Li-Quan Zhu

Localization of S Genes on Extended DNA Fibers (EDFs) in Brassica oleracea by High-Resolution FISH

The compactness of plant chromosome and the structures of plant cell wall and cytoplasm pose a great resistance to fluorescence in situ hybridization (FISH), and consequently many new methods for improving spatial resolution are being exploited to overcome these problems. However, for plants with small chromosomes like rice and Brassica, there are still many difficulties. In this article a new and effective technique for preparation of extended DNA fibers (EDFs), using a series of treatments to prophase I chromosomes of Brassica oleracea PMCs, is presented. This technique allows longitudinal extension of the chromosomes 30-107 times longer than those of their metaphase counterparts. The length of the extended DNA fibers is between 89 microm and 273 microm, and the space resolution is 42.8-53.0 kb. Stretching ratios were assessed in a number of FISH experiments with super-stretched chromosomes from meiotic prophase I nuclei of B. olerecea. Through FISH to EDFs of pachytene chromosomes hybridized in situ with SRK (S-locus receptor kinase) and SPII (S-locus protein II) probes, for the first time we localized the accurate positions of S-locus and quantitatively analyzed the features of S genes in B. oleracea genome to show all S genes were single-copied. In addition, the length between two linked genes was measured to be about one micron. As a result, the highest space resolution which was about 4 kb was obtained.

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.

Interaction Between Two Self-Incompatible Signal Elements, EXO70A1 and ARC1

Abstract ARC1 and EXO70A1 are important signal elements of self-incompatibility in Brassica . To characterize the interaction of ARC1-EXO70A1 during the course of self-incompatibility, the coding sequences of ARC1 and EXO70A1 were cloned from Brassica napus L. and B. oleracea L. var. acephala . Sequence analysis showed that ARC1 consisted of 663 amino acids in B. oleracea and 661 amino acids in B. napus , with a 45-amino-acid difference between them. Sequence alignment showed 95.9% similarity, with 93.9% exact match between BoARC1 and BnARC1. Only a 6-amino-acid difference was detected between BoEXO70A1 and BnEXO70A1, with 99.4% similarity and 98.9% exact match according to further sequence alignment. The homology between EXO70A1 alleles was higher than that between ARC1 alleles. Yeast 2-hybrid results indicated that a strong interaction existed between ARC1 and EXO70A1, which could activate the expressions of 4 reporter genes ( ADE2 , HIS3 , AUR1-C , and MEL1 ) in diploid yeast. However, there was very weak interaction between EXO70A1 and a 316-C-terminal-deletion mutant of ARC1 (ARC1 N ), which only activated the expressions from 3 reporter genes ( ADE2 , AUR1-C , and MEL1 ). This indicated that the interaction interface between ARC1 and EXO70A1 might not reside within the Armadillo (ARM) repeat domains of ARC1. The N-terminal domains of ARC1 play an essential role in the interaction of ARC1 with EXO70A1. The influence of the differences in amino-acid composition between BoARC1 and BnARC1 on the interaction between ARC1-EXO70A1 was not detected with a yeast 2-hybrid system, which may indicate that the binding interface between ARC1 and EXO70A1 was not altered by sequence differences between the 2 proteins in these Brassica species.

Expression of ARC1 in Vitro and Test of Interaction Between ARC1 and SRK from Brassica oleracea L. in Signal Transduction Pathway of Self-Incompatibility

Abstract ARC1 is an arm repeat containing a downstream gene of S-locus receptor kinase (SRK). It interacts with SRK probably in signal transduction pathway of self-incompatibility. In this study, the interaction between SRK and ARC1 proteins was tested using the expression vectors pET43.1a-ARC1 and pET43.1a-SRK E1 . The coding sequences of ARC1 gene were amplified from stigma mRNA of Brassica oleracea L. and inserted into the expression vector pET43.1a to construct the recombinant plasma pET43.1a-ARC1. The recombinant plasmid was transformed to Escherichia coli (BL21) and the expression of the recombinant protein was detected using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using the co-immunoprecipitation theory and characteristic of 6× His Tag combined with Ni+ in pET43.1a-ARC1, a new method was proposed for testing the interaction between proteins. With this method the interaction between ARC1 and SRK was analyzed, showing that ARC1 and SRK could interact with each other to combine and form a complex. This study provides the theoretical and technical bases for further analyzing the mechanism of interaction between ARC1 and SRK proteins and for probing into interaction between ARC1 and downstream targets.

