2 resultados para endosperm development
em Chinese Academy of Sciences Institutional Repositories Grid Portal
Resumo:
籽粒的灌浆是将光合器官合成的有机物贮存在籽粒中的过程。这一过程直接决定了籽粒的产量及品质。先前研究表明灌浆籽粒中贮存物质的累积是各种代谢活动和细胞学过程协同作用的结果,但灌浆的分子机制目前还不是非常清楚。水稻是研究籽粒灌浆的优良模式材料,不仅因为它是世界上最重要的淀粉食物来源,更重要的是其全基因组的测序完成为分子机制的研究带来极大的便利。我们对发育水稻籽粒的观察表明在开花后6 天,籽粒就已完成了胚的分化和胚乳的细胞化;此后籽粒经历了一个显著的细胞增大过程,并在开花后12 天左右达到成熟籽粒的大小;而籽粒的灌浆过程起始于开花后6 天,这个过程一直持续到开花后20 天。因此,我们将开花后6 天到20 天的籽粒分为8 个连续的发育阶段进行动态的蛋白质组分析,396个蛋白点的表达在灌浆过程中发生了两倍以上变化。质谱鉴定得到的345 个差异表达的蛋白划分为10 个不同的功能类别。其中新陈代谢类(45%)和蛋白合成/终点(destination)类(20%)两个功能类别中就包括了大多数的差异表达蛋白,预示着这两类蛋白在籽粒发育中的重要性。蛋白功能群的表达分析显示与淀粉合成和乙醇发酵相关的蛋白在发育过程中大幅度的上调,而与碳代谢中心过程(糖酵解和三羧酸循环)相关蛋白呈现明显的下调趋势。大多数的功能类或(亚类)也呈现出下调的表达趋势,如细胞生长/分裂类,蛋白合成类,水解类,信号传导类和转录类。蛋白表达分析的结果表明蛋白的表达随籽粒的发育发生了显著的变化,这些变化与籽粒在不同阶段的发育和代谢过程密切相关并协调一致,是细胞从生长分裂过渡到以淀粉合成为中心的物质基础。同时也说明代谢重点由中心碳代谢向乙醇发酵的转变对于籽粒的发育和淀粉的合成与累积具有重要意义。 籽粒发育的研究表明在长到成熟籽粒大小后(开花后12 天),籽粒的代谢集中到淀粉累积途径上,一直持续到进入脱水期(18 天),绝大多数淀粉合成相关蛋白在这期间到达表达的顶点。为了解淀粉累积关键时期淀粉合成关键部位(胚乳)的发育规律,我们进一步应用DIGE 技术对这一淀粉累积关键时期(灌浆中后期,开花后12 到18 天)的蛋白表达特性进行分析。细胞学的观察发现胚乳在灌浆后期先后经历了过氧化氢的爆发、半透明胚乳的形成以及胚乳细胞死亡事件。相应的DIGE 分析显示有321 个蛋白点在胚乳的后期发育中发生了显著的表达变化。细胞学的观察结合DIGE 分析显示胚乳的后期发育是一个典型的衰老过程:细胞结构的崩溃;氧化自由基的爆发;脱水干燥;蛋白、脂类和DNA 由同化作用向异化作用的代谢转化。与代谢转化相伴随的细胞营养的重新分配是胚乳后期发育的一个显著过程。DIGE分析全面展示了参与营养重新分配相关蛋白在后期发育中的表达变化,为细胞学中观察到的有机物向淀粉的转化提供了清晰的蛋白水平的证据支持。在鉴定的差异表达蛋白中有2/3 的蛋白是已知的对氧化电位变化敏感的蛋白,表明由H2O2 爆发形成的氧化压力将引起氧化还原调控从而对胚乳的后期发育进行全面的影响。而其中与碳元素代谢相关的代谢途径中尤其富含氧化还原电位敏感的蛋白,表明后期的营养重新分配以及淀粉的累积受到氧化还原电位的紧密调控。另一方面,H2O2 的爆发激发了胚乳中的抗氧化体系。由抗氧化蛋白(如thioredoxin、抗坏血酸和超氧化物歧化酶等)、氧化还原敏感蛋白、代谢中间产物以及glyoxalase 构成的抗氧化体系在胚乳后期发育中协同作用调节氧化还原电位的变化,从而控制胚乳细胞衰老的节奏。另外,我们发现与RraA 相关的转录本的调控在胚乳发育末期急剧上调,在调控的代谢途径、调控时间以及调控的部位与氧化还原调控相重叠,并且支持RraA 活动有利于胚乳细胞对氧化压力的适应。所有这些结果表明内生的过氧化氢(或氧化自由基)在胚乳的后期发育和淀粉累积中起到核心的调控作用。
Resumo:
高等植物种子胚乳贮藏蛋白是种子发芽时的主要氮源,也是人类和动物食用植物蛋白的主要来源。大麦种子胚乳贮藏蛋白主要是醇溶蛋白(hordeins),占大麦胚乳总蛋白的50–60%。根据大麦醇溶蛋白的大小和组成特点,大麦醇溶蛋白被划分为三种类型:富硫蛋白亚类(B,γ-hordeins)、贫硫蛋白亚类(C-hordeins)以及高分子量蛋白亚类(D-hordeins)。B组和C组醇溶蛋白是大麦胚乳的两类主要贮藏蛋白,它们分别占大麦总醇溶蛋白成分的70–80%和10–12%。遗传分析表明,大麦B、C、D和γ-组醇溶蛋白分别是由位于大麦第五染色体1H(5)上的Hor2、Hor1、Hor3和Hor5位点编码。Hor2位点编码大量分子量相同但组成不同的B组醇溶蛋白(B-hordein)。B-hordein的种类、数量和分布是影响大麦酿造、食用及饲养品质的重要因素之一。为深入了解B-hordein基因家族的结构和染色体组织,探明Hor2位点基因表达的发育调控机制,最终达到改良禾谷类作物籽粒品质的目的,本研究以青藏高原青稞为材料,采用同源克隆法,分别克隆B-hordein基因和启动子,通过原核生物表达验证B-hordein基因功能,并利用实时定量PCR探索B-hordein基因表达时空关系,取得如下研究结果: 1. 以具有特殊B组醇溶蛋白亚基组成的9份青藏高原青稞为材料,根据GenBank中三个B-hordein基因序列(GenBank No. X03103, X53690和X53691)设计一对引物,通过PCR扩增,获得23个B-hordein基因克隆并对其进行了序列分析。核苷酸序列分析表明,所有克隆均包含完整的开放阅读框。有11个克隆都存在一个框内终止密码子,推测这11个克隆可能是假基因。