Molecular analysis of the gene encoding a rice starch branching enzyme

Summary The sequence of a rice gene encoding a starch branching enzyme (sbe1) shows extreme divergence from that of the rice gene, that is homologous to bacterial glycogen branching enzyme (sbe2). sbe1 is expressed abundantly and specifically in developing seeds and maximally in the middle stages of seed development. This expression pattern completely coincides with that of the waxy gene, which encodes a granule-bound starch synthase. Three G-box motifs and consensus promoter sequences are present in the 5 flanking region of sbe1. It encodes a putative transit peptide, which is required for transport into the amyloplast. A 2.2 kb intron (intron 2) precedes the border between the regions encoding the transit peptide and the mature protein, and contains a high G/C content with several repeated sequences in its 5 half. Although only a single copy of sbe1 is present in the rice genome, Southern analysis using intron 2 as a probe indicates the presence of several homologous sequences in the rice genome, suggesting that this large intron and also the transit peptide coding region may be acquired from another portion of the genome by duplication and insertion of the sequence into the gene.     

Creation of a potato mutant lacking the starch branching enzyme gene StSBE3 that was generated by genome editing using the CRISPR/dMac3-Cas9 system

The potato tuber starch trait is changed depending on the composition of amylose and amylopectin. The amount of amylopectin is determined by the activity of the starch branching enzymes SBE1, SBE2, and SBE3 in potato. SBE3, a homolog of rice BEI, is a major gene that is abundant in tubers. In this study, we created mutants of the potato SBE3 gene using CRISPR/Cas9 attached to the translation enhancer dMac3. Potato has a tetraploid genome, and a four-allele mutant of the SBE3 gene is desired. Mutations in the SBE3 gene were found in 89 of 126 transformants of potato plants. Among these mutants, 10 lines contained four mutant SBE3 genes, indicating that 8% efficiency of target mutagenesis was achieved. These mutants grew normally, similar to the wild-type plant, and yielded sufficient amounts of tubers. The potato starch in these tubers was similar to that of the rice BEI mutant. Western blot analysis revealed the defective production of SBE3 in the mutant tubers, suggesting that these transformants were loss-of-function mutants of SBE3.     

玉米雄性不育资源的发掘与利用

植物雄性不育是指植物雄性生殖器官不能产生正常有功能花粉的现象.玉米(Zea mays L.)是重要的粮食作物之一,也是较早利用杂种优势的作物之一.当前,生产上广泛种植的玉米品种类型主要是单交种.我国玉米杂交种的播种面积常年稳定在6.2亿亩左右,年用种量10亿公斤以上,常年制种面积高达250多万亩.利用传统的人工去雄或机...     

中国17个优良玉米自交系的分子标记杂合性及其与杂交种性状的关系研究

在玉米单交种育种中 ,鉴定高产杂交种和具有优良特性的自交系是一个重要的问题。研究以 1 7个优良玉米自交系为亲本 ,按照双列杂交配组合 ,利用 RAPD技术分析了 1 7个自交系的多态性以及 RAPD标记与 9个重要农艺性状 (包括产量 )的关系。基于 RAPD标记计算的相似系数聚类将 1 7个自交系分为 5个类群 ,经分析与系谱亲缘关系基本一致。杂交种性状及其特殊配合力与亲本间的遗传距离是高度相关的 ,与聚类前比较 ,聚类后平均遗传距离与平均产量、平均特殊配合力的相关系数显著提高 ,类间平均产量高于类内平均产量。RAPD技术可揭示优良玉米自交系的系谱亲缘关系 ,将自交系划分成不同的类群 ,从而为选择类间自交系杂交 ,进行亲本选配和分子标记辅助育种提供一种方法。     

玉米及马齿苋叶片SOD活性诱导研究

以玉米幼苗及马齿苋作材料,通过甘露醇、H2O2、臭氧和强光等胁迫后,用NBT光化还原抑制法测定叶中SOD活性的变化。臭氧和强光能诱导玉米叶片SOD活性增加。0.5mol／L甘露醇处理玉米叶片12h,SOD活性上升,至48h后下降;在该甘露醇溶液中另外加入10^-2mol/L H2O2;处理12h后SOD活性基本不变。对玉米叶片单独用10^-2mol/L H2O2诱导,30min内SOD活性上升到最高值,随着处理时间的延长又逐渐下降。用耐强光、耐干旱的野生马齿苋作为材料,与玉米相比,其叶片SOD基础活性低于后者;若予以正午强光结合渗透胁迫2h,其叶中SOD活性增幅超过玉米,从种间比较的角度旁证了SOD在抗逆性中的作用。推测植物中存在比活性氧更为直接的物理诱导机制。     

