Studies On Introduction The Gene Encoding Small Heat Shock Protein In The Endoplasmic Reticulum Into Tomato

Low-temperature may cause damages which include many aspects such as the change of the membrane lipids phase, the increase of membrane's permeability and the level of reactive oxygen species, the decomposition of chlorophyll, the accumulation of some secondary metabolites, and so on, to plants. All these phenomena result from the damages of some enzymes which involved in the metabolism of plants cells.By genetic engineering method, the ER-sHSP-LeHSP21.3 gene was introduced into tomato. Driven by the cauliflower mosaic virus (CaMV) 35S promoter, the ER-sHSP over-expressed constitutively. The growth and the phenotype of transgenic plants can be used for researching the function of ER-sHSP in improving tomato's cold resistance and the ER-sHSP chaperones function in vivo.After degested by Kpnl and Xbal enzymes from the pBS-ER plasmid, the gene ER-sHSP-LeHSP21.3 was inserted into the pROKII vector to construct an eukaryotic expressing vector. The tomato's cotyledon were transformated with this vector via Agrobacterium tumefciens-mediated method. PCR result indicated that ER-sHSP gene had been integrated into tomato successfully.The Tl seeds are screening on the MS medium which contains 50ug/ml Kan, and there are about 70% of the transgenic lines showed the Kan-resistance with a ratio of 3:1. However, the other lines didn't show the ratio consistent with the Mendelism. It might prove that most of the transgenic plants only have one insert.Put the transgemc seedlings and controls together at low-temperature ranged from 5℃-15 ℃ for at least 3 weeks, and then determine their physiology indexes:1: the growth of seedlings: at low-temperature, the growth potential of the transgenic seedlings was drastically higher than the controls and the height changed more distinctness in transgenic plants than in the controls.2: the accumulation of anthocyanins: after treated by low-temperature for 3 weeks, thecolor of the controls' leaves turned red and could be observed from the appearance which indicated the accumulation of anthocyanins was remarkable, but there nearly has no changes on the color of transgenic plants' leaves .3: chlorophyll content: low-temperature can restrain the biosynthsis of chlorophyll and destroy its structure. The contents of chlorophyll a, chlorophyll b and chlorophyll a+b in controls were significantly lower than in transgenic seedlings. The ratio of chlorophyll a/b had the similar result.4: Photosynthetic characters: the Pn, Gs and transpiration rate both in control and in the transgenic seedling increased during the growth, and the Ci decreased, but the change scope in transgenic seedling is more great than in control.5: MDA content: the contents of MDA in controls were significantly higher than in transgenic seedlings.The results suggest that low-temperature can caused damages to the control. However, with the existence of large number of ER-sHSP which act as molecular chaperones, the transgenic tomato deduced the damage effect of cold stress at the same instance. So, we can concluded that over-expressed constitutively ER-sHSP can improved the tomato's cold-resistance indeed.