The Study Of Salt Tolerance Of Transgenic AtNHX1, BetA And TsVP Cotton And Cultivation Of Transgenic GhPEAMT-betA Cotton

The analysis of salt tolerance in overexpressing AtNHX1-betA-TsVP gene cottonSalinization is a widespread soil problem in the world and severely limits crop yields.Salinization affected nearly 6%of all the world's land and has a tendency to further expand.Cotton is the world's most important source of natural fiber and major oil crops.Cotton production areas of China are mainly concentrated in the northwest inland and coastal saline-alkaline land.It is significant for promoting the large-scale planting of cotton in the saline-alkali land and solving the problem of "cotton and grain crops compete for fertile land" to raise the salt tolerance of cotton,which has important practical significance.Glycine betaine(GB)is an osmotic protection substance that can effectively protect plants against salt stress,drought stress,low temperature stress and so on.Choline dehydrogenase encoded by the betA gene from Escherichia coli can catalyze the reaction of choline into GB.H+-Ppaselocalized on the tonoplast membrane of the plant is encoded by the TsVP gene cloned from Thellungiella salsuginea,which can provide the power for Na+ partitioning in the vacuole to maintain ionic homeostasis.The Na+/H+ antiporter encoded by the NHX1 gene is localized on the tonoplast membrane,whichis responsible for the transport of Na+ into the vacuole under adverse concentration gradient conditions.Overexpression of AtNHXl gene from Arabidopsis thaliana can significantly improve stress tolerance of plant.In this experiment,three genes responsible for two different metabolic pathways were co-transformed into cotton to achieve the purpose of obtaining a cotton variety with significantly increased salt tolerance.The AtNHXl-betA-TsVP gene was transformed into cotton using agrobacterium-mediated method.Three T3 generation lines(ABT1,ABT2,ABT3)with good agronomic traits in saline land were selected as experimental materials.The T83 line(T6 generation)with TsVP gene,B29 line(T6 generation)with betA gene and Lumianyan 21(WT)were used as control.The difference in salt tolerance of different cotton lines was examined from a variety of perspectives including gene expression level,seed emergence under salt stress,determination of physiological and biochemical indexes at the seedling and bud stage,and agronomic traits of cotton in saline-alkali lands.In this experiment,the AtNHX1,betA and TsVP gene of AtNHX1-betA-TsVP transgenic plants were detected by PCR and the expression level of those genes were detected by q-PCR.The results showed that AtNHXl-betA-TsVP gene were successfully transferred into cotton and expressed normally.In the experiment of cultivating cotton in sand,all cotton lines began to suffer from salt stress at the four leaf stage.The many physiological parameters were determined on the Oth,1st,7th,14th and 21st day after treatment with 250 mM NaCl.The study found that there were no significant differences in all the cotton chlorophyll content,plant dry weight,relative water content(RWC),saturated osmotic,Na+,net photosynthetic rate(Pn),stomatal conductance(Gs),Fv/Fm soluble sugar,ion leakage,malondialdehyde content(MDA),and superoxide dismutase(SOD)activity.Compared with WT and overexpressing betA,TsVP gene cotton,the transgenic cotton ABT3 with AtNHX1-betA-TsVP gene had significantly higher chlorophyll content,plant dry weight,RWC,Na-,Pn,Gs,Fv/Fm soluble sugar,SOD activity,and significantly lower saturated osmotic,ion leakage,MDA after suffering from salt stress.The results showed that the salt tolerance of transgenic cotton with AtNHX1-betA-TsVP gene was the best among all cotton lines in the four-leaf stage to the bud stage under the condition of cultivating cotton in sand.Under hydroponic experiment,all cotton lines began to suffer from 0 mM,150 mM,and 250 mM NaCl at the four leaf stage.The plant materials from the 0th and 18th days after treatment with different concentrations of NaCl stress were used to determine a number of physiological indicators.The study found that there was no significant difference in RWC,saturated osmotic,dry weight,Pn and Fv/Fm of all the cotton before suffering from salt stress.Compared with WT and overexpressing betA,TsVP gene cotton,the transgenic cotton ABT3 with AtNHX1-betA-TsVP gene had significantly higher RWC,plant dry weight,Pn and Fv/Fm,and significantly lower saturated osmotic after suffering from salt stress.The results showed that the salt tolerance of transgenic cotton with AtNHX1-betA-TsVP gene was the best among all cotton lines under hydroponic condition.In the experiment of seed emergence under salt stress,the emergence rate,plant height and dry weight of each strain of cotton were measured.The experimental results showed that there were no significant difference in the emergence rate and dry weight of each strain of cotton when suffering from 0 mM NaCl.The emergence rate,plant height,and dry weight of the transgenic cotton lines were significantly better than that of WT,and the emergence rate,plant height,and dry weight of transgenic cotton with AtNHX1-betA-TsVP gene were significantly better than that of transgenic betA,TsVP gene cotton,when suffering from 200 mM NaCl.From this we can see that the transgenic AtNHX1-betA-TsVP cotton has stronger salt tolerance than the transgenic TsVP cotton,transgenic betA cotton and WT at the seed emergence stage.We performed the measurement and analysis on the data of seed cotton yield and other agronomic traits,Pn,and Fv/Fm of different cotton lines in Dongying saline-alkaline land.The results of the data analysis showed that transgenic AtNHXl-betA-TsVP cotton had higher Pn,Fv/Fm,seed cotton yield,and better performance of field agronomic traits than the transgenic TsVP cotton,transgenic betA cotton and WT.The experimental results showed that the transformation of AtNHXl-betA-TsVP gene into cotton could further improve the salt tolerance of cotton.Cultivation of transgenic cotton with GhPEAMT-betA geneThe GhPEAMT gene was responsible for the phosphoethanolamine-N-methyltransferase,a key enzyme in the biosynthesis of choline,which could catalyze the reaction of phosphoethanolamine to phosphocholine by to three-step methylation.The choline dehydrogenase encoded by the betA gene could catalyze the production of betaine aldehyde from choline,an important enzyme in the synthesis of betaine.Transformation of the GhPEAMT-betA gene into cotton might increase the betaine content of cotton cells,which was expected to improve the stress tolerance of cotton.The GhPEAMT,betA genes were constructed on the plant expression vector and transformed into cotton cells using agrobacterium-mediated transformation in this experiment.Transgenic GhPEAMT-betA cotton was obtained by preliminary screening of glyphosate and PCR identification.