Characterization of the regulatory elements of the maize P-rr gene by transient expression assays

The maize P-rr gene conditions floral-specific flavonoid pigmentation, especially in the kernel pericarp and cob. We analyzed the P-rr promoter by transient expression assays, in which segments of the P-rr promoter were fused to the GUS reporter gene and introduced into maize cells by particle bombardment. A basal P-rr promoter fragment (–235 to +326) gave low, but significant, levels of GUS reporter gene expression. Interestingly, two widely spaced segments containing enhancer-like activity were found. When tested individually, both the proximal (–1252 to –236) and distal (–6110 to –4842) segments boosted expression of the basal P-rr promoter::GUS construct about five-fold. A 1.6 kb segment of the P-rr promoter (–1252 to +326) containing the proximal enhancer and the 5-untranslated leader driving the GUS reporter gene showed preferential expression in BMS and embryogenic suspension cell cultures vs. endosperm-derived suspension cell cultures. These results demonstrate the application of transient assay techniques for the identification of regulatory elements responsible for floral-specific regulation of the complex P-rr gene promoter in maize.     

Characterisation of a gene encoding wheat endosperm starch branching enzyme-I

 A genomic DNA fragment from Triticum tauschii, the donor of the wheat D genome, contains a starch branching enzyme-I (SBE-I) gene spread over 6.5 kb. This gene (designated wSBE I-D4) encodes an amino acid sequence identical to that determined for the N-terminus of SBE-I from the hexaploid wheat (T. aestivum) endosperm. Cognate cDNA sequences for wSBE I-D4 were isolated from hexaploid wheat by hybridisation screening from an endosperm library and also by PCR. A contiguous sequence (D4 cDNA) was assembled from the sequence of five overlapping partial cDNAs which spanned wSBE I-D4. D4 cDNA encodes a mature polypeptide of 87 kDa that shows 90% identity to SBE-I amino acid sequences from rice and maize and contains all the residues considered essential for activity. D4 mRNA has been detected only in the endosperm and is at a maximum concentration mid-way through grain development. The wSBE I-D4 gene consists of 14 exons, similar to the structure for the equivalent gene in rice; the rice gene has a strikingly longer intron 2. The 3′ end of wSBE I-D4 was used to show that the gene is located on group 7 chromosomes. The sequence upstream of wSBE I-D4 was analysed with respect to conserved motifs. Received: 14 January 1998 / Accepted: 14 July 1998     

Genomic organization and promoter activity of the maize starch branching enzyme I gene

Starch branching enzymes (SBE) which catalyse the formation of α-1,6-glucan linkages are of crucial importance for the quantity and quality of starch synthesized in plants. In maize (Zea mays L.), three SBE isoforms (SBEI, IIa and IIb) have been identified and shown to exhibit differential expression patterns. As a first step toward understanding the regulatory mechanisms controlling their expression, we isolated and sequenced a maize genomic DNA (−2190 to +5929) which contains the entire coding region of SBEI (Sbe1) as well as 5′-and 3′-flanking sequences. Using this clone, we established a complete genomic organization of the maize Sbe1 gene. The transcribed region consists of 14 exons and 13 introns, distributed over 5.7 kb. A consensus TATA-box and a G-box containing a perfect palindromic sequence, CCACGTGG, were found in the 5′-flanking region. Genomic Southern blot analysis indicated that two Sbe1 genes with divergent 5′-flanking sequences exist in the maize genome, suggesting the possibility that they are differentially regulated. A chimeric construct containing the 5′-flanking region of Sbe1 (−2190 to +27) fused to the β-glucuronidase gene (pKG101) showed promoter activity after it was introduced into maize endosperm suspension cells by particle bombardment.     

Interaction of divalent metal ions with the NADP+-malic enzyme from maize leaves

The effect of several metal ions on NADP⁺-malic enzyme (EC 1.1.1.40) purified from Zea mays L. leaves was studied Mg²⁺, Mn²⁺, Co²⁺ and Cd²⁺ were all active metal cofactors. The malic enzyme from maize has a moderately high intrinsic preference for Mn²⁺ relative to Mg²⁺ at pH 7.0 and 8.0 Negative cooperativity detected in the binding of Mg²⁺ at pH 7.0 and 8.0 and in the binding of Mn²⁺ at pH 7.0 suggests the existence of at least two binding sites with different affinity. All of the activating metal ions have preference for octahedral coordination geometry and have ionic radii of 0.86–1.09 Å. The ions that act as inhibitors are outside this range and/or are incapable of octahedral coordination. Ba²⁺, Sr²⁺, Cd²⁺, Ca²⁺, Be²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Co²⁺, Hg²⁺ showed mixed-type inhibition. The reciprocal of their K₁ values follow the order of their apparence in the Irving-Williams series of stability that derives in part from size effects. It is suggested that the size of the ions may play a partial role in determining the strength of the metal interaction.     

