Cloning And Functional Analysis Of Unkown Mitochondria Genes In Brassica Napus L

Mitochondria are a semi-autonomous organelles with their own systems for DNA and protein syntheses in plant cells. Mitochondria can directly involving in the catabolism of fatty acids, oxidate organics and generate energy of ATP, while ATP is the basis for all the plant physiological and biochemical reactions. Functional reseach of mitochondrial genes is a significant exploration prospect for basic and crop improvement research. Currently, the mitochondrial genome of Brassica napus L. have been completed, and a large number of unknown open reading frames were found. But the unknown genes with very important practical significance for rapeseed breeding have not been fully studied.In order to reseach the function of these unknown genes, the following studys were carried out:1. After aligning the mitochondrial genome sequences between pol and nap varieties (Brassica napus L.), fouteen open reading frames (ORFs) were screened out, as ORF117, ORF118, ORF122, ORF123, ORF132, ORF188, ORF286, ORF293, ORF222, ORF224, ORF261, ORF265, ORF305, and ORF322.2. Fourteen fusion genes were obtained by fusing a transit peptide (TP) coding sequence to the 5’end and 6xHis codens to the 3’end of the ORFs. The fusing genes were cloned to the pEarleyGate-100 plasmid to construct the fourteen plant expression vectors harboring the expression cassette 35S::TP-ORF117,35S::TP-ORF118,35S::TP-ORF122,35S::TP-ORF123,35S::TP-ORF132,35S::TP-ORF188,35S::TP-ORF222,35S::TP-ORF224, 35S::TP-ORF264,35S::TP-ORF265,35S::TP-ORF286,35S::TP-ORF293,35S::TP-ORF305, and 35S::TP-ORF322. Meanwhile, a control vector containing 35S::TP-GFP was constructed by replacing the ORFs with the green fluorescent protein gene (GFP). The fusion genes were transformed into Arabidopsis thaliana by Agrobacterium tumefaciens mediated floral-dip transformation.3. Transgenic Arabidopsis plants were screened by using herbicide combining PCR method and total 279 homozygous transgenic Arabidopsis lines were established. In order to explore the genes’functions, we investigated the physiological and biochemical characteristics of some transgenic lines. The results showed that the ORF188 is homologous to a subunit of ATP synthetase. The length of the root with five-day culture, mitochondrial ATP synthase activity and ATP content, seed oil content were longer or higher in transgene overexpressed plants than in control ones. In addition, the transgenic plants were more insensitive to 30μM Cu2+ poison stress than control.4. The transgenic line 35S::ORF222 has roots longer than that of control. On the contrary, the transgenic line 35S::ORF224 is shorter than normal. However, their sensitivities to stress are still unknown.5. We didn’t find the homologous sequences for the other genes. The transgenic linses generated by transformation of these genes don’t show phenotypic change. But we found some mutant Arabidopsis, which is vital for other research.In this work, a preliminary screening and verification of unknown mitochondrial ORFs were carried out, which laid a solid foundation for further research of these genes in the near future.