| Plant cytoplasmic male sterility (CMS) is a widespread phenomenon in flower plants, which is often used in producing hybrid seeds. Most studies have established that this maternally inherited defect is often associated with mitochondrial genome rearrangement. Although approximately 10 ORFs have been identified to be associated with CMS systems in the family of Brassicaceae, only several direct evidences could prove that these genes caused male sterility. Pol CMS has been regarded as the first and with great practical value CMS in Brassica. However, few studies were done on the molecular mechanism of sterility and restoration of fertility compared with other crops such as rice, maize.In this study, the expression of pol CMS associated genes and fertility restoration-related genes were determined by quantitative real-time PCR, ectopic and over-expression to investigate their possible functions in the development of male reproductive organs.First, the full length of orf224 and 3’ truncated region were cloned into pET32a. The growth of recombinant E.coli BL21 was repressed in the expression of 3’-region of orf224 with IPTG; while the growth was quite normal in the expression of orf224. This finding indicated that 3’-region of orf224 may encode a toxic peptide that is toxic to host bacteria. To further confirm the effect of orf224 on the development of male organs, we constructed vectors with full length of orf224 and the 3’-region fused to a mitochondrial transit peptide sequence respectively. The promoters we used were 35S and the APETALA3 of Arabidopsis. After transformed into Arabidopsis thaliana (Col-0), a lot of transgenic plants had been obtained and the male fertility needs to be determined.The subcellular localization of ORF224 and the N-terminus were targeted to the mitochondria and nucleus, and the C-terminus of ORF224 was mainly localized in the cytoplasm, with no signal in the nucleus and organelle. ORF224 has two transmembrane domains, and the N-terminus of ORF224 has a mitochondria signal anchor peptide. Without N-terminus, ORF224 cannot effectively locate to the mitochondria. The result of the subcellular localization of PPR agreed well with theoretical prediction. PPR mainly localized in the cellular matrix, mitochondria, and some unknown cell organelles. In addition, there was weak signal in the nucleus.qRT-PCR was performed to detect transcriptional differences of pol CMS fertility-related genes in 92A (sterile line),92B (maintainer line),09-399 (restorer line) and F1. The results showed that orf224 co-transcribed with the atp6 gene in 92A and F1 in the different period of the pollen development. However, there was more complete co-transcript in 92A than in F1 (About 40% was reserved at maturity stage in 92A, only a quarter of it in F1). These data showed that orf224-atp6 co-transcript largely degraded in F1 due to the present of Rfp in the pollen development.Comparising the sequence of fertility restoration-related PPR genes from 92A, 92B and 09-399, there were 9 SNPs in 09-399 and other lines that influenced 8 amino-acids in coding information. The over-expression vector of PPR gene from 09-399 was constructed and transformed 92A after pollinated with 92B by Agrobacterium-mediated infection. The fertility restoration status of transgenic plants needs to be identified.This work concentrates on pol CMS and fertility-related genes in Chinese cabbage. The preliminary result shows that the expression difference and stability of orf224-atp6 in 92A and F1 may be the main cause of male sterile. Fertility restoration-related PPR gene in 09-399 with 9 SNPs may greatly influence target sites and activity in cleavage of orf224-atp6 transcript, but these remain further investigations. |
PDF Full Text Request