Optimization Of A Novel Transformation System Of Brassica Napus And Transformation Hemoglobin Gene SLR2097 Into Brassica Napus

Brassica napus is one of the most important oil crops.Conventional breeding has made tremendous contribution to the improvement of economic trait.The increasing demand on production of diferent oils such as edible oil,industrial oil needs new technology in rapeseed breeding to further enhance both the productivity and adaptability of new cultivars.So it was necessary to adopt new technology to transfer the new genes into rapeseed.Genetic engineering offered new possibilities to improve the crop in various special characters that were difficult to obtain by traditional breeding procedures.Important traits,such as rapeseed yield,quality,disease and insect tolerance, male sterility,and the other special characaters,have been introduced into the crops by genetic transformation,and genetic transformation was also used to detect the function of different genes.However,it was very difficult to use the present Agrobacterium-mediated transformation in many genotypes because there existed serious low transformation frequency,low reproducibility and long-term transformation. Therefore,it is essential to found an efficient Agrobacterium-mediated transformation system that has short period.Hemoglobin existed in most organisms including animals,plants,protozoa,algae, bacteria and fungi.Hemoglobin in animal can bound and transport oxygen in order to improve provision of oxygen,fluence of electrons in respiratory chain,vigor of ATPase; and hemoglobin in microbacteria can promote growth of microbacteria,synthesis of protein and production of metabolite.Recently,a paper reports that Vitreoscilla hemoglobin can enhance growth of tobacco,the chlorophyll content and the nicotine content of it.Using cotyledonary nodes as explants,an efficient system for Agrobacterium-mediated transformation of Brassica napus cv."Ningyou12" was developed through optimizing the infection conditions and the concentrations of phytohormone.And then hemoglobin gene SLR2097 of Synechocystis was transformed into Brassica napus with the optimized transformation system in order to research the influence of SLR2097 gene on Brassica napus cv."Ningyou12".The major study contents and conclusions are as follows:1.Cotyledonary nodes of Brassica napus cv."Ningyou12" were infected with Agrobacterium tumefactiens containing expression vector pCAMBIA1303-GFP in order to optimize genetic transformation system.Different combinations of optical densities and infection durations and different concentrations of acetosyingone and silwet L77 were evaluated for the efficient infection of Agrobacterium.Maximum transformation frequency was 6.96%when cotyledonary nodes were infected for 10 min with Agrobacterium suspension of which the OD was 0.8 and then co-cultivated on the medium with 200μM acetosyingone and 0.02%silwet L77.In addition,concentrations of 6-benzyladenine and naphthalene acetic acid were also optimized.Maximum green plantlet frequency was 94.38%when cotyledonary nodes were cultivated on the medium containing 4 mg/L 6-BA and 0.04 mg/L NAA after co-cultivation.Evidences for transformation were confirmed by PCR analysis,Southern blotting analysis.The expression of GFP gene in transgenic plants was also observed with fluorescence microscope.2.The literatures indicated that hemoglobin can improve growth of transgenic tobacco and increase the chlorophyll content and the nicotine content of transgenic tobacco.The open reading frame of hemoglobin gene SLR2097 was cloned from genomic DNA of Synechocystis sp.PCC 6803.Sequence analysis indicated that the open reading frame of hemoglobin gene SLR2097 was only 375 bp,which encoded a 123 amino acid polypeptide chain belonging to the truncated hemoglobin family. Molecular weight of its deduced protein was 13.9kDa and theoretical pI of its deduced protein was 5.27.Multiple sequence alignment demonstrated that hemoglobin gene of Synechocystis shared low sequence identity with another twelve organisms,and there was only a common motif 'F-[L]-x(4)-[G]-G-[T]-x(2)-[Y]-x-[G]-[R]-x-[M]-x(3)-H' among thirteen species.3.Expression vector pCAMBIA1300-CaMV35S was constructed successfully. And then SLR2097 was ligated into the vector pCAMBIA1300-CaMV35S.Finally,the pCAMBIA1300-CaMV35S containing SLR2097 was transformed into A.tumefactiens strain LBA4404.The hemoglobin gene SLR2097 of Synechocystis was transformed into Brassica napus with the optimized transformation system.And transgenic plants were identified with PCR analysis.Phenotype analysis of transgenic Brassica napus indicated that exogenous expression of hemoglobin gene SLR2097 led to the earlier maturity.