Expression Analysis And Functional Verification Of SoTUA Transgenic Sugarcane

Sugarcane is an important economic crop in the world,and China is the fourth largest sugar producing country in the world.Sugarcane is cultivated in tropical and subtropical regions,which is adapted to warm climatic conditions.Low temperature not only limits its geographical distribution,but also reduces cane and sugar yield,so it’s one of the major climatic damages in commercial sugarcane production.Therefore,it is of great significance to cultivate cold resistant varieties.The development of transgenic technology provides an effective method to improve the cold resistance of sugarcane.In this study,SoTUA transgenic sugarcane lines and control(WT)were used as the experimental material.Four transgenic lines(L1,L2,L3 and L4)and a WT control were treated under the cold stress(4℃)to detect the relative expression of SoTUA gene and SoTUA protein and to investigate their physiological and biochemical performance.In addition,the agronomic characters of transgenic lines L5,L6,L7,L8,L9,L10 and L11 and WT control were measured at seedling stage,tillering stage,elongation stage and maturity stage,respectively,to lay a foundation for further research on SoTUA gene.The main results are as follows.1.SoTUA transgenic sugarcane lines were identified by PCR amplification and sequencing of GFP marker gene on vector.The bands consistent with the positive control were obtained,and the GFP gene sequencing was carried out,indicating that the So TUA transgenic sugarcane plants were obtained.2.Four SoTUA transgenic sugarcane lines(L1,L2,L3 and L4)and WT control were treated under cold stress to detect the relative expression of So TUA gene and SoTUA protein by using qRT-PCR and Western blot.The results of qRT-PCR assay showed that SoTUA gene expression in the transgenic sugarcane lines was higher than the control before treatment,but it was strongly induced in all the tested lines after cold treatment started.Except for L1,all the lines showed the SoTUA gene expression peak at 2 d after cold treatment,and the change pattern was first increased,then decreased and increased again.The SoTUA gene expression in the transgenic sugarcane lines was higher than the control during cold treatment.Western blot was used to detect SoTUA protein expression,and the results show that SoTUA protein expression was higher in the transgenic lines than the control before treatment.Except for Ll,all the lines were down-regulated at 0-2 d after cold treatment.The protein expression patterns were different,L1 increased first and then decreased,L2 and L4 first decreased then increased,L3 showed decreasing slowly,and the control showed a pattern of"fall-raise-fall-raise",but the SoTUA protein expression was higher in the transgenic sugarcane lines than the control during cold treatment.3.Under cold stress,the soluble sugar and soluble protein contents in the four SoTUA transgenic sugarcane lines were higher than the control,the soluble sugar content in these lines changed in different modes;soluble protein raised strongly at the early treatment stage in response to low temperature,the overall change trend was first increased and then decreased.The proline content changed in a mode of"S"type,except for L4,other transgenic lines were higher than the control.MOD content in all the lines increased,especially in the control.POD activity first decreased,then increased and again decreased,and there was no significant difference between the transgenic sugarcane lines and the control except for L2.The modified pole ordination method and subordinative function method were used to evaluate the cold resistance of the transgenic lines and control,respectively,the results showed the cold resistance of four transgenic lines were stronger that the control.4.The agronomic traits investigation on 7 transgenic lines(L5,L6,L7,L8,L9,L10 and L11)and WT control revealed that the plant height was higher in L5,L6 and L11 than the control at all the four stages.The stem diameters showed higher in L5,L6,L8 and L11 than the control at all the four stages,and L6 was significantly higher than the control at all the four stages.Except for L6 and L7,the chlorophyll content showed higher in the transgenic sugarcane lines than the control at all the four stages.The leaf area showed higher in L5,L8 and L9 than the control at all the four stages.Except for L6 and L9,the plant height,stem diameter,leaf area and chlorophyll content in the transgenic sugarcane lines were significantly higher than or equivalent to those in the control,indicating that the SoTUA gene transformation did not cause adverse effects on the agronomic traits in the transgenic sugarcane plants.