Engineering high amylose and resistant starch in maize by CRISPR/Cas9-mediated editing of starch branching enzymes

To improve the amylose content(AC)and resistant starch content(RSC)of maize kernel starch,we employed the CRISPR/Cas9 system to create mutants of starch branching enzyme I(SBEI)and starch branching enzyme IIb(SBEIIb).A frameshift mutation in SBEI(E1,a nucleotide insertion in exon 6)led to plants with higher RSC(1.07%),lower hundred-kernel weight(HKW,24.71±0.14 g),and lower plant height(PH,218.50±9.42 cm)compared to the wild type(WT).Like the WT,E1 kernel starch had irregular,polygonal shapes with sharp edges.A frameshift mutation in SBEIIb(E2,a four-nucleotide deletion in exon 8)led to higher AC(53.48%)and higher RSC(26.93%)than that for the WT.E2 kernel starch was significantly different from the WT regarding granule morphology,chain length distribution pattern,X-ray diffraction pattern,and thermal characteristics;the starch granules were more irregular in shape and comprised typical B-type crystals.Mutating SBEI and SBEIIb(E12)had a synergistic effect on RSC,HKW,PH,starch properties,and starch biosynthesis-associated gene expression.SBEIIa,SS1,SSIIa,SSIIIa,and SSIIIb were upregulated in E12 endosperm compared to WT endosperm.This study lays the foundation for rapidly improving the starch properties of elite maize lines.

Cloning and Expression of the Promoter of Maize Starch-Branching Enzyme sbeⅡb Gene

[Objective] The aim was to construct promoter of maize starch-branching enzyme sbe Ⅱb gene specifically expressed in seed.[Method] The promoter sequence of maize starch-branching enzyme sbe Ⅱb gene was amplified by LA-PCR and then cloned into pMD18-T vector.Subsequently,the promoter was cloned into pBI121 vector to construct plant expression vector pBI121-sbe Ⅱb.Recombinant plasmid pSBE-GUS was constructed by connecting GUS gene into pBI121-sbe Ⅱb and transformed into tobacco mediated by Agrobacterium tumefaciens,before detection by PCR and Southern blot.The biochemical analysis and fluorescence detection of GUS activity were performed in different parts of the tobacco by Gene Gun Method and Agrobacterium tumefaciens transformation respectively.[Result] The homology between promoter sequence cloned in this experiment and the corresponding sequence announced in the GenBank reached 98.52%.Four transformed tobaccos were obtained by PCR and Southern blot after co-culture and selective culture.A small number of blue spots appeared in leaves,and the spots could barely been seen in the stems and roots,while a large number of blue spots were found in seeds.So,it could be concluded that the promoter of sbeⅡb gene was specifically expressed in seeds.[Conclusion] Promoter of maize starch branching enzyme sbe Ⅱb specifically expressed in seeds was successfully cloned.

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.

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.

Changes in Activities of the Key Enzymes Related to Starch Synthesis in Rice Grains During Grain Filling and Their Relationships with the Filling Rate and Cooking Quality

With 10 rice cultivars (lines) as materials, the changes in activities of adenosine diphosphoglucose pyrophosphorylase(ADPGPase), starch synthase (SSase) and starch branching enzyme (Q-enzyme) in the grains during grain filling, and theirrelationships with the filling rate, gel consistency (GC), alkali spreading value (ASV) and amylose content (AC) werestudied. The results showed that changes in activities of ADPGPase, SSase and Q-enzyme exhibited a single peak duringgrain filling, and the time of the activity peaks for the former two enzymes was earlier than that of the maximum grain-fillingrate (Tmax), and the time reaching the peak for Q-enzyme was synchronous with Tmax. The activities at early grain fillingstage, and the mean and maximum activities of each enzyme during grain filling period were positively and significantly orvery significantly correlated with the mean and maximum grain filling rate and starch content (mg grain-1) in the grains.Activities of ADPGPase at all grain filling stages and those of Q-enzyme at the early and mid filling stages were notsignificantly correlated the cooking quality (GC, ASV and AC). SSase activities at the early filling stage were significantlyand negatively correlated with GC and ASV, and positively correlated with AC. Activities of SSase at mid and late grainfilling stages and Q-enzyme at the late filling stage were significantly and positively correlated with GC and ASV, andnegatively correlated with AC. Spraying zeatin or abscisic acid at early grain filling stage could obviously regulate theactivities of ADPGPase, SSase and Q-enzyme in the grains.

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.

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.

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.

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.

