一个陆地棉bZIP蛋白cDNA的克隆及表达分析

利用PCR筛选方法从陆地棉纤维cDNA文库中筛选到一个全长cDNA序列,命名为GhbZIP。其编码产物长度为645个氨基酸残基,序列中含有两个未知功能的保守区域DUF630和DUF632,而DUF632区中有一个类似碱性亮氨酸拉链基元;此外氨基酸序列中还存在一个富脯氨酸区和一个富苯丙氨酸区,因此该蛋白具有植物碱性亮氨酸拉链蛋白的结构特征。亲水性分析表明,GhbZIP为一个典型的膜蛋白。GhbZIP基因主要是在开花3d之后在胚珠和纤维细胞中表达,这表明该基因可能与棉纤维伸长过程中的基因表达调控有关。     

A novel subtilisin-like protease gene from Arabidopsis thaliana is expressed at sites of lateral root emergence.

Differential screening of a cDNA library for mRNA species that specifically accumulate during auxin-induced lateral root formation in Arabidopsis thaliana led to the isolation of the AIR3 cDNA clone. The corresponding single copy gene consists of 10 exons which encode a protein that possesses all the characteristics of subtilisin-like proteases. The promoter of the AIR3 gene was fused to the gusA (beta-glucuronidase) reporter gene and introduced into Arabidopsis. Expression was almost completely restricted to the outer layers of the parental root at sites of lateral root emergence and could be observed even before protrusion of the newly formed root tip. In the presence of external auxin, GUS activity was visible throughout the parts of the root that are competent for lateral root formation. By digesting structural proteins in the extracellular matrix of cells located above sites of lateral root formation, AIR3 might weaken cell-to-cell connections and thus facilitate lateral root emergence.     

棉花胚珠内种皮特异基因GhIAA16的分离鉴定

棉花(Gossypium hirsutum L.)纤维是由胚珠外珠被表皮细胞发育形成的一种单细胞表皮毛.为了分析鉴定与棉花胚珠发育相关的基因,本文通过cDNA阵列方法分离了25个在开花前后棉花胚珠中差异表达的基因.其中一个基因与拟南芥IAA16具有很高的同源性,并命名为GhIAAl6.该基因编码一个208个氨基酸组成预测蛋白.分子生物学分析表明它在棉花基因组中以单拷贝形式存在,而且在棉花胚珠内种皮(endothelium)中特异表达.GhIAAl6是棉花中第一个分离到的内种皮特异表达的基因,本文对它在棉花胚珠发育中的可能功能进行了讨论.     

Developing Fiber Specific Promoter-Reporter Transgenic Lines to Study the Effect of Abiotic Stresses on Fiber Development in Cotton

Cotton is one of the most important cash crops in US agricultural industry. Environmental stresses, such as drought, high temperature and combination of both, not only reduce the overall growth of cotton plants, but also greatly decrease cotton lint yield and fiber quality. The impact of environmental stresses on fiber development is poorly understood due to technical difficulties associated with the study of developing fiber tissues and lack of genetic materials to study fiber development. To address this important question and provide the need for scientific community, we have generated transgenic cotton lines harboring cotton fiber specific promoter (CFSP)-reporter constructs from six cotton fiber specific genes (Expansin, E6, Rac13, CelA1, LTP, and Fb late), representing genes that are expressed at different stages of fiber development. Individual CFSP::GUS or CFSP::GFP construct was introduced into Coker 312 via Agrobacterium mediated transformation. Transgenic cotton lines were evaluated phenotypically and screened for the presence of selectable marker, reporter gene expression, and insertion numbers. Quantitative analysis showed that the patterns of GUS reporter gene activity during fiber development in transgenic cotton lines were similar to those of the native genes. Greenhouse drought and heat stress study showed a correlation between the decrease in promoter activities and decrease in fiber length, increase in micronaire and changes in other fiber quality traits in transgenic lines grown under stressed condition. These newly developed materials provide new molecular tools for studying the effects of abiotic stresses on fiber development and may be used in study of cotton fiber development genes and eventually in the genetic manipulation of fiber quality.     

Tissue-specific and developmental regulation of cotton gene FbL2A. Demonstration of promoter activity in transgenic plants.

