Study On The Stress Resistance Of Transgenic Wheat With Gene Involved In Glycinbetaine Synthesis

Hot-dry windy and drought stresses are the most important environmental constraining factors for wheat production. The hot-dry windy climate is the product of low humidity, high temperature and wind stress occuring simultaneously. Moverover, water deficit is one of the global environmental problems. For a long time, the researchers abroad and at home have carried out extensive study on how to improve wheat resitance to hot-dry windy and drought stresses. With the development of plant genetic engineering, lots of genes have been identified and cloned. Genetic engineering technology has become an important strategy to create new wheat varieties with enhanced stress tolerance.Wheat is one of the most important crops and distributed widely all over the world. With the deterioration of natural environment and the continual increase of population, the contradictions between the increased demand and the decreased supply of wheat come into prominence. Genetic engineering technology provides a promising way for new wheat breeding against abiotic stresses and with increased wheat production. However, due to the huge genome and the genotype-depedence in in-vitro culture, the progress of wheat genetic engineering was far slower than other crops. It is important to create abiotic stress-tolerant wheat cultivars by genetic engineering.In our lab, genes of glycinebetaine (GB) biosynthesis have been introduced into elite wheat varieties (Jinan17 and Jimai 19). The genes used in this study are betA [encoding choline dehydrogenase (CDH)] from E.coli, ApGSMT2 and ApDMT2 [encoding glycine sarcosine methyltransferase (ApGSMT) and dimethylglycine methyltransferase (ApDMT) respectively] from a halotolerant cyanobacterium (Aphanothece halophytica). The transgenic Jinan 17 with betA gene and the transgenic Jimai 19 with ApGSMT2 and ApDMT2 were used to carry out hot-dry windy and drought tolerance studies. Analysis of hot-dry windy resistance of betA-expressing wheat plantsDuring grain filling period, three homozygous transgenic lines (BL1, BL2, BL3) from Jinan 17 of T3 generation and the WT (Jinan 17) were treated under hot-dry windy conditions for three days. After the stress treatment, the transgenic wheat plants grew much better with more green and extended flag leaves compared with the WT, and the GB contents of transgenic lines were obviously higher than that of WT.Under hot-dry windy stress, the chlorophyll contents of the flag leaves reduced in all lines, while the transgenic lines had higher chlorophyll contents than WT all along. The hot-dry windy stress inhibited the net photosynthesis, stomatal conductance and transpiration rate, while the inhibition extent to transgenic lines was lower compared with the WT. During the treatment, the temperature of flag leaves rose gradually, while the temperature of transgenic lines was lower than WT. The lower flag leaf temperature would be beneficial to retain physiological function and grain filling rate in transgenic plants. In comparision with the WT, the maximal photochemical efficiency of PSII (Fv/Fm) maintained a relatively higher value after three days of stess treatment in transgenic lines. Synchronously, the activities of sucrose phosphate synthase (SPS) and sucrose synthase (SS) in flag leaves of transgenic lines were higher than that of the WT. The results above were consistent with the data of soluble sugar and sucrose contents in flag leaves, and suggested that the transgenic plants could synthesize more carbohydrates under hot-dry windy stress. This would be benefitial for grain filling.After the hot-dry windy stress treatment, the plant height showed no differences between all lines. The spike lengths were shorter and the grain numbers per spike were less than those plants grown under normal conditions all the time. The plants fade and the flag leaves crimpled in all lines under stress, while the green areas of the flag leaves were remarkably larger in the transgenic lines. The 100-kernel weight and the grain yield per plants decreased greatly in all lines, but they were significantly higher in BL2 and BL3 than in WT. These results indicated that the transgenic wheat lines exhibited improved hot-dry windy tolerance. Analysis of drought resistance of ApGSMT2 and ApDMT2 co-expressing wheat plantsThe T2 generations of ApGSMT2 and ApDMT2 transgenic lines were identified by PCR amplication after herbicide selection (the selectable marker gene is epsp, which encode glyphosate resistant enzyme). Seeds of WT and the homozygous