高油、高淀粉玉米吸硫特性及施硫对其产量、品质的影响

研究了高油、高淀粉玉米硫素的吸收分配及施硫对籽粒产量、品质的作用。结果表明：在11250kg/hm^2的产量水平下,开花前硫素吸收量占总吸收量的百分比平均为57.53%,花后对硫的吸收较强;高油1号、长单26、掖单13每公顷吸硫量分别为：32.46kg、34.94kg,34.20kg。施硫能增加穗粒数和粒重（高油玉米粒重增加不显著）,并显著提高其产量,施硫22.5kg/hm^2（S1）能使籽粒含油率和蛋白质含量分别比对照提高9.05%和6.88%,蛋白质中清蛋白、球蛋白和谷蛋白含量提高,品质改善;施硫量增至90kg/hm^2(S2)时,籽粒含油率和蛋白质含量仍有提高,但蛋白质中醇溶蛋白含量增加,蛋白质品质降低。     

Colchicine, an efficient genome-doubling agent for maize (Zea mays L.) microspores cultured in anthero

The construction of maize genotypes with high haploid induction capacity made it possible to study the effect of colchicine on maize androgenesis in vitro. Anther cultures of three hybrids were treated with 0.02% and 0.03% colchicine for 3 days at the beginning of microspore induction. Colchicine added to the induction medium had no negative influence on the androgenic responses (anther induction, induction of structures of microspore origin and their regeneration ability) of the genotypes examined. However, significantly higher fertility was observed in plants originating from colchicine-treated microspores, especially at 0.03%. Cytological examinations showed that colchicine treatment before the first microspore division efficiently arrested mitosis and resulted in homozygous doubled-haploid microspores. Under the experimental conditions, the antimitotic drug had no later effect on the division symmetry of the microspore nucleus, and unequal divisions remained dominant. Callus formation from the induced microspores seemed to be more typical (ranging between 60–70%), but embryo frequency was increased by approximately 10%, especially at the higher colchicine concentration. These results suggest that the mechanism of colchicine action in premitotic maize microspores may differ from that previously observed in wheat. Received: 15 June 1998 / Revision received: 17 September 1998 / Accepted: 3 December 1998     

野桑蚕酚氧化酶原基因cDNA的分子克隆及其特征

酚氧化酶在昆虫的免疫防御机制中起着重要作用。利用RT-PCR和RACE方法,克隆了野桑蚕酚氧化酶原基因,获得了其cDNA序列。该序列长2 134 bp,含有一个2 082 bp的完整开放阅读框,编码一个由693个氨基酸残基组成的蛋白质。推导的氨基酸序列与其他鳞翅目昆虫PPO2基因相应氨基酸序列有较高的同源性,该序列具有它们的PPO基因所共有的典型特征。组织特异性表达分析表明了该基因在野桑蚕5龄幼虫的血细胞、体壁、头部、精巢、卵巢、脂肪体和中肠等组织及其不同的发育阶段均有表达。这些结果为进一步研究野桑蚕酚氧化酶原基因的功能提供了分子基础。     

Distribution of enzyme activities within the developing maize (Zea mays) kernel in relation to starch, oil and protein accumulation

The association of enzyme activities in developing kernels with specific storage product accumulation at maturity was analyzed in different parts of Zea mays inbred OH43 kernels. Maize kernels were harvested at 20 and 55 days post-pollination and dissected into basal region, pericarp, embryo, lower endosperm, middle endosperm and upper endosperm. Mature (55 days pos(-pollination) kernel parts were analyzed for starch, total protein, zein and oil content. Immature (20 days post-pollination) kernel parts were assayed for activities of 15 enzymes of sugar and amino acid metabolism. Statistical analyses of the data suggested that glucokinase (EC 2.7.1.2) fructokinase (EC 2.7.1.4) and phosphofructokinase (EC 2.7.1.1 11) activities were primarily associated with oil accumulation, whereas ADP'-glueose pyrophosphorylasc (EC 2.7.7.27) and sucrose synthase (EC 2.4.1.13) activities were associated with starch accumulation. The results suggest that oil biosynthesis utilizes inveitase-mediated sucrose degradation in a pathway not requiring pyrophosphatc. whereas starch biosynthesis utilizes a sucrose synthase-mediated pathway of sucrose degradation in a pathway requiring pyrophosphatc. Additional groups of enzyme activities were associated with each oilier but not with any specific storage product and appeared to be associated with general metabolic activity.