A gene (FbL2A) that is preferentially expressed in cotton (Gossypium barbadense L. cv Sea Island) fiber was isolated and characterized. Genomic and cDNA analyses suggest multiple FbL2A genes in cotton. The gene is developmentally regulated and is activated during late primary and early secondary wall synthesis stages. FbL2A encodes a polypeptide of 43.4 kD and a predicted isoelectric point of 5.97. The nucleotide-derived protein is highly hydrophilic except for a hydrophobic N terminus and has a compositional bias for glutamic acid (26.3 mol%) and lysine (18.9 mol%). Sixty-two percent of the putative protein is composed of repeat motifs. A 55-amino-acid peptide region is repeated four times in a concatenate fashion within the protein. The function of the protein in the fiber cells is not known. A 2.3-kb DNA fragment 5' from the FbL2A gene is shown to direct expression of heterologous proteins in transgenic cotton in a fiber-specific and developmentally regulated fashion. The FbL2A promoter was used to express in transgenic cotton genes encoding acetoacetyl-coenzyme A reductase and polyhydroxyalkanoic acid synthase, which are involved in the synthesis of the thermoplastic polymer polyhydroxybutyric acid. Transgenic plants containing both enzymes produced polyhydroxybutyric acid in fiber. Thus, the FbL2A promoter is useful in genetic engineering schemes to modify cotton fiber.     

棉花胚珠内种皮特异基因GhIAA16的分离鉴定

棉花(Gossypium hirsutum L.)纤维是由胚珠外珠被表皮细胞发育形成的一种单细胞表皮毛。为了分析鉴定与棉花胚珠发育相关的基因,本文通过cDNA阵列方法分离了25个在开花前后棉花胚珠中差异表达的基因。其中一个基因与拟南芥IAA16具有很高的同源性,并命名为GhIAA16。 该基因编码一个208个氨基酸组成预测蛋白。分子生物学分析表明它在棉花基因组中以单拷贝形式存在,而且在棉花胚珠内种皮(endothelium)中特异表达。GhIAA16是棉花中第一个分离到的内种皮特异表达的基因,本文对它在棉花胚珠发育中的可能功能进行了讨论。     

棉纤维蔗糖合酶基因SS3上游调控序列的克隆及其表达分析

棉纤维蔗糖合酶基因SS3在棉纤维发育过程中起着重要作用.采用YADE技术克隆了该基因5′上游1717bp的调控区,该调控区含有典型的启动子核心元件TATA box ,以及TATC box、G box、GCN4 -motif、Prolamin box、Skn 1 likemotif、TCA element、HSE和O2 site等各种顺式调控元件和其他一些反应元件.将此序列和报告基因GUS融合在烟草、棉花中表达.组织化学分析结果显示棉花SuSyR序列启动GUS基因在烟草的子房、胎座、种子以及在棉花花蕾与棉铃中表达.在棉花花蕾蕾长为3mm、6mm、9mm和15mm花蕾中表达主要存在于雄蕊及雄蕊管、胎座等器官;在棉铃中,1DPA棉铃的花柱、花药、子房及胚珠中出现了蓝色,6DPA棉铃的子房及胚珠被染成蓝色,在2 0DPA的棉铃中蓝色只出现在胚珠及其纤维中、在胚珠中只有珠心被染成蓝色,在4 0DPA胚珠中只有纤维呈蓝色.研究结果揭示,棉花的SuSyR调控序列启动GUS基因主要在子房、胚珠和纤维等器官和主叶脉、茎微管束等输导组织中表达,在棉花中尤为明显,表明棉纤维蔗糖合酶基因SS3除参与棉花蕾铃发育、纤维素的合成外,还参与了光合产物的运输与分配过程.     

Differential expression of the Arabidopsis genes coding for Em-like proteins

Late embryogenesis abundant (lea) genes are a large and diverse group of genes highly expressed during late stages of seed development. Five major groups of LEA proteins have been described. Two Em genes (group I lea genes) are present in the genome of Arabidopsis thaliana L., AtEm1 and AtEm6. Both genes encode for very similar proteins which differ basically in the number of repetitions of a highly hydrophilic amino acid motif. The spatial patterns of expression of the two Arabidopsis Em genes have been studied using in situ hybridization and transgenic plants transformed with the promoters of the genes fused to the beta-glucuronidase reporter gene (uidA). In the embryo, AtEm1 is preferentially expressed in the pro-vascular tissues and in meristems. In contrast, AtEm6 is expressed throughout the embryo. The activity of both promoters disappears rapidly after germination, but is ABA-inducible in roots of young seedlings, although in different cells: the AtEm1 promoter is active in the internal tissues (vasculature and pericycle) whereas the AtEm6 promoter is active in the external tissues (cortex, epidermis and root hairs). The AtEm1 promoter, but not AtEm6, is also active in mature pollen grains and collapsed nectaries of young siliques. These data indicate that the two Em proteins could carry out at least slightly different functions and that the expression of AtEm1 and AtEm6 is controlled at, at least, three different levels: temporal, spatial and hormonal (ABA).