Characterization of phosphorus starvation-induced gene BnSPX3 in Brassica napus

Plant and Soil - The molecular studies of Pi starvation in Brassica napus have been limited. To explore the molecular responses and mechanisms involved in Pi starvation in Brassica napus, two SPX...     

甘蓝型油菜Toc33基因编码区的分离及蛋白序列的比较

以甘蓝型油菜新鲜嫩叶为实验材料提取其总DNA,以其为模板,根据拟南芥Toc33基因编码区序列设计引物,PCR扩增甘蓝型油菜叶绿体外膜蛋白转运机器的构件蛋白基因Toc33,得到两条扩增带,测序结果显示克隆到的两个片段分别长1370bp、1490bp,将这两个片段分别命名为Bn Tpc33-1,Bn Toc33-2,序列比较发现它们之间的同源性为78％,其中外显子的同源性为96％,而内含子的同源性仅为60％。为研究Toc33与同一基因家族的Toc34基因功能间的关系,对拟南芥、油菜、诸葛菜等植物的Toc33、Toc34蛋白序列进行比较分析并构建了分子系统进化树。     

油菜蔗糖转化酶基因的电子克隆和生物信息学分析

运用电子克隆技术获得油菜中一个蔗糖转化酶基因eDNA序列,同时根据此段序列设计引物以油菜eDNA为模板进行扩增。经测序得到证实。采用生物信息学方法,对该基因编码蛋白从氨基酸组成、基本理化性质、跨膜结构域、信号肽导肽、疏水性／亲水性、二级结构、亚细胞定位等方面进行了预测和分析。结果表明：该基因eDNA序列长度为2150bp,包含一个1779bp开放阅读框,编码592个氨基酸;该编码蛋白含有蔗糖转化酶的多个典型的保守结构域。同源比对分析显示,该基因编码的氨基酸序列与拟南芥等植物的蔗糖转化酶基因具有高度的相似性,进一步确定该蛋白为蔗糖蛋白酶。研究结果为该基因进一步的实验克隆,表达分析,功能鉴定奠定基础。     

Hydrolysis products of glucosinolates in Brassica napus tissues as inhibitors of seed germination

Enzymatic hydrolysis of glucosinolates, a class of compounds found in Brassica species, results in a number of products with potential to inhibit seed germination. To investigate the impact of both volatile and water-soluble allelochemicals, germination bioassays using Lactuca sativa seeds were conducted with root and combined leaf and stem tissues of Brassica napus. Tissues in which glucosinolates were hydrolyzed to remove volatile glucosinolate degradation products were compared with intact tissues and water controls. Only tissues containing glucosinolates produced volatiles that inhibited germination. Volatiles were trapped and identified using GC-MS. Volatiles produced in greater quanitity from intact tissues than from tissues without glucosinolates were almost exclusively glucosinolate hydrolysis products. Water-soluble components also inhibited germination. Chemical analysis of extracts confirmed the presence of glucosinolate hydrolysis products, but indicated the involvement of additional allelochemicals, especially in leaf and stem tissues. Results support the proposal that glucosinolate-containing plant tissues may contribute to reductions in synthetic pesticide use if weed seeds are targeted.Abbreviations ITC isothiocyanates - CN organic cyanides - OZT oxazolidinethione - iRoot intact root tissue - iL&S intact leaf and stem tissue - hRoot hydrolyzed root tissue - hL&S hydrolyzed leaf and stem tissue     

Agrobacterium-mediated transformation of Brassica napus and Brassica oleracea

Agrobacterium-mediated transformation is widely used for gene delivery in plants. However, commercial cultivars of crop plants are often recalcitrant to transformation because the protocols established for model varieties are not directly applicable to them. The genus Brassica includes the oil seed crop, canola (B. napus), and vegetable crop varieties of Brassica oleracea, including cauliflower, broccoli and cabbage. Here, we describe an efficient protocol for Agrobacterium-mediated transformation using seedling explants that is applicable to various Brassica varieties; this protocol has been used to genetically engineer commercial cultivars of canola and cauliflower in our laboratory. Young seedling explants are inoculated with Agrobacterium on the day of explant preparation. Explants are grown for 1 week in the absence of a selective agent before being transferred to a selective medium to recover transgenic shoots. Transgenic shoots are subjected to an additional round of selection on medium containing higher levels of the selective agent and a low-carbohydrate source; this helps to eliminate false-positive plants. Use of seedling explants offers flexible experiment planning and a convenient explant source. Using this protocol, transgenic plants can be obtained in 2.5 to 3.5 months.     

