| Glycinebetaine(GB)is important one kind of compatible osmolyte.Some plant accumulated the osmoprotectant GB to maintain the cell normal turgor,the structure and function of enzyme,protein complex,and the integrity of membrane responding the stress such as low temperature,salt and drought.The betA gene from E.coli. encodes chroline dehydrogenase(CDH)which has both the function of Choline Monooxygenase(CMO)and betaine aldehy dedehydrogenase(BADH). Transformation of the single gene betA can endow the transgenic plants the capability of accumulating GB.Low temperature and soil salinization are important factors affecting the quality and yield of cotton.There is important significance to improve the salt and low temperature tolerance of cotton for increasing the yield and plant area of cotton.The tolerance to salt and low temperature stress of four T3 progeny of transgenic lines from Lumianyanl9 was investigated.The result shows that the tolerance of transgenic plants improved significantly.The expression of betA increased the accumulation of GB in transgenic cottonThe expression of betA increases the level of GB in transgenic cotton leaves significantly.The level of GB in the leaves of transgenic lines LM1,LM2,LM3,LM4 and wild-type Lumianyanl9 are detected by NMR method.The concentration of GB in the leaves of wild-type was 99.29μmolg-1DW,the level of GB in the transgenic lines were between 125.98-214.34μmolg-1DW under normal growing condition.After ten days treated by 250mM NaCl,the concentration of GB increased significantly,the four transgenic lines accumulated 321.06,357.32,355.51 and 276.09μmolg-1DW respectively,significantly higher levels of GB than the wild-type plant with 176.00μmolg-1DW GB.After 24 hours' treatment with 5℃,the GB content in transgenic lines LM1,LM2,LM3 and LM4 reached 170.78,205.93,248.21 and 189.46μmolg-1DW,they were 1.56,1.88,2.27 and 1.73 times compared to that in wild-type plants.It is possible that significant increase of the level of GB in transgenic lines resulted from the enhanced expression of betA under the stress of salt or low temperature.Analysis of salt tolerance of betA-expressing cottonThe germination rates of the transgenic lines LM1,LM2,LM3 and LM4 treated by 250mM NaCl for 9 days are 52.04%,66.04%,60.44%and 42.40%respectively, each of which is significantly higher than that of the wild-type Lumianyan19.The biomass of the lines LM2,LM3 is significantly higher than that of the wild-type.After 21 days salt stress under 250mMNaCl,the relative water content of the wild-type plants reduced from 80.79%to 48.54%,whereas that of the four transgenic lines are 62.90%,64.93%,64.44%and 63.53%,the water loss rates of the transgenic lines more slowly than that of the wild-type plants.The content of other osmoprotectant such as soluble sugar,free amino acids in the leaves of the transgenic cotton are obvious higher than those in wild-type.The solute potential of the lines LM1,LM2, LM3,LM4 is 8.86%,13.89%,3.50%and 8.61%lower than that of the wild-type.The results indicated that more solutes were accumulated in transgenic plants to enable cells to absorb more water and to maintain cell normal shape and turgor under stress. The cell membrane ion leakage,the content of malondialdehyde of lines LM2,LM3 is 23.08%,23.56%and 38.72%,36.03%lower than that of the wild-type respectively, which suggested less membrane damage and lipid peroxidation occurred in these transgenic lines.After osmotic stress,net photosynthesis,stomatal conductance and transpiration rate of the four transgenic lines were significantly higher than those of wild-type,the effect to the Photosynthesis System of transgenic cottons is less than that of the wild-type.The seed cotton yield per plant of the lines LM2,LM3 is significantly higher than that of the wild-type on the salinized land(salt content 0.45%),which were of great value in cotton breeding and production. Analysis of low temperature tolerance of betA-expressing cottonThe germination rates of the lines LM1,LM2,LM3 and LM4 at the 13thday after seeding at 15℃condition are 67.33%,78.20%,70.11%and 74.25%,each of which is significantly higher than 53.24%which is the germination rate of the wild-type Lumianyanl9.After 24h low temperature treatment at 5℃,the relative water content of the wild-type plants reduced from 87.94%to 53.43%,whereas that of the transgenic line LM3 is 66.28%,which is significantly higher than that of the wild-type plants,the water-retaining capability of the transgenic lines leaves is better than that of the wild-type plants.The cell membrane ion leakage of the four transgenic lines LM1,LM2,LM3,LM4 is 22.59%,37.95%,40.28%and 23.11%respectively, which is lower than that of the wild-type,the content of malondialdehyde of the transgenic lines LM2,LM3 is 32.96%and 39.65%,which is less than that of the wild-type.The results suggested that less membrane damage occurred in these transgenic lines which have stronger low temperature tolerance.The SOD activity in these transgenic lines is also higher than that of wild-type plants under stress.The results implied that the transgenic plant has strong activity of cell protective enzyme, powerful ability to scavenging free radicals under low temperature stress.After low temperature stress,net photosynthesis,stomatal conductance,intercellular CO2 concentration and transpiration rate of the four transgenic lines were significantly higher than those of wild-type plants,the net photosynthesis of the LM2 and LM3 is 13.81%and 31.61%higher than that of the wild-type plants,the damage to the Photosynthesis System of transgenic cottons by low temperature stress is less than that of the wild-type.The above physiological analysis of cotton plants transformed with betA gene demonstrated that a proportion of the transgenic lines(LM2,LM3)accumulated higher levels of glycinebetaine and acquired significantly higher tolerance to salt and low temperature stresses.In addition,the transgenic lines with improved stress tolerance provided valuable cotton materials for cotton stress tolerance breeding.
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