Study On Buchloe Dactyloides Sexaul Reproductive Characteristic And Cloning Of DREB Trascription Factor Involved In Drought Resistance

Buffalo grass (Buchloe dactyloides) is one extensive managing turf grass that has strong resistance against adverse environment. The 20th century 40's, this grass was introduced as the conservation of water and soil plant from US to GansuTianshui, displays a better compatibility. At present, buffalo grass variety comparatively sole in our country, the seed output lower, the major part seed dependence from the overseas, and the seed price extremely expensive. People use the vegetative reproduction method to construct the lawn usually, but require a lot of work time-consuming, the long-distance and range application extremely difficult, therefore breeds new variety that the green time to belong, the seed output high is the research main direction. In addition, fully uses this resistance strong plant resources thoroughly to develop the molecular mechanism which the plant arid forces and to combat drought the gene separation and the clone, enhances the arid patience and the compatibility of plant, regarding the preventing and controlling loess plateau soil erosion, protect the ecological environment, as well as enhances the ecological agriculture technology. There has vital significance in the theory and the practical.Studied the flower organ shape, blossomed habit, acceptation of stigma of Buchloe dactyloides and the influence on the seed production with different manage measure. The result indicates, the flower organ of male and female of buffalo grass differs greatly on the external shape, characteristics of blooming is different, but meet basically in florescence; The acceptation of female stigma is strongest in the latter 10 hours to blossom, after 24 hours weaken gradually; The artificial wrap bag pollination should choose in beginning of blooming period, also carries on in the morning, thus can obtain the high yield and good quality of seed; Applies fertilizer and irrigates should choose in the female earing earlier period and the earing period, blooming period not suitably carries on.For the purpose of selecting new species of Buchloe dactyloides that have high value in seed production, sod production and cross breeding, ten grass breeding materials and three hybrid species of Buchloe dactyloides that with different characteristics were comparatived. Testing indexes including of reproduction phenophase, appearance of vegetative and flower organ, pollen quality, seed yield, sex differentiation ratio of F1 generation, turf quality and stolon spreading rate. The result indicates, the seed quality is fine of female NO.1, and the yield is relatively high, so can as the maternal material of the Buchloe dactyloides seed selection. Pollen quantity of the male NO.1 is large, and pollen quality is good, so can be regarded as the male parent material of the crossbreeding. The female NO.3, NO.5, NO.6 and the polygamy can be regarded as the new variety of vegetative propagation and sod production. The heterosis of three hybrid species is obviously, but there have different in sex differentiation ratio among them.In present study, we obtained a DREB gene from Buffalo grass by RT-PCR using degenerate primers and RACE. In this study, by the multiply alignment of amino acid sequences and nucleotide acid sequences of several plant DREB in GenBank, a pair of degenerate primers was designed against the conserved regions that was used for RT-PCR to amplify a 209bp cDNA segment from the Buffalo grass. Base on the sequence of the cDNA segment, primer was designed for 3'RACE and a 653bp coda segment was acquired; from the sequence of 3'end of the gene, primer was designed for 5'RACE and a 426bp cDNA segment was acquired. The full-length cDNA was gained by overlapping sequences and the analysis of the sequence indicated that it contained an open reading frame, comprising 254 amino acid with predicted molecular mass of 38.75 kDa and isoelectric point 5.85 was obtained. The amino acid sequence compared by Blast revealed high homology with that of other plant DREB, and the similarity to DREB of Cynodon dactylon(AAS46285)was the highest with 89%. The result indicated the gene cloned from Buffalo grass is a DREB gene, which we have named as BdDREB2. The GenBank accession number is EF512460.Semi-quantitive RT-PCR was performed to reveal transcript level of BdDREB2 in different tissues and under different abiotic stresses. The results indicated BdDREB2 is abundant in root, rather than in leaf and stem, furthermore, the transcript level of BdDREB2 was up-regulated by 20% PEG and 3% NaCl and reached its peak after 6 hours, but cold treatment could not induce.The coding region of BdDREB2 was inserted into the expression vector pBI121 and the re-constituted vector was named as pBI- BdDREB. Then pBI- BdDREB was introduced into Agrobacterium tumefaciens LBA4404. Tobacco leaf discs were transformed with LBA4404 containing recombinant plasmid. The results of PCR identification of regeneration plants showed that some tobacco plants had the positive bands. In addition, the results of RT-PCR showed that the DNA of BdDREB2 could be transcribed into mRNA in positive transgenic tobacco.