transgenic lines were sowed in vermiculite pots, and the resistance to herbicide was examined at the 3-4 leaves stage. Statistics x2 test was taken to deduce whether the genetic behavior of exogenous gene in transgenic lines accorded with the Mendelian Law of segregation. The results indicated that the foreign genes were stably exsisted in the twenty-one T2 transgenic lines. PCR and RT-PCR identification of the plants seleced by herbicide were carried out. Most cf the plants resisted to herbicide were also PCR positive. Real-time RT-PCR assay indicated that the expression levels of ApGSMT2 and ApDMT2 were different between transgenic lines, and the expression level in the line AL6 was the highest.Young seedlings of wheat plants are sensitive to water deficit. At this stage, the tillering number per plant and the grain yields could be reduced greatly when suffered with drought stress. Three transgenic lines (AL1,AL4,AL6) with higher expression levels of target transgenes were chosen to analyze the drought stress tolerance at 'young seedlings stage, using the wild type (Jimail9) as the control.The morphological changes of young seedlings suffered from sustaining drought treatment were observed. The transgenic wheat plants grew better under drought treatment, with more tillerings compared with the WT. Under severe drought stress conditions, most of the transgenic plants wilted later than the WT. After re-watered, they showed a significantly higher survival ratio and recovered more rapidly compared with the WT. After seven days of moderate drought stress, the expression levels of ApGSMT2 and ApDMT2 in transgenic plants increased, and the GB contents also increased simultaneously. Moreover, the GB contents were correlated positively with the expression levels of target transgenes.Wheat seedlings at 5-leaf stage were subjected to drought stress in soil pots for seven days. The WT plants showed disastrous water deficit and the leaves wilted severely. However, most of the transgenic plants performed better. The relative water contents (RWC) in transgenic lines were significantly higher than the WT. Correspondingly, the ion leakage and lower MDA levels in the transgenic plants were significantly lower than the WT. This indicated less cell membrane damage and lipid peroxidation in the ApGSMT2 and ApDMT2 co-expressing plants. The more solutes accumulation in transgenic plant cells would be beneficial to retain water uptake to maintain cell turgor under osmotic stress. Under drought stress condition, soluble sugar and free amino acid concentration in all lines increased. However, the contents of total soluble sugar and proline were significantly higher in the transgenic lines than in the WT. After drought treatment, the chlorophyll content in the transgenic seedlings was higher than the WT and it was significantly different between AL6 and WT. The values of net photosynthesis, stomatal conductance and transpiration rate were the same to the chlorophyll contents. The data of Fv/Fm in transgenic plants maintained moderately higher value on the 7th day of drought treatment, which revealed that the PSII complexes were not damaged seriously. The results suggested that co-expressing ApGSMT2 and ApDMT2 for GB accumulation exhibited enhancd resistance to drought stress compared with WT.Considering that ApGSMT2 and ApDMT2 catalyze the glycine-methylation biosynthetic pathway of GB from glycine, it is necessary to analyse the changes of free amino acid levels before and after drought stress, which would be benifit to evaluate the application potential of the target genes. After drought stress, the amounts of both Gly and Ser in transgenic plants were significantly lower than those in the WT, and the amounts of Gly had significant difference between the transgenic plants and WT. The results suggested that considerable amounts of Gly were used to synthesis GB, and the reaction rate from Gly to Ser were elevated. As Gly exits abundantly in plant cells and can be synthesized in vivo, the pathway is not constrained by the substrate concentration as much as choline-oxidation biosynthetic pathway of GB. The dominace could promote the development of GB genetic engineering with the utilization of glycine-methylation biosynthetic pathway. In conclusion, transgenic lines of hot-dry windy and drought resistance have been obtained in this work; this would provide valuable wheat materials for wheat breeding. At the same time, this study has also provided important information for better understanding of the hot-dry windy climate and drought stress resistance molecular mechanisms in wheat.