Bioinformatics Screening And Verification Of Candidate Restorer Gene In New CMS Lines 1193A With Sinapis Arvensis L. Cytoplasm Of Brassica Napus L.

Cytoplasmic Male Sterility(Cytoplasmic Male Sterility,CMS)is the most effective and convenient way to use rapeseed heterosis,and it is of great significance to the production and utilization of rapeseed.At present,a large number of studies have shown that the cloned restore genes are basically members of the PPR gene family,with PPR motif.According to the characteristics of restore gene,in this study,the PPR gene family of Brassica napus was screened and classified by bioinformatics,and the clustering,chromosome distribution,subcellular localization prediction and functional annotation were analyzed.Combining molecular markers and bioinformatics analysis candidate restore gene was screened of the CMS with Sinapis arvensis L cytoplasm,and cloned in the sterile line and restorer line.The difference of spatiotemporal expression of the fertile gene in the sterile line and restorer line was analyzed by qRT-PCR.The main results obtained are as follows:1079 PPR family of protein sequences were obtained from the genome of Brassica napus by Hidden Markov model and the Arabidopsis thaliana PPR family model was used to classify them.Brassica napus PPR protein is divided into two subfamilies: P subfamily and PLS subfamily,PLS subfamily is divided into PLS,E,E + and DYW four categories,of which PLS subfamily type accounted for 68% of all BnPPR protein.The multiple sequence alignment and cluster analysis of the BnPPR protein showed that it had complex sequence composition.The PPR domain exhibits a high degree of conservatism,the composition of non-PPR domains is very different.The chromosomal localization analysis of the BnPPR gene showed that the total number of PPR genes in the A subgemone of the Brassica napus L.was approximately equal to that of the C subgenome,but there are some differences in the number of PPR genes on different chromosomes.The subcellular localization of BnPPR protein showed more than 75% of the BnPPR protein was localized in a semi-autonomous organelle,more than 15% are located in the cytoplasm,very few in the nucleus and other endoplasmic reticulum,in which,the total number of BnPPR proteins on mitochondria and chloroplasts was about 800.Functional annotations of the BnPPR gene family show that the BnPPR gene family has complex biological functions.In the biological process,BnPPR gene mainly involves the biological cell processes,metabolic processes,development,reproduction and cell structure and so on;Molecular function annotations show that the most important molecular function of the BnPPR gene is manifested in binding activity and catalytic activity.Combined the results of previous studies with bioinformatics analysis,the potential recovery gene BnPPR_C09 of CMS lines 1193 A with Sinapis arvensis L cytoplasm was screened.2514 bp cDNA fragments,BnPPR_C09b and BnPPR_C09a were amplified by molecular cloning in the cytoplasmic male sterile line and restorer line.Sequencing revealed that BnPPR_C09b appeared a single T-base deletion occurred at 1725 bp,resulting in frameshift mutations in subsequent sequence.The results of bioinformatics analysis showed that protein BnPPR_C09b encoded lacks 238 amino acid residues than BnPPR_C09a,which contains the ATP13 structure that plays an important role in mitochondrial structure and function,suggesting that pollen abortion of 1193 A is due to early termination of protein translation in the CMS.BnPPR_C09b and BnPPR_C09a were able to transcribe RNA,and have protein coding activity,BnPPR_C09b encoded fusion protein size of about 65 KDa,which was smaller than BnPPR_C09a protein 95 KDa,indicating that BnPPR_C09b gene encoded protein was terminated prematurely.The results of the spatio-temporal expression analysis of BnPPR_C09 gene in various tissues of the CMS and restorer lines showed that the expression of the gene in the reproductive organs was higher than that of the vegetative organs,the gene expression of the male sterile line is higher in the vegetative growth process and the flower,while the expression level in young buds of restorer lines was higher than that of male sterile lines.