推测的氨基酸序列分析表明,所有大麦B-hordein具有相似的蛋白质基本结构,均包括一个高度保守的信号肽、中间重复区以及C-端结构域。不同大麦种重复区内重复基元的数目有较大差异。青稞材料Z07–2和Z26的B-hordeins仅具有12个重复基元结构,更接近于野生大麦。这些重复基元数目的差异导致了重复区序列长度和结构的变异。这种现象极可能是由于醇溶谷蛋白基因在进化过程中染色体的不平衡交换或复制滑动所造成的。对所克隆基因和禾本科代表性醇溶谷蛋白基因进行聚类分析,结果表明所有来自栽培大麦的B-hordeins聚类成一个亚家族,来自野生大麦的B-hordeins以及普通小麦的LMW-GS聚类成另外一个亚家族,表明这两个亚家族的成员存在显著差异。此外,我们发现B-hordein基因推测的C-末端序列具有一些有规律的特征:即具有相同C-末端序列的B-hordein基因在系统发生树中聚类为同一个亚组(除BXQ053,BZ09-1,BZ26-5分别单独聚为一类外)。这个特征将有助于我们对所有B组醇溶蛋白基因家族成员进行分类,避免了在SDS-PAGE电泳图谱上仅依靠大小分类的局限性。 2. 根据上述克隆的青稞B-hordein基因的5’端序列设计三条基因特异的反向引物,以青稞Z09和Z26的基因组DNA为模板,采用SON-PCR和TAIL-PCR技术分离克隆出8个B-hordein基因的上游调控序列(命名为Z09P和Z26P)。序列分析表明,推测的TATA box位于–80 bp,CAAT–like box位于–140 bp处。此外,Z09P和Z26P中有六个序列在–300 bp处均存在一个由高度保守的EM基序和类GCN4基序构成的胚乳盒(Endosperm Box,EB),在约–560 bp处存在一个胚乳盒类似结构。而Z09P-2和Z26P-3不存在保守的胚乳盒或其类似结构,预示着这两个启动子所调控的基因表达可能受不同类型反式作用因子的调节,推测该启动子对基因的表达调控具有多样性。 3. 将B-hordein基因的开放阅读框定向克隆到表达载体pET-30a中,将其导入大肠杆菌表达菌株BL21中进行外源基因的诱导表达以验证所克隆基因的功能。结果表明仅含重组子pET-BZ07-2和pET-BZ26-5的BL21细菌有目的表达蛋白产生。在诱导3 h时的蛋白表达量最高;3 mM IPTG诱导的蛋白表达量要高于1 mM IPTG诱导的表达量。这为分离纯化B-hordein蛋白以及进一步研究其对大麦籽粒品质的影响奠定基础。 4. 根据从青稞Z09和Z26中分离克隆的B-hordein基因序列设计一对基因特异的引物,同时,选择大麦α-微管蛋白基因(GenBank no. U40042)为看家基因并设计特异引物,利用实时荧光定量PCR检测了青稞籽粒4个胚乳发育时间段的B-hordein基因表达,荧光定量结果显示:两份材料中B-hordein基因的表达量均随发育过程的进行而逐渐升高。Z09中B-hordein基因在开花后7天开始转录,而Z26开花4天后就有低水平B-hordein的表达,这表明Z26中B-hordein基因可能比Z09表达的较早或者Z09中B-hordein基因表达水平较低以致于不能被检测到。此外,在4个不同的胚乳发育时期中,Z26中B-hordein基因的表达量均高于Z09材料。在开花12天到18天的过程中,Z09和Z26中B-hordein基因的表达水平有一个急剧性的升高。这说明在不同胚乳发育时期,Hor2位点的B-hordein等位基因变异体存在mRNA的差异表达。 Seed endosperm storage proteins in higher plants are the main resources of nitrogen for germinating and plant proteins for human and animals. Barley prolamins (also called hordeins) are the major storage proteins in the endosperm and account for 50–60% of total proteins. Hordeins are classically divided into three groups: sulphur-rich (B, γ-hordeins), sulphur-poor (C-hordeins) and high molecular weight (HMW, D-hordeins) hordeins based on the size and composition. B-hordeins and C-hordeins are two major groups and each respectively account for about 70-80% and 10-12% of the total hordein fraction in barley endosperm. Genetic analysis showed that B-, C-, C-, γ-hordeins are encoded by Hor2, Hor1, Hor3 and Hor5 locus on the chromosome 1H (5). Hor2 locus is rich in alleles that encode numerous heterogeneous B-hordein polypeptides. It is reported that B-hordein species, quantity and distribution are significant factors affecting malting, food and feed quality of barley. To understand comprehensively