Research On Heat Tolerance Identification And Cloning Of Heat Tolerant Gene Fragment In Non-Headingchinese Cabbage (Brassica Campestris SSP. Chinensis Makino)

According to the observations under natural high temperature in summer many years, 10 non-heading Chinese cabbages (Brassica campestris ssp. chinensis Makino) materials, that were significant differences in heat tolerance, were used in this research. The research found out a comparatively accurate heat tolerance identification method of non-heading Chinese cabbages seedling; and studied the physiological and biochemical characters and morphology under normal temperature and heat stress of non-heading cabbages at seedling stage and adult stage, and the correlation between the above indexes and the heat injury index of non-heading Chinese cabbages seedling. At same time we also cloned the characterization of BOHSP612 small heat shock protein gene from non-heading Chinese cabbage, which provided an effective tool for the further study of BOHSP612 gene function.The results are showed as flows:1.Heat tolerance identification of non-heading Chinese cabbage at seedling stage10 non-heading Chinese cabbages materials were continuously treated with 6 different high temperatures for 7 days in the artificial climate box in autumn 2009, spring and summer 2010 in order to be identified their heat tolerance.The results indicated that different degrees of heat injure symptoms like leaf backscrolling, chlorosis appeared in all of the tested materials, and the symptoms became severe with the temperature increasing and the time prolongation.The different heat tolerance of non-heading Chinese cabbage could be significantly reflected by the heat injury index at seedling stage with heat treatment under 37℃/27℃(d/n) for four days in spring and autumn.However,37℃/27℃(d/n) treated in summer could not be used by distinguishing the different heat tolerance of non-heading Chinese cabbage which seedlings growed in nature temperature. 2.Heat tolerance identification of non-heading Chinese cabbage at adult stage10 non-heading Chinese cabbages materials above were used in this experiment when they were at adult stage and they were seedinged in the field in spring and summer 2010. After 35 days, we investigated some morphological indexes under normal temperature and heat stress of non-heading cabbages, and the correlation between the above indexes and the heat injury index of non-heading Chinese cabbages seedling. The results indicated that only the width of leaf of all morphologieal indexes at adult stage in spring is signifieantly and positively correlated with the heat injury index at seedling stage, and others show less correlation with the heat-resistant ability. In summer, however, most of all morphologieal indexes at adult stage are positively correlated with the heat injury index at seedling stage except the number of leaves, among them width of plant, length of leaf and width of leaf are signifieantly and positively correlated with the heat injury index at seedling stage, and the length of leafstalk shows a hightly significant positive correlation with the heat injury index at seedling stage.3.The physiological and biochemical indexes on heat tolerance identification of non-heading Chinese cabbageOn the base of the result of the heat tolerance identification of non-heading Chinese cabbage at seedling stage,6 non-heading Chinese cabbages materials were continuously treated with 37℃/27℃(d/n) high temperature for 10 days in the artificial climate box in order to be measured content of chlorophyll, malondiadehyde(MDA) and proline(Pro), activity of protective enzymes and membrane permeability. The results indicated that after heat-shock treatment, the Pro content and activity of protective enzymes in the leaves of heat resistant varieties had a higher increase than in the heat sensitive varieties, and the different heat tolerance of non-heading Chinese cabbage could be significantly reflected by the these indexes with heat treatment for 4 (Pro),6 (SOD, CAT),8 (SOD, POD) days; the increase of MDA content and membrane permeability was higher in the heat sensitive varieties than in the heat resistant varieties, and the different heat tolerance of non-heading Chinese cabbage could be significantly reflected by the these indexes with heat treatment for 6 (membrane permeability),8 (MDA) days. Meanwhile, the content of total chlorophyll and chlorophyll-a in the leaves of heat sensitive varieties had a higher decrease than in the heat resistant varieties, and the different heat tolerance of non-heading Chinese cabbage could be significantly reflected by the these indexes with heat treatment for 4 (chlorophyll-a),6 (total chlorophyll) days. But in the research, the change rule of chlorophyll-b is very complex, and shows less correlation with the heat-resistant ability.4.Cloning of heat tolerant gene specific fragment of non-heading Chinese cabbagesAccording to the consensus domain of brassica oleracea HSP gene, a gene fragment DNA of heat shock protein named BOHSP612 were cloned from Brassica campestris ssp. Chinensis cultivar'Gaohuaqing'. The BOHSP612 gene contained 612 nucleotide. BLAST analysis of its nucleotide sequence found a high homology with the portion of nuclear genome of the other crops of Brassica rapra, Arabidopsis thaliana, Soybean and Peanut, heat shock associated protein mRNA, hypothetical protein mRNA and pseudo gene.