Research Of Temperature-induced Lipocalin (TIL1) Required For The Development Of Female Gametophyte In Arabidopsis Thaliana

The lipocalin protein family is a large group of small extracellular proteins and widely distributed in bacteria, protozoa, arthropods animals and plants. In Arabidopsis, there is only one kind of lipocalin protein called as temperature-induced lipocatin 1 （TIL1）, which is located at the plasma membrane. Its encoding gene （TIL1） expresses in various tissues, including roots, stems, leaves, flowers and siliques, showing no abvious effect on the seed germination, vegetative growth, flowering time, leaf senescence and seed yield. Previous researches showed that the TIL1 gene was mainly involved in response of Arabidopsis to adversity stresses, such as temperature, light, high salt and oxide.In this research, the observation of the mutant Salk₁36775c of the TIL1 encoding gene inserted by T-DNA found that the silique setting rate was decreased and the seeds were obvious aborted. Different modern biological technologies were used to explore the mechanism that the TIL1 gene involved in the female gametophyte development. The major results are as follows:1. The observation of silique setting rate showed that the seeds of the til 1-1 mutant seed was abviously aborted, the undeveloped ovules looked like white points, and the seed setting rate decreased by 20%. The same lower fertility phenotypes were observed on the mutant till-2 and til 1-3. It was speculated that the TIL1 gene is involved in the seed development, due to abortion at the early development stage of the zygotic or gametophyte embryos in the till-1 mutant.2. Analysis of semi-quantitative RT-PCR and real-time fluorescence quantitative Q-PCR showed that the TIL1 gene was knocked out by the T-DNA insertion, resulting in the loss of its function, and the lower fertility phonotype of the til1-1 mutant.3. The silique setting rate was investigated on the offsprings of the reciprocal crosses between the pistils of the till-1 mutant with the stamens removed and wide-type pollens, and between the homozygous ms-1 pistils and the pollens of the til1-1 mutant. The result indicated that the lower fertility phenotype of the til1-1 mutant took place during the gametophyte development, instead of the early stage of the zygotie embryo development.4. The results of Alexander staining and pollen cultivation in vitro results proved showed that the development of male gametophyte was normal, and the lower fertility phenotype of the mutant was not because of the male gametophyte.5. The ovule transparent experiment showed that the function megaspore cell of the til 1-1 mutant formed normally and went through three continuous cycles of mitosis, but the defect took place during the further development, resulting in abortion of the mature female gametophyte.6. The till-1 mutant was transformed by the fusion expression vector combing the wild-type Arabidopsis TIL1 gene and green fluorescent protein gene GFP. The lower fertility phenotype of the transformants was recovered, and the fusion gene highly expressed in the ovules, as well as in the vascular tissue of seedling stem and root.7. Although the expression of the mutated gene At2g33830 and the mutated gene At2g27420 were found up-regulated and down-regulated in the till-1 mutant respectively, the lower fertility phenotypes were not found in the siliques of the corresponding mutants Salk₀98437c and Salk₀85797c, indicating that these two genes were not directly relative to the abortion caused by the mutation of the TIL1 gene.All the above results indicated that the mutation of the TIL1 gene causes lower fertility phenotype in Arabidopsis. The TIL1 gene is involved in the ovule development and seed formation. The male gametophytes of the till-1 mutant develope normally, and have normal fertility. The megaspores of the gametophytes form normally, and go through three cycles of continuous mitosis, but the mature female gametophytes are defective, resulting in the abortion phenotype of the til1-1 mutant.