Cloning And Functional Verification Of ARF1?TUA3 And AP4-? Gene In Lonicera Confusa

The southwest karst area is distributed in eight provinces or cities in the southwest of China. In recent years, the soil erosion and rocky desertification have been very serious in southwest karst area. The use of genetic engineering for rocky desertification repair by clone relative genes from plants adaptable of this environment is one of the important means of vegetation restoration in karst area. As a typical economic crop in the southwest-karst-area, Lonicera confusa can adapt to calcium-rich karst environment very well. It will be of great importance to clone the genes of Loniceraconfusa which associated with calcium transportation in vivo and culture transgenetic plants adaptable of karst-area. Based on three calcium-inducing gene fragments which had been found by using genefishing technology during preliminary reaserch, the complete coding box was cloned, and then the genes were transformed into the E.coli BL21 and Arabidopsis thaliana to carry a functional verification. The main results of this paper are as follows:First of all, the entire coding frame of ARF1, TUA3 and AP4-? gene fragment was amplified from cDNA of Lonicera confuse leaf and then was analyzed through bioinformatics technique.Secondly, recombinant vectors constructed with ARF1, TUA3, AP4-? gene and prokaryotic expressing vector pET-28 a were express in E.coliBL21, which was used for prokaryotic expression analysis. The results show that the strain of transgenic strains had a shorter lag phase growth and higher growth compared with empty vector at high concentrations of Ca2 +.Thirdly, the plant expressing vectors recombined with ARF1, TUA3, AP4-? gene were constructed severally. Then agrobacterium-mediated transformation was used to introduce the recombinant vectors into Arabidopsis thaliana and then trangetic plants were identified. The transgenic and wild-type Arabidopsis thaliana were treated with high-calcium stress and it was shown that plants with recombinational TUA3 and AP4-? gene got enhanced high-calcium tolerance, but the ARF1 genetic plant has not done benefits to the calcium-tolerant of Arabidopsis obviously.Finally, under the stress of high calcium, the physiological indexes in transgenic plants and wild type plant were respectively determined. It is indicated that the chlorophyll, soluble protein, soluble sugar content is decreased in Arabidopsis with transferred TUA3 and AP4-? gene but still remaining at normal levels. And the CAT activity has been at a high level of activity in these two plants. It is of great significance to maintain high cytoplasm concentration, eliminate excessive 02- and maintain the stability of cell membrane systems and enzyme system.