The Interaction Study Between Plant U-box Protein And S-locus Receptor Kinase Protein Based On Transcriptome Analysis In Brassica Oleracea L.

Self-incompatibility(SI) is one of the important mechanisms to prevent selfing, promote hybridization, and maintain genetic diversity of species. Brassica crops such as Brassica oleracea L. is sporophytic self-compatibility, which is determined by multiple alleles of S-locus. When the pollen falling on the stigma, the SCR in the pollen can be recognized and combined with extracellular domain of SRK specifically, causing transformation of SRK conformation and autophosphorylation, and release the Thioredoxin-H-like-1/2 combined with the kinase domain of SRK in the cell. Then ARC1 combines with SRK, and be phosphorylated. Being an E3 ubiquitin ligase, activated ARC1 can transmit the cell signal though a series of pathway, which leads to germinate blocked of the self-pollination.Studies have shown that antisense inhibition of ARC1 gene expression can partly broke the self-incompatibility in B. napus and A. lyrata. ARC1 gene was found deleted in A. thaliana though gene analysis. And then A. thaliana with SCR-SRK transgenes are rendered fully self-compatible. Maybe ARC1 isn`t necessary for Self-incompatibility, some proteins in the stigma cell can interact with SRK, and transmits SI signal instead of ARC1.While self-pollinating, the activity of PUB proteins in the stigma had risen sharply. So the unknown substrate protein interacted with SRK could be a member of plant U-box family. We chose A4 type of Brassica oleracea L. for materials, at the florescence we got the c DNA of the self-pollination 0 min stigma and self-pollination 30 min stigma. Then 25 PUB genes were found by transcriptome technology, their expressions are apparently different after self-pollination. 4 genes named as BoSU03、BoSU05、BoSU07、BoSU11,are choson for further study. The gene expression level is cahnged after self-pollination though qRT-PCR. We designed the primers according their homologous genes, then constructed their T cloning vector. In the study, yeast two-hybird system and GST pull-down are utilized to analysis the interaction of the PUB proteins and SRK. The main research results are as following:(1)Screening PUB proteins though technology of transcriptome technology and qRT-PCRThe expressions of BoSU03、BoSU05、BoSU07 increased, though BoSU11 decreased in the analysis of transcriptome technology. The expressions of BoSU03、BoSU05、BoSU07、BoSU11 had decreased wia qRT-PCR. The reason could not be determined, but it shows that the changes of gene expression level are related to self-pollination.(2)The gene clones and gene analysis of PUB proteinsAfter Bioinformatically analyzing PUB genes sequences, BoSU03 、 BoSU05 、BoSU07、BoSU11 are cloned. The primers of PUB are added with restriction digest sites, so that the sequence can get rid of U-box domain, and keep ARM domain.Though bioinformatically analyze the PUB genes, PUB proteins don`t contain any transmembrane domain or nuclear localization signal(NLS). There are ARM repeats in the C-terminal just like ARC1. And the PUB proteins contain a lot of phosphorylation sites, such as cAMP and cGMP dependency phosphorylation site, protein kinase C phosphorylation site and casein kinase Ⅱ phosphorylation site. Constructed phylogenetic trees, and found that PUB gene and ARC1 both belong to plant U-box family with close relationship.(3) Identify of the interaction between PUB protein and SRK with yeast two-hybrid systemBuilded yeast expression vectors of PUB genes 、 ARC1 and SRK: pGADT7-BoSU03 、 pGADT7-BoSU05 、 pGADT7-BoSU07 、 pGADT7-BoSU11 、pGADT7-BoSU12、pGADT7-BoSU13、 pGADT7-ARC1、pGBKT7-SRK. Though the text of toxicity and autoactivation detection, these yeast expression vector are not toxic to yeast cell, and failed be able to activate the expression of reporter genes. The co-transformed yeast AH109 on the plant of SD/-Leu/-Trp and SD/-Ade/-His/-Leu/-Trp/x-α-gal/25 mmol 3-AT, could grow and change its color to blue. It shows that the interaction between PUB protein and SRK.Yeast β-Galactosidase activity of transformed yeast can be quantitative measured by Yeast β-Galactosidase Assay Kit, the value respected the intensity of interaction. The values of BoSU03、BoSU05、BoSU07 and BoSU11 are 15.37 Miller Units、12.71 Miller Units、15.36 Miller Units、15.61 Miller Units, much higher than the value of negative control.(4)Identify of the interaction between PUB protein and SRK with GST pull-downTake advantage of the characteristic of 6×His tag of fusion protein combined with Ni+, the purified fusion proteins pET43.1a-BoSU07 and pET43.1a-BoSU11 respectively were incubated with pGEX-4T-1-SRK, fished interacting proteins of SRK, and established a new method for separation of the MLPK interacting proteins in vitro. The result of incubation products by SDS-PAGE electrophoresis showed that compound is eluted by Ni+ though the interactions between pET43.1a-BoSU07、pET43.1a-BoSU11 and pGEX-4T-1-SRK.In the conclusion, BoSU07、BoSU11can both interact with SRK. Whether these PUB proteins is the downstream signaling proteins needs more further study.Screening PUB proteins which are the downstream signaling proteinsof SRK, could complete the signal transduction pathway of SI, and have important meaning for SI mechanisms.