Variance Analysis Of Stamen Petalody Mutation And Its Maintainer Line In Non-Heading Chinese Cabbage

Non-heading Chinese cabbage(Brassica campestris ssp. chinensis Makino), is originated in China, and is one of the most favorite vegetables in Asia. In recent years, the demand of non-heading Chinese cabbage is growing. However, it’s the toughest and the most urgent task for the breeders to breed fine cultivars of non-heading Chinese cabbage, because its weak duration in storage, frangible in transportation and absence in special cultivars, Molecular marker assisted breeding technology is one of the immidate, convenient and labor saving tools, by which we can screen majority of traits in almost every stage to help breeders find out useful genes. Petalody stamens causes one entirely male sterility type in non-heading Chinese cabbage. This can be a good research material for the breeders, and can be of great value in cross breeding in non-heading Chinese cabbage.In this paper, the stamen-petalody line W053and its maintainer line W052of non-heading Chinese cabbage were used as experiment material. Firstly, physiological characteristics of the two materials were determined. This established a base for further research on the molecular mechanism of the stamen-petalody characteristic in non-heading Chinese cabbage; and then we explored the differences between the two materials at molecular level through the cDNA-AFLP technique, to establish the cDNA-AFLP protocol, which was suitable for the stamen-petalody line of non-heading Chinese cabbage; Finally, we tested and verified the expression of differential gene through the fluorescence quantitative PCR technology. The results were summarized as follows:1. Contrast analysis on physiological characteristics between stamen-petalody line and its maintainer lineDistinct organs at early flowering stage of the two lines W053and W052were used to test the contents of proline, soluble sugar, soluble protein and activities of SOD, POD, CAT. The results indicated that soluble protein and sugar contents was the highest in flower, then in big buds, then in small buds, and the lowest in leaves, and the contents in maintainer line were markedly higher than those in mutant. Proline content in big buds was the highest, then in the flower and then in small buds, and the lowest in leaves. Proline contents in flower buds and leaves in maintainer line were higher than those in mutant, but lower than the mutant in flower. The activity of SOD activity in mutant was markedly higher than the one in maintainer line, and both showed highest in flower, and then in big buds, and then in small buds, and that in the leaves was the lowest. The activities of POD and CAT activities in mutant were significantly higher than those in maintainer line, and showed the highest in buds, and then in flower, while showed the lowest in leaves in both materials.These results suggested that there were significant differences in physiological characteristics between stamen-petalody line and its maintainer line of non-heading Chinese cabbage.2. Analysis of the differentially expressed genes between the stamen-petalody line and its maintainer line by cDNA-AFLPThe young flowerbuds that were detached at early flowering stage in sunny morning. A cDNA-AFLP (complementary deoxyribonucleic acid amplified fragment length polymorphism) technique was employed to find the polymorphisms of transcriptional profiling of flower bud between the two materials. The specific process of this technique is as follows:1) Extraction of the plant total RNA and the synthesis of the double strands cDNA;2) Digestion of the double strands cDNA fragments and link to the specific synthetic adaptors;3) Pre-amplification of the digestion fragments and selective amplification;4) Polyacrylamide gel electrophoresis (PAGE) of the selective amplification products.The results showed that12pairs of primers did not obtain band in the chosen64pairs of selective amplification primers. There were1346transcript-derived fragments between100bp-1000bp amplified in two lines with52pairs of primer combinations. Eight polymorphic loci in two lines and averagely26bands per primer combination were amplified.Then, we established the cDNA-AFLP protocol which was suitable for gene differential display of non-heading Chinese cabbage stamen-petalody line. 3. Expression analysis of the gene related to stamen-petalody mutation in non-heading Chinese cabbageRoot, stem, leaf were sampled at the seedling stage, flower-bud differentiation stage, squaring stage and flowering stage, respectively. Leaf, small flower bud, big flower bud and flower were specialized sampled at the early flowering stage. Total RNA was extracted and single-strand cDNA was synthesized. The cDNA product was used for template in the experiment. Fluorescence quantitative PCR method was used to study the expression level of the differential fragment ERFB001which was recovered from the polyacrylamide gel electrophoresis (PAGE), and gas chromatography method was used to test the production of ethylene, the activities of ACC synthase and ACC oxidase.Bioinformatics analysis indicates that the differential fragment ERFB001was highly identical to an ethylene response transcription factor ERF109in Arbidopsis thiliana. The fluorescence real-time quantification PCR results showed that there was no remarkable difference in expression level of the differential gene between stems and roots at seedling stage, flower bud differentiation stage, squaring stage and flowering stage, but it was slightly higher in leaves. The expression level of the differential fragment ERFB001in distinct organs at early flowering stage was the highest in big buds, then in small buds, then in flowers and was the lowest in leaves, and the expression level in mutant were markedly higher than those in maintainer line.The production of ethylene continued to rise in mutant, but the change in maintainer line was unconspicuous, and the productions in mutant were markedly higher than those in maintainer line. The activity change of ACC synthase and ACC oxidase was similar to production of ethylene, and to the maximum in flower.These results indicated that the differential fragment ERFB001may be an acceptor for the ethylene, which can be stimulated by the ethylene, and regulate the expression of the downstream genes, and thus involved in the regulation of adaptbility of the stamen petalody line under low carbohydrate and high oxidation level for it’s internal environment.