桃SFB2m参与S-RNase泛素化降解途径

Peach SFB2m participates in the ubiquitination degradation of S-RNase

【目的】自交亲和特性的产生可由多种因素导致,其中S-RNase被泛素化标记后移动到26S蛋白酶体中被降解这一途径,是显现自交亲和的重要原因。探究桃SFBs在S-RNase泛素化降解过程中的作用,为桃自交亲和机制的研究提供参考。【方法】通过生物信息学方法对PpSFBs和PpSLFLs进行基因定位和共线性分析,使用PCR确定PpSFBs在花器官中的特异表达位置,利用BiFC验证PpSFBs与S-RNase的互作后,通过S-RNase体外泛素化实验和寡核苷酸转染沉默PpSFBs实验来探究PpSFBs在S-RNase泛素化降解途径中的作用。【结果】PpSFBs和PpSLFLs无共线性关系;PpS1/2/4-RNase在花柱中特异性表达,PpSFB1m/2m/4m在花粉中特异性表达,PpSLFL1/2/3在不同桃品种中差异性表达,PpSLFL2在龙1-2-4品种中不存在;通过BiFC技术证明,PpS1/2/4-RNase分别与PpSFB1m/2m/4m和PpSLFL1/2/3互作。体外泛素化实验证明,PpSFB2m具有与PpSLFL2相似的功能,能泛素化PpS2-RNase。寡核苷酸转染实验证明PpSFB1m/2m/4m和PpSLFL2/3沉默后可显著抑制花粉管生长。【结论】PpSFB2m在桃花粉管中发挥与PpSLFLs类似的功能,可泛素化标记S-RNase,泛素化的S-RNase移动到26S蛋白酶体中被降解失活,致使桃自交亲和性状的产生。

【Objective】In flowering plants,the presence of self-incompatibility mechanism allows plants to avoid self-pollination for fruit set and therefore obtain higher genetic diversity,thus ensuring population continuation in the face of complex and variable environments.A variety of fruit trees in the Rosaceae family showed self-incompatibility,such as apple,pear and apricot.In contrast,diploid peach,which belongs to the same family Rosaceae,shows significant self-compatibility in production,and the mechanism behind this remains partially blank.In this study,we investigated the role of peach SFBs in the degradation of S-RNase ubiquitination to provide a reference for the study of peach self-compatibility mechanism.【Methods】Gene localization and synteny analysis of PpSFB1m/2m/4m and PpSLFL1/2/3 were performed by bioinformatics.The genome annotation file of Ppersica_ChineseCling_v1.0 was obtained from the Rosaceae Genome Data GDR(https://www.rosaceae.org)website.Blastp(v2.10.1+)was applied to match the 6 genes to the genome as well as the 6 genes for self-matching,respectively,with a threshold control E-value<1e-5,the best match for each gene was obtained.The gene localization display of the best matches of the genes on the genome was made using TBtools(v1.108).The selfcomparison screening bit score>100 was the result of having synteny and applying shinyCircos(v2.0)for visualization;PCR reactions of pollen,style and petal cDNAswere peformed using specific primers for PpS1-RNase,PpS2-RNase,PpS4-RNase,PpSFB1m,PpSFB2m,PpSFB4m,PpSLFL1,PpSLFL2 and PpSLFL3.The amplification products were obtained and the obtained PCR products were subjected to agarose gel electrophoresis to detect gene expression,and peach Actin gene expression was used as a control.For example,PCR was performed on pollen cDNA of Long 1-2-4 using the PpSFB1m specific primers,and the amplified products were obtained and subjected to agarose gel electrophoresis.The PpSFB1m was considered to be expressed in Long 1-2-4 pollen if bands of the corresponding size were present in the agarose gel plots;We used BiFC to verify whether PpSFBs and S-RNase have an interaction relationship,and explore whether PpSFB2m can ubiquitously label PpS-RNase in vitro assay;and the oligonucleotide transfection was used to silence PpSFBs to explore the important role of the PpSFB1m/2m/4m in the elongation of peach pollen tubes.【Results】The main results of this study were as follows:the PpSFB1m/2m/4m were localized on chromosome 3,the PpSLFL1 was localized at the end of chromosome 2,the PpSLFL2 and the PpSLFL3 were both localized at the end of chromosome 6,and the two gene positions were adjacent to each other,and the six genes of the PpSFB1m/2m/4m and the PpSLFL1/2/3 were analyzed for synteny,and the results showed that there were three fragment replication genes,located on chromosome 2 and chromosome 6,respectively,and there was no synteny between the PpSFB1m/2m/4m and the PpSLFL1/2/3.Structural domain analysis of the six genes showed that all the six genes had F-box structural domains,which might perform similar biological functions;To clarify the gene expression patterns of PpS-RNases and PpSFBs and PpSLFLs,three varieties Long 1-2-4(S1S2),Fei Cheng Hong Li(S2S4)and Bai Nian He(S2S2)were selected for experiments in this study,and RNA was extracted from the styles,pollen and petals of the three varieties and reversed to cDNA.Afterwards,the presence of gene expression was detected using PpS1-RNase,PpS2-RNase,PpS4-RNase,PpSFB1m,PpSFB2m,PpSFB4m,PpSLFL1,PpSLFL2 and PpSLFL3 specific primers.PpS1/2/4-RNase was specifically expressed in the style,the PpSFB1m/2m/4m were specifically expressed,the PpSLFL1/2/3 differentially were specifically expressed in different peach varieties.It is worth noting that the PpSLFL2 was absent in Long 1-2-4 variety;It was demonstrated by BiFC technique that PpS1/2/4-RNase interacted with PpSFB1m/2m/4m and PpSLFL1/2/3,respectively.PpS1-RNase interacting with PpSLFL3,PpS2-RNase interacting with PpSLFL1/2,PpS4-RNase interacting with PpSFB2m/4m showed very strong interactions.In vitro ubiquitination experiments showed a significant drag band of PpS2-RNase when PpSLFL2 was added compared with the control,which represented PpS2-RNase being added with different lengths of ubiquitin.Similarly,PpS2-RNase showed a similar drag band phenomenon when PpSFB2m was added,implying that PpSFB2m ubiquitinated PpS2-RNase,demonstrating that PpSFB2m had a similar function to PpSLFL2 and could ubiquitinate PpS2-RNase;In this study,we designed and synthesized antisense oligonucleotide chains of the PpSFB1m/2m/4m as well as the PpSLFL1/2/3,and used the sense oligonucleotide chains of the PpSFB1m/2m/4m and the PpSLFL1/2/3 as controls for gene transient silencing of trhe peach pollens of the different varieties,followed by incubation using self S-RNase.The experimental results showed that the growth of peach pollen tubes lacking the PpSFB1m/2m/4m and the PpSLFL2/3 gene expression was significantly inhibited compared with the S-ODN(sense oligonucleotide chains)treated group and CK,indicating that the PpSFB1m/2m/4m and the PpSLFL2/3 would play important roles in the growth of peach pollen tubes.【Conclusion】The PpSFB2m could also function as part of the SCF complex similar to the PpSLFL,recognizing PpS-RNase and mediating its ubiquitinated degradation,and this gene would play an important role in peach pollen tube elongation.More in-depth studies are still needed to elucidate the mechanism of peach self-compatibility.