Molecular Cloning And Expression Of AnGolS1 In Ammopiptanthus Nanus And Obtainment Of Its Transgenic Tomato Plants

Tomato is one of the most widely grown vegetable in the world and the suitable temperature for plant growth are 13-33℃. In our country, low-temperature in winter and spring is an important limiting factor for the yield and quality of tomato in protected cultivation. However, cultivated tomato lack resistance resources to low-temperature stress. Thus, in this experiment, an EST fragment of galactinol synthase was separated from Ammopiptanthus nanus which grows in the extreme low temperature environment by low-temperature induced suppression subtractive hybridization library, and we obtained the full-length sequence of AnGolSl by RACE methods (GenBank accession:GU942748). Meanwhile, the characteristic of gene and encoding protein were also analyzed and gene expression pattern was studied under low-temperature. On the basis of these works, this gene was transferred into cultivated tomato in order to improve low-temperature tolerance. The main results and conclusions are as follows:(1) According to the characteristic of pET30a(+) vector possessing HIS tag, pET30a(+)-AnGolSl recombinant plasmid was constructed by enzyme digestion and ligation methods. The AnGo1S1 fusion protein was expressed by using IPTG induced and then purified by Ni-NTA His-Bind Resin and then catalyzed substrate reaction. The production content of galactinol was analyzed after different reaction time by HPLC. The results reveal that AnGo1S1 very quickly catalyzed substrate reaction, so it is likely to an early-responsive enzyme and its validity enzyme activity is 40 min.(2) The expression of AnGolSl in the seedling roots, stems and leaves were analyzed under different stresses using qRT-PCR technology. The results revealed that the expression of AnGolSl was only efficient up-regulated by low-temperature stress, especially the expression in stems was 334.5 folds than control. However, the expressions of AnGolSl were not obviously up-regulated under drought and salt stresses. These results suggest that AnGolSl only responses to low-temperature stress.(3)A.nanus seedlings were treated by low-temperature and then its roots, stems and leaves were gathered, embedded using paraffin and sliced. Meanwhile, the probe vector was constructed and the DIG-labeling probes were synthesized and then used in ISH experiment. Under light microscope, It was found that the hybridization signals obviously accumulated in fascicular cambium stems after low-temperature treatment, but was not evidently observed surrounding other cells such as xylem, phloem and cortex. Moreover, the hybridization signals in xylem roots and leaves were significantly strong. It suggestes that fascicular cambium cells in stems might undertake to synthesize, transport and coordinate for AnGolSl.(4) According to the requirement of Gateway transgenic technology, the transgenic recombinant plasmid was constructed and then is transferred agrobacterium, which was used to disseminate tomato cotyledons. The tomato seedlings which were sifted by resistance culture medium cultivating calluses are obtained. After, the transgenic plants were detected by PCR amplification and living fluorescence. Finally,25 TO generation AnGolSl transgenic tomato plants were obtained and four plants may be multicopy plants, which provide important experiment materials for next study.