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Kenaf is one of the important fiber crop, its good fiber quality and a wide range of uses, has important application value, therefore be regarded as a multi-purpose crop of potential development in the 21st century. And other crops, the use of cytoplasmic male sterile line to cultivate kenaf hybrids become the main research direction of kenaf breeding. Cytoplasmic male sterility (Cytoplasmic Male Sterility, CMS) crops crop utilization of heterosis, the CMS mechanism is very complex, so the CMS has also become a hot and difficult issues of domestic and foreign scholars. A large number of studies have shown that the cytoplasmic male sterility and mitochondrial DNA variation are closely linked. The work of our group pre by SRAP markers confirmed mitochondrial nad gene fragments in Sterile and Maintainer Lines differences. To the relationship between the better of cytoplasmic male sterility and mitochondrial DNA, the kenaf sterile line P3A and corresponding maintainer lines P3B experimental material to establish a method for separation of high-quality kenaf mitochondrial DNA, focusing on cloning may kenaf cytoplasmic male sterility related genes nadl, nad2 nad3; semi-quantitative RT-PCR analysis of gene nadl-9; conducted a preliminary exploration of the kenaf regeneration system to establish the main results are as follows: (1) red ephedra of seedlings material, combined with the density gradient centrifugation and by differential centrifugation method of extracting the mitochondrial DNA (mtDNA), the results show that the sucrose density gradient centrifugation method than the Percoll density gradient centrifugation method is more suitable for the separation of kenaf mitochondria. MtDNA extracted by 1.0% agarose gel electrophoresis, and ultraviolet spectrophotometer detector and design chloroplast and nuclear specific gene primers detecting whether or not the presence of contamination, the results show that this method of extraction of mtDNA high purity, there is no pollution. (2) the nad genes conserved sequences degenerate primers based on the NCBI database using PCR amplification Sterile and Maintainer Lines were obtained nad1 nad2, nad3 genes conserved sequence, length 817bp, 1052bp, 235 bp, the the gene sequence of these fragments, and sugarbeet and bamboo, tobacco, watermelon than their homology respectively 98.2%, 96.7%, 97%, and the amplified fragment of the correct fragment. And the same gene sequence comparison between the two lines, individual bases. Nad1 gene two different bases, respectively P3A 115 by A → G 411 A → G, P3B deletion of an A in the first 247 nucleotides, the two series 99.6% homology . nad2 gene in P3A 189 missing a T base by G → A 206, 477 A → G, 863 A → G, 936 by the C → T homology 99.5%. And nad3 gene is only one base difference, i.e. 94 G → A homology of 99.6%. Semi-quantitative RT-PCR analysis of the draw, the nad1, nad2 nad3 nad4 nad5 gene expression levels did not differ significantly between the two lines. nad4L, nad6 CMS lines higher expression levels remained relatively Department. nad7, nad9 sterile line of lower expression levels remained relatively Department. (3) Kenaf regeneration system to establish preliminary studies, the culture conditions of 25 ± 1 ℃, illumination time 14 h / d, light intensity the 21001x about conditions, kenaf embryo explant material induced callus suitable medium formula: processing A6: MS 0.5mg/L6-BA 1.0 mg ,4-D can induce strong callus, its amount, yellow-green, tight texture, callus induction frequency of up to 100%. Processing callus differentiation medium MS basic medium, add 0.5-2.5mg/mL 6-BA and 0.1-0.5mag/mL the NAA, along with the increase in the concentration of 6-BA, NAA lower fewer points higher concentrations increase in Green Point, 6-BA and NAA hormones with induced differentiation of cells, enough to deal with the B20 optimum differentiation medium: MS 2mg/L6-BA 0.5mg/LNAA to be further tests, but to induce adventitious buds rates and ratios.
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