the structure and organization of B-hordein gene family in hull-less barley and explore the developmental control mechanisms of Hor2 locus gene expression and eventually to better exploitation in crop grain quality improvement, we isolated and cloned B-hordein genes and promotors of hull-less barley from Qinghai-Tibet Plateau by PCR, and testified their expression founction in bacteria expression system and explore their spatial and temporal expression pattern by quantitative real time PCR. Our results are as followed, 1. Twenty-three copies of B-hordein gene were cloned from nine hull-less barley cultivars of Qinghai-Tibet Plateau with special B-hordein subunits and molecularly characterized by PCR, based on three B-hordein genes published previously (GenBank No. X03103, X53690 and X53691). DNA sequences analyses confirmed that the six clones all contained a full-length coding region of the barley B-hordein genes. Eleven clones all contain an in-frame stop codon and they are probably pseudogenes. The analysis of deduced amino acid sequences of the genes shows that they have similar structures including signal peptide domain, central repetitive domain, and C-terminal domain. The number of the repeats was largerly variable and resulted in polypeptides in different sizes or structures among the genes. Twelve such repeated motifs were found in Z07–2 and Z26, and they are close to those of the wild barleys, and it is most probably caused by unequal crossing-over and/or slippage during replication as suggested for the evolution of other prolamins. The relatedness of prolamin genes of barley and wheat was assessed in the phylogenetic tree based on their polypeptides comparison. Our phylogenetic analysis suggested that the predicted B-hordeins of cultivated barley formed a subfamily, while the B-hordeins of wild barleys and the two most similar sequences of LMW-GS of T. aestivum formed another subfamily. This result indicated that the members of the two subfamilys have a distinctive difference. In addition, we found the B-hordeins with identical C-terminal end sequences were clustered into a same subgroup (except BXQ053,BZ09-1 and BZ26-5 as a sole group, respectively), so we believe that B-hordein gene subfamilies possibly can be classified on the basis of the conserved C-terminal end sequences of predicted polypeptide and without the limit of SDS-PAGE protein banding patterns. 