅡb基因启动子的克隆和表达载体构建

以玉米基因组DNA为模板,通过LA-PCR技术扩增了玉米淀粉分支酶sbeⅡb基因启动子序列,并将其克隆到pMD18-TVector上,对重组子进行PCR检测和限制性内切酶分析并测序。结果表明,该启动子和Genbank中发表的玉米淀粉分支酶sbeⅡb基因启动子同源性达98.52%,克隆片段长为934bp。再将经BamHⅠ和HindⅢ双酶切得到的启动子片段克隆到相同酶酶切的pBI121载体上,构建植物表达载体pBI121-sbeⅡb,并进行酶切鉴定和PCR检测。结果显示,启动子基因sbeⅡb已成功整合到植物表达载体pBI121上。序列中发现高等植物启动子所特有的基本核心序列和种子特异表达所需的特殊调控元件。

玉米淀粉分支酶基因片段的克隆和植物表达载体的构建

应用聚合酶链式反应技术(PCR)扩增了玉米淀粉分支酶的cDNA基因片段,并将其克隆到pMD18-Tvector载体上,对重组子进行了PCR检测和限制性内切酶分析,测定了DNA全序列。结果表明:克隆片段全长为935bp。将反义+正义基因片段插入到pBI12135S启动子下,构建成表达质粒pCJSBE2b。通过花粉管通道法将其导入玉米自交系,获得了转基因植株。