| Many plant adaptations to stresses involve mitochondria Mitochondria have been reported to be the main cellular organelles affected by low temperatures. Mitochondria processes energy exchange by oxidation and phosphorylation, which can afford energy for a set of acts of cell, Mitochondria is the energy metabolization center, Reactive oxygen species during respiration can ajust oxidoreduct reaction ,gene expressioaThe mitchondria small heat shock protein is usually the most heat responsive of the Mitchondria HSPs.Mitochondria sHSP studies in vitro indicate which act as molecular chaperones ,over-expressed constitutively mitchondria sHSP can improved the tobacco's thermotolerance. Moreover, it has been reported that mitochondria sHSP can been induced by cold stress.In fact,it has been reported that chloroplast sHSP ,cytosolic sHSP and ER sHSP are related to the ability of cold-resistance.But it does not know if the mitochondrion sHSP has the same protective functions against cold sresses too.By genetic engineering method, An integrated vector pROK II of the mitochondria sHSP gene and NPTII gene(a kanamycin resistant gene) with CaMV35S promoter was constructed and introduced into tomato mediated by Agrobacterium tumefaciens LBA4404.Transgenic tomato were screened by their ability of growing on media containing kanamycin. The genomics DNA of the transformants was extracted and assayed by PCR with NPT II primer and the results indicated that the mitochondria sHSP gene has been integrated into the genomics of the tomato.Western-blot indicated that mitochondria sHSP gene has expressed in tomato. The Tl seeds are screening on the MS medium which contains 50ug/ml Kan, some transgenic seeds from a transgenic line can grow in gear which showed Kan-resistance, others transgenic seeds from a transgenic line were limited to Kan,which shown symptoms in common to controls, the results indicated that the mitochondria sHSP gene has been integrated into the genomics of the tomato.Put the transgenic seedlings and controls together at low-temperature(in winter,on natural condition,the top temperature was 15? and the lowest temperature was 2癈)and a set of parameterswere measured after 2weeks.The results were shown as follows:1) Conductance rateAt low temperature, membrane of plant can been damaged, conductance rate of the transformants were lower distinctly than controls,which showed that ability of cold-resistance of transgenic tomato were stronger than control.2) Effectson chlorophyll contentsThe chlorophyll is the important pigment for the photosynthesis of plants.The measurement results showed that the chlorophyll contents in transgenic tomato were highter than that of the control,which indicated the damage extent that the chlorophyll in transformants suffered was down to that of the control plants at low temperature.4) Effects on photosynthesis:The important parameters of photosynthesis such as net photosynthetic rate, transpiration rate, and stomatal conductance were measured and the results was that the transgenic tomato were highter than that of the control plants, which indicated that the photosynthesis of transgenic tomato were stronger than control clearly.5) Qulity of mitochondriaScarfskins of the transgenic tomato and the control plants after 2weeks at low temperature were soared in hepes buffer containing Rhodamine 123,by observing under Leica DMR microscope,the results showed that Qulity of mitochondria in cilia and stoma of the transgenic tomato were higher than control,which indicated that mitochondria of transgenic tomato had a lower damage.Moreover,the type of the transgenic tomato and the control had obvious differerance after 3 weeks at low temperature ,leaves of control dried out and desquamated,contrarily, leaves of the transgenic tomato were greener and growed in gear comparatively.All the results suggested that the mitchondria sHSP gene has been integrated into the genomics of the transgenic tomato and the cold-tolerance ability of the transformant were inhanced. |
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