N-terminal domains of ARC1 are essential for interaction with the N-terminal region of Exo70A1 in transducing self-incompatibility of Brassica oleracea

Self-incompatibility (SI) is an important mating system to prevent inbreeding and promote outcrossing. ARC1 and Exo70A1 function as the downstream targets of the S-locus receptor kinase and play conservative roles in Brassica SI signaling. Based on the sequence homology, Exo70A1 is divided into four subdomains: leucine zipper (Leu(128)-Leu(149)), hypervariable region (Ser(172)-Leu(197)), SUMO modification motif (Glu(260)-Ile(275)), and pfamExo70 domain (His(271)-Phe(627)). ARC1 contains four domains as follows: leucine zipper (Leu(116)-Leu(137)), coiled-coil domain (Thr(210)-Val(236)), U-box (Asp(282)-Trp(347)) motif, and ARM (Ala(415)-Thr(611)) domain. Bioinformatics analysis, yeast two-hybrid screening and pull-down assays show that leucine zipper and coiled-coil motifs of ARC1116-236 are required for the interaction with Exo70A1, while the addition of ARM motif results in loss of the interaction with Exo70A1. Meanwhile, the N-terminal of Exo70A1 without any domains shows a weak interaction with ARC1, and the level of LacZ expression increases with addition of leucine zipper and reaches the maximum value with hypervariable region and SUMO modification motif, indicating that hypervariable region and SUMO modification motif of Exo70A1172-275 is mainly responsible for the binding with ARC1, whereas pfamExo70 domain has little affinity for ARC1. Lys(181) located in the Exo70A1 hypervariable region may be the ubiquitination site mediating the interaction between ARC1 and Exo70A1. Therefore, both the leucine zipper with coiled-coil structure of ARC1116-236, and the hypervariable region and SUMO modification motif of Exo70A1172-275 are the core interaction domains between ARC1 and Exo70A1. Any factors affecting these core domains would be the regulators of ARC1 mediating ubiquitin degradation in self-incompatible system.

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。

Test of Interaction Between SRK and THL1 from Brassica oleracea L. in Self-Incompatibility Signaling Process

Abstract The S -locus receptor kinase (SRK) and thioredoxin-like protein 1 (THL1) from Brassica oleracea L. probably interact with each other in self-incompatibility signal process. To testify the interaction, the coding sequences of SRK kinase domain (termed SRK E1 ) and THL1 were amplified from stigma mRNA of B. oleracea . They were, then, inserted into the expression vector pET43.1a and yeast expression vector pPIC9K to construct the recombinant plasma pET43.1a-SRK E1 , pPIC9K-SRK E1 , and pET43.1a-THL1. The recombinant proteins were expressed in Escherichia coli (BL21) and Pichia pastoris (GS115), respectively. The recombinant THL1 was approved with oxidation/reduction activity by testing the ability of THL1 reducing insulin. Using a new method for testing the interaction between proteins, SRK E1 and THL1 were identified to combine with each other and form a stable complex.

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.

Localization of MLPK and SSP for Self-Incompatibility of Brassica oleracea by Fluorescence in situ Hybridization

Abstract To understand the physical locations of self-incompatibility (SI) genes and their linkage relationship, this study conducted fluorescence in situ hybridization (FISH) in Brassica oleracea genome using the probes of MLPK and SSP genes on prometaphase chromosomes, early pachytene chromosomes, and extended DNA fibers. The results indicated that the MLPK probe was hybridized onto the short arm of a pair of homologous prometaphase chromosomes, and the average percent distance from the centromere to the signal point was 53.41±3.16. The SSP probe was hybridized onto the long arm of a pair of homologous prometaphase chromosomes with the satellite, and the average percent distance from the centromere to the signal point was 78.36±4.26. Hybridization signals from the 3 types of cytological targets with different FISH resolutions showed that both MLPK and SSP might be located at a single-copy locus in B. oleracea genome. Repeated FISH analysis indicated that both MLPK and 5S rDNA probes were hybridized onto the same chromosomes. According to the karyotype standard, MLPK is primarily inferred on chromosome 2, and SSP on chromosome 7. The results presumably revealed that neither MLPK nor SSP should be linked to S-locus, and they were located on different chromosomes of B. oleracea.