2. The specific primers were designed according to the published sequences of barley B-hordein genes from Z09 and Z26. Using total DNA isolated from them as the templates, eight clones (designated Z09Pand Z26P) of upstream sequences of the known B-hordein genes was obtained by TAIL-PCR and SON-PCR. Sequences analysis shows that the putative TATA box was present at position –80 bp and CAAT-like box at position –140 bp. Besides, a putative Endosperm Box including an Endosperm Motif (EM) and a GCN4-Like Motif was found at position –300 bp in six clones, and another Endosperm-like box was found at positon –560 bp. While the Endosperm Box or Endosperm-like box was not found in Z09P-2 and Z26P-3. This may indicate that gene expression drived by the two promtors was probably controlled by different trans-acting factors and the genetic control mechanism of corresponding gene expression may be diverse. 3. The B-hordein genic region coding for the mature peptide was cloned into expression vector pET-30a and transformed into bacterial strain BL21 for identifying gene expression fountion. Protein SDS–PAGE analysis showed that only the transformed lysate with the pET-BZ07-2 and pET-BZ26-5 constructs produced proteins related to B-group hordeins of barley, and the mounts of proteins induced by 3 mM IPTG and 3 h were higher than other conditions. This established a base for isolating and putifying B-hordein and further exploring their effects on barley grain quality. 4. The gene-specific primers of B-hordein genes from Z09 and Z26 were used for the quantification of B-hordein gene expression. The α-tubulin gene from Hordeum vulgare subsp. vulgare (GenBank accession number U40042) was used as a control gene. The result shows the transcription of the B-hordein genes in Z09 was found 7 days after flowering, while the transcription of the B-hordein genes in Z26 was found 4 days after flowering, but at a very low level, and it suggested that the B-hordein genes in Z26 probably expressed earlier than those in Z09, or the B-hordein genes in Z09 expressed at so a lower level than Z26 that it can not detected. In addition, B-hordein genes in Z26 accession showed higher expression levels than those in Z09 in four developing stages. Furthermore, a progressive increase in the expression levels of the B-hordein genes between 12 and 18 days after anthesis was observed in both Z09 and Z26. It implies that the B-hordein allelic variants encoded by Hor2 locus exist the differential expression in mRNA levels of during barley endosperm development.