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

水稻灌浆期籽粒中淀粉合成关键酶的活性变化及其与灌浆速率和蒸煮品质的关系

以10个水稻品种(系)为材料,研究了灌浆期籽粒中腺苷二磷酸葡萄糖焦磷酸化酶(ADPGPase)、淀粉合成酶(SSase)和淀粉分支酶(Q-酶)活性变化及其与灌浆速率、胶稠度(GC)、碱化值(ASV)和直链淀粉含量(AC)的关系。结果表明,籽粒中ADPGPase、SSase和Q-酶活性变化呈单峰曲线,前两个酶活性峰值出现的时间在最大灌浆速率的时间(Tmax)之前,Q-酶活性峰值的时间与Tmax趋于同步。上述各酶在灌浆前期的活性、灌浆期的最大活性和平均活性与平均灌浆速率、最大灌浆速率和籽粒中淀粉的含量(mg·粒-1)均呈显著或极显著正相关。灌浆各期籽粒中ADPGPase活性以及灌浆前、中期的Q-酶活性与蒸煮品质(GC,ASV和AC)的相关均不显著;灌浆前期SSase活性与GC和ASV呈显著负相关,与AC呈显著正相关,灌浆中期和后期的SSase活性以及灌浆后期的Q-酶活性与GC和ASV呈显著正相关,与AC呈显著负相关。灌浆前期喷施玉米素或脱落酸对籽粒中的ADPGPase、SSase和Q-酶活性有明显的调控作用。

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。

水稻灌浆期籽粒中3个与淀粉合成有关的酶活性变化

在水培和盆栽条件下 ,研究了 6个水稻品种 (含籼 /粳杂交组合、新株型品系 )灌浆期强、弱势粒中 ADPG焦磷酸酶 (EC 2 .7.7.2 1)、淀粉合成酶 (EC 2 .4 .1.2 1)和淀粉分枝酶或 Q-酶 (EC 2 .4 .1.18)的活性变化及其与灌浆充实的关系。 3个酶的活性变化与籽粒灌浆动态相关联 :淀粉酶最高活性出现的时间稍前或同步于最大灌浆速率的时间 ;ADPG焦磷酸酶活性峰值的时间在籽粒达最大重量前的 3～ 6 d;Q-酶最高活性在籽粒重量接近最大时出现。灌浆期各个酶活性的平均值、最高值以及灌浆前期(花后 3～ 12 d)酶的活性与平均灌浆速率、最大灌浆速率、粒重及谷粒充实率均呈显著或极显著的正相关 ,尤以 Q-酶的相关值最大。籼 /粳杂交稻组合籽粒中酶的活性的高低与其亲本有关。在抽穗期通过剪叶、疏花或施用氮素等调节灌浆初期的源库关系或植株体内的营养水平 ,能增加或降低籽粒中特别是弱势粒中酶的活性。这些结果表明 :上述 3个酶尤其是 Q-酶对籽粒灌浆起着重要的调控作用 ;在育种上注意选择籽粒中酶活性高的亲本 ,可望培育出籽粒充实好的品种或杂种后代 ;通过栽培等措施提高灌浆前期籽粒中酶的活性 ,可提高结实率和粒重。

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。

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。

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。

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%。

植物淀粉分支酶基因的研究进展

淀粉分支酶(Starch branching enzyme,SBE)是直接参与淀粉生物合成的关键酶,它催化-α1,6糖苷键分支点形成支链淀粉。本文综述了SBE基因的分类、结构、同种型的功能、各同种型在不同组织和不同生育时期的表达特异性以及它们不同的功能、基因表达与淀粉合成的关系、SBE的应用等方面的研究进展,旨在为利用基因工程技术调控植物淀粉的含量与特性提供相关信息。

RNAi沉默淀粉分支酶sbe3基因对水稻直链淀粉的影响

本研究利用RNAi技术,通过RT-PCR方法克隆出水稻淀粉分支酶sbe3基因片断,经两步组装法将sbe3基因片断分别正向、反向组装入pRNAi-ubi,成功构建sbe3基因RNAi转化载体pRNAi-ubi/sbe3。在农杆菌介导下,对粳稻中花11未成熟胚诱导的愈伤组织进行转化,通过PCR、Southern杂交鉴定获得一批转基因植株。半定量RT-PCR鉴定出转化苗T1代种子中sbe3基因表达被明显抑制,但只引起转基因水稻胚乳中直链淀粉含量少量的提高,说明采用RNAi仅沉默sbe3基因对水稻胚乳直链淀粉含量没有显著影响。