关键词: 野牛草, 有性繁殖, DREB, 基因克隆

Abstract: Buffalo grass (Buchloe dactyloides (Nutt.) Engelm.) is an extensive managing turf grass that has strong resistance against adverse environment. In the 20th century 40’s, this grass was introduced as the plant for water and soil conservation from US to Tianshui, Gansu Province and displayed good compatibilities. At present, the variety of buffalo grass is comparatively simplex in our country, the seed output is low and the major amount of seeds are dependent on the import from overseas, and the seed price is extremely expensive. The vegetative reproduction method is usually used to establish the lawn, but it requires a lot of time-consuming works and is not valid for the long-distance application. Therefore, it is a main research direction to breed new varieties with longer green time and higher seed output. In addition, fully use of the strong drought resistance characters of this plant resource and its molecular mechanism as well as the related gene separation and clone would enhance the the arid patience and compatibility of the plant for better perfomences on the desert prevention and control, ecological environmental protection, productivity, and ecological agriculture technologies in the loess plateau areas. The flower organ shape, blossom habit, acceptation of stigma, and the effects of different management regimes on the seed production of B. dactyloides were studied by this research. The result indicates that the flower organ is different greatly in the external shapes and blooming characteristics between male and female, but similar in florescence periods. The acceptation of female stigma is strongest in the latter 10 hours to blossom, and weaken gradually after 24 hours. In order to obtain the high yield and good quality of seed, the artificial wrap bag pollination should be applied in the beginning period of blooming of male plants and in the morning also. Fertilization and irrigation should be employed in the earlier earing period and the earing period of female plants, not suitabl during blooming period. For the purpose of selecting new varieties of B. dactyloides with high values in seed production, turf performance, and cross breding potential, ten breding materials and 3 hybrids F1 generation of B. dactyloides with different characteristics were compared in 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 results indicate that the female NO.1 had relatively higher seed quality and yield and could be used as the maternal material for B. dactyloides seed selection. Pollen quantity and pollen quality of the male NO.1 were better and it could be regarded as the male parent material of the crossbreeding. The female NO.3, NO.5, NO.6, and the polygamy could be regarded as the new varieties for vegetative propagation and sod production. The heterosis of the three hybrids was obviously, but there were differences in the sex differentiation ratio among them. We also obtained a DREB gene of B. dactyloides induced by drought and high salt concentrations by RT-PCR using degenerate primers and RACE. At first, a pair of degenerate primers was designed against the conserved regions by the multiply alignment of amino acid sequences and nucleotide acid sequences of several plant DREB in GenBank, then a 209 bp cDNA segment was amplified using RT-PCR. Base on the sequence of the cDNA segment, a primer was designed for 3’ RACE and a 653 bp coda segment was acquired. According to the sequence of 3’ end of the gene, a primer was designed for 5’RACE and a 426 bp cDNA segment was acquired. The full-length cDNA 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. The amino acid sequence compared by Blast revealed a high homology with that of other plant DREB, and the similarity to DREB of Cynodon dactylon (L.) Pars. (AAS46285) was the highest at 89%. The results indicate the gene cloned from Buffalo grass was a DREB gene which we have named as BdDREB2 (Buchloe dactyloides Dehydration Responsive Element Binding protein 2). 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 indicate that BdDREB2 was 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 not reduced by cold treatment. 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 most 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.

Key words: Buchloe dactyloides (Nutt.) Engelm., Sexual reproductive, DREB, Gene clone