Isolation and characterization of a polygalacturonase gene highly expressed in Brassica napus pollen

A cDNA clone, Sta 44-4, corresponding to a mRNA highly expressed in Brassica napus cv. Westar stamens, was isolated by differential screening and characterized. Northern blot and in situ analyses demonstrated that Sta 44-4 is synthesized in pollen beginning at the late uninucleate stage and reaches a maximum in trinucleate microspores. Sta 44-4 displayed significant sequence similarity to known pollen polygalacturonase genes. The B. napus pollen polygalacturonase gene was shown to be part of a small gene family and to display some polymorphism among different cultivars.     

Classification and expression of a family of cyclin gene homologues in Brassica napus

In order to investigate the role of cell division in plant development, we isolated several plant genes which encode homologues of animal and yeast cell cycle regulators known as cyclins.Through the use of degenerate primers and the polymerase chain reaction (PCR) we isolated a Brassica sequence which showed homology to the cyclin box functional domain found within cyclin proteins. Southern blot analysis indicated that Brassica napus has a large number of genes containing cyclin box-related sequences. This was further supported by the isolation of cyclin box sequences from six different genomic clones. In addition, we have isolated two different cyclin cDNA clones, BnCYC1 and BnCYC2, from a Brassica napus shoot apical cDNA library. Both of the cDNA clones contain a destruction box regulatory domain similar to animal mitotic cyclins.Northern blot analysis using BnCYC2 shows mRNA levels which correlate well with the level of cell division in various tissues. Messenger RNA abundance was highest in 1–3 mm leaves, root tips and shoot apices. The mRNA detected using BnCYC1 was restricted to young leaves and the shoot apex, suggesting divergent, organ-specific roles for cyclin family members. The results demonstrate that the plant cyclin gene family is more extensive than previously demonstrated and consists of genes expressed in all dividing tissues as well as a subset of developmentally specific members.     

Relationships between the rapid axonal transport of newly synthesized proteins and membranous organelles

Rapid axonal transport is generally viewed as being exactly analogous to the secretory process in nonneuronal cells. The cell biology of rapid axonal transport is reviewed, the central concern being to explore those aspects that do not fit into the general secretory model and which may thus represent specific neuronal adaptations. Particular attention is paid to the relationship between the transport of newly synthesized proteins and of the membranous organelles that act as carriers. Sites in the transport sequence at which the behavior of axonal transport may differ from the secretory model are at the initiation of axonal transport at the trans-side of the Golgi apparatus, within the axon where molecules are deposited from the moving phase to a stationary phase, and at nerve terminals or axonal lesions where transport reversal takes place.     

Integrative analysis of genome‐wide lncRNA and mRNA expression in newly synthesized Brassica hexaploids

Polyploidization, as a significant evolution force, has been considered to facilitate plant diversity. The expression levels of lncRNAs and how they control the expression of protein‐coding genes in allopolyploids remain largely unknown. In this study, lncRNA expression profiles were compared between Brassica hexaploid and its parents using a high‐throughput sequencing approach. A total of 2,725, 1,672, and 2,810 lncRNAs were discovered in Brassica rapa, Brassica carinata, and Brassica hexaploid, respectively. It was also discovered that 725 lncRNAs were differentially expressed between Brassica hexaploid and its parents, and 379 lncRNAs were nonadditively expressed in this hexaploid. LncRNAs have multiple expression patterns between Brassica hexaploid and its parents and show paternal parent‐biased expression. These lncRNAs were found to implement regulatory functions directly in the long‐chain form, and acted as precursors or targets of miRNAs. According to the prediction of the targets of differentially expressed lncRNAs, 109 lncRNAs were annotated, and their target genes were involved in the metabolic process, pigmentation, reproduction, exposure to stimulus, biological regulation, and so on. Compared with the paternal parent, differentially expressed lncRNAs between Brassica hexaploid and its maternal parent participated in more regulation pathways. Additionally, 61 lncRNAs were identified as putative targets of known miRNAs, and 15 other lncRNAs worked as precursors of miRNAs. Some conservative motifs of lncRNAs from different groups were detected, which indicated that these motifs could be responsible for their regulatory roles. Our findings may provide a reference for the further study of the function and action mechanisms of lncRNAs during plant evolution.