He-Cui Zhang

Identification of Interacting Motifs Between Armadillo Repeat Containing 1 (ARC1) and Exocyst 70 A1 (Exo70A1) Proteins in Brassica oleracea

In order to identify the functional domains which regulate the interaction between the self-incompatibility proteins armadillo repeat containing 1 (ARC1) and exocyst 70 A1 (Exo70A1) in Brassica oleracea, fragments containing selected motifs of ARC1 (ARC1210, ARC1246, ARC1279, ARC1354) and site-specific mutants with substitutions at possible interaction sites (ARC1354m, ARC1664m) were PCR amplified and inserted into pGADT7, while coding sequences from Exo70A1 (Exo70A185, Exo70A1) were subcloned into pGBKT7. The interactions between the protein products produced by these constructs were then analyzed utilizing a yeast two-hybrid system. Our data indicate that both ARC1210 and ARC1246 interact strongly with Exo70A185 and Exo70A1, while ARC1279, ARC1354, ARC1354m and ARC1664m exhibited a weak interaction, indicating that the recognition sites are located within the 210 N-terminal amino acids of ARC1 and the 85 N-terminal amino acids of Exo70A1. This was further verified by GST pull-down analysis. This supports a model in which the N-terminal leucine zipper of ARC1 and the first 85 N-terminal amino acids of Exo70A1 mediate the interaction between these two proteins. Bioinformatic and phylogenetic analysis demonstrated that these motifs were highly conserved across different species, indicating that the interaction characterized in B. oleracea may operate in a wide array of cultivars.

Fluorescence in situ hybridization on plant extended chromatin DNA fibers for single-copy and repetitive DNA sequences

The compactness of plant chromosomes and the structure of the plant cell wall and cytoplasm provide a great obstacle to fluorescence in situ hybridization (FISH) for single-copy or low-copy DNA sequences. Consequently, many new methods for improving spatial resolution via chromosomal stretching have been employed to overcome this technical challenge. In this article, a technique for extracting cell-wall free nuclei at mitotic interphase, then using these nuclei to prepare extended DNA fibers (EDFs) by the method of a receding interface, whereby slide-mounted chromatin produces EDFs in concert with gravity-assisted buffer flow, was adopted as a result of the low frequency of EDF damage produced by this procedure. To examine the quality of these EDFs, we used single-copy gene encoding S-locus receptor kinase and multi-copy 5S rDNA (ribosomal DNA) as probes. The resulting EDFs proved suitable for high-resolution FISH mapping for repetitive DNA sequences, and the localization of a single-copy locus.

Cloning and Expression Characteristics of EXO70A1 from Brassica oleracea , Brassica campestris , and Brassica napus : Cloning and Expression Characteristics of EXO70A1 from Brassica oleracea , Brassica campestris , and Brassica napus

从甘蓝、大白菜与甘蓝型油菜中分离出 EXO70A1 基因，对该基因的序列进行生物信息学分析, 然后转化酵母Y187, 应用半定量RT-PCR检测 BoEXO70A1 基因的表达特性。结果表明, 3种芸薹属植物 EXO70A1 序列长度均为1 917 bp，相似性97.1%, 它们的gDNA序列均为单一序列，长度分别为3 797、3 752和3 770 bp，一致度达91.0%，均由12个外显子及11个内含子组成，除了第4、第5、第6、第8个内含子外，其余内含子的保守性低于外显子; 推导的3种蛋白质序列(BnEXO70A1、BrEXO70A1和BoEXO70A1)的相似度与一致性分别达99.8%与98.1%，其二级结构、三维结构及理化特性高度相似。 EXO70A1 基因的11个内含子的剪切位点均符合“GU-AG”法则，剪切受体(AG)的前20~50个碱基存在一段保守的序列“CU(A/G)A(C/U)”; 3种芸薹属植物与拟南芥 EXO70A1 基因的12个外显子的对应序列长度完全相同，所构成的编码区的序列一致性达90.1%，相应的蛋白质序列的相似度与一致性分别达99.8%与93.7%; 分子进化分析表明, EXO70A1在整个EXO70蛋白家族中及不同的植物间表现出较高的保守性; BoEXO70A1 在酵母细胞Y187呈现弱表达; EXO70A1 在甘蓝的雄蕊、幼茎、幼嫩花瓣、雌蕊、幼根及叶片中均能表达，可能属于组成型表达基因，但是其表达量在不同发育时期的不同器官中存在差异，授粉前雌蕊中最高，雄蕊中最低。由此可知， EXO70A1 在芸薹属植物中整体高度保守, 但在酵母转化株和甘蓝各器官中的组成型表达有所差异，推测 EXO70A1 在植物细胞中具有多种重要的功能。

Identifications of DNA Sequences Encoding Key Region of SCR Interacting with SRK Extracellular Domain by Using Yeast Two-Hybrid System: Identifications of DNA Sequences Encoding Key Region of SCR Interacting with SRK Extracellular Domain by Using Yeast Two-Hybrid System

The S-locus cystein-rich protein(SCR)is the male-determining factor of self-incompatibility in Brassica.In this study, the SCR fragments with different lengths amplified from Brassica oleracea L.were ligated with pGBKT7 to construct recombinant bait plasmids,which were then transformed into yeast Y2HGold cells for detecting their interaction with the S-locus receptor kinase extracellular domain(eSRK)in Brassica oleracea by using the yeast two-hybrid system.The results showed that recombinant vectors were not activated autonomously.The full length SCR could interact with eSRK,and the core region in the SCR was located between 97 and 186 bp.Moreover,the result also indicated that the splicing site of signal peptides of this haplotype SCR and its several adjacent amino acid residues could affect the interaction.These conclusions add some novel insights into the mechanism research of self-incompatibility in Brassica.