Genetic Stability Analysis And Functional Verification Of Sugarcane SoACLA-1 Gene

Sugarcane is the main sugar crop in China and drought is one of the most challenging agricultural problems limiting sustainable sugarcane production.ATP-citrate lyase(ACL)is a key enzyme in plant growth and development,and one of its functions is to improve plant stress resistance.SoACLA-1 gene is a subunit gene encoding ATP-citrate lyase protein of sugarcane.In previous study,our research group had constructed the eukaryotic expression vector PUBTC-SoACLA-1 with corn ubi as the promoter,and transferred the vector into the sugarcane variety ROC22 by agrobacterium transfection method to obtain the sugarcane clone with SoACLA-1 gene.In this study,the wild type sugarcane variety ROC22 and T1 and T2 generations of above transgenic sugarcane strains were used as materials to study the effect of overexpression of SoACLA-1 gene on sugarcane related physiological and biochemical indexes under drought stress and rewatering,as well as the expression of SoACLA-1 gene and several other genes responding to drought stress in different stress days,so as to provide a reference for the evaluation of genetic stability of sugarcane transformed with SoACLA-1 gene and the verification of the function of gene.The main results are as follows.1.The introduced SoACLA-1 gene was found to be lost in both T1 and T2 generation transgenic sugarcane.The bar gene was amplified by PCR and sequenced,and the results showed that 25 lines of 31 positive transgenic sugarcane strains could be detected with bar gene in T1 generation,while 21 lines could be detected with bar gene in T2 generation,that is,the transgenic positive rate was 80.6%in T1 generation and 84%in T2 generation.2.RT-PCR and herbicide resistance screening further verified the transgenic materials.RT-PCR was used to detect the transformed SoACLA-1 gene in sugarcane,and the results showed that the introduced SoACLA-1 gene could be efficiently expressed at mRNA level.After herbicide resistance screening,it was found that all the SoACLA-1 transgenic sugarcane strain with the marker gene bar had herbicide resistance.3.SoACLA-1 gene can improve the drought resistance of T1 generation of transgenic sugarcane.At the early elongation stage,the SoACLA-1 transgenic RT1,RT2,RT3,RT4 and the wild type(WT)sugarcane were subjected to drought stress treatment at 0,3,6,9 days and rehydration 3 days.The membership function in fuzzy mathematics was used for comprehensive evaluation with four indexes related to drought resistance,that is,chlorophyll,malondialdehyde,proline and soluble sugar contents.The results showed that five sugarcane strains in T1 generation expressed the drought resistance from strong to weak as RT2>RT4>RT3>RT1>WT,and the resilience ability after drought release from strong to weak was RT1>RT2>RT4>RT3>WT.The transgenic sugarcane strain RT2 maintained the highest SOD activity in the whole process of drought stress and the complex water were among the transgenic strains and WT.For POD,genetically modified strain RT4 showed higher activity than the other strains for the whole process of drought stress,while RT2 showed fast increasing activity at the middle and late stage of drought stress,and it was significantly higher than the WT(P<0.05).At 9 d after drought stress,the SoACLA-1 gene expression level was higher in the transgenic strains than the WT.4.SoACLA-1 gene can improve the drought resistance of transgenic sugarcane in T2 generation,and the stress-induced expression of SoACLA-1 gene in transgenic sugarcane resulted in up-regulation of the genes responding to plant drought stress.At the end elongation of sugarcane,drought stress treatment was carried out with the transgenic sugarcane strains RT2 and RT4 and wild type sugarcane(WT),and leaf samples were taken at 0,6,9 days of drought stress and rehydrated for 3 days.and four indexes related to drought resistance,that is,contents of chlorophyll,malondialdehyde,proline and soluble sugar,were comprehensively evaluated using membership function in fuzzy mathematics.The results showed that the order of drought resistance from strong to weak was RT4>RT2>WT,and the drought resilience ability from strong to weak was RT2>RT4>WT.The SOD activity was significantly higher in the transgenic sugarcane strain RT2 than WT at drought stress 9 d and rehydration 3 d,while the POD activity was significantly higher in RT2 and RT4 than WT at 9 d of stress.The results of real-time fluorescence quantitative PCR showed that the expression levels of SoACLA-1 and SCDR4 were the highest at 6 d,and significantly higher in both RT2 and RT4 than WT.The expression levels of P5CS and dhy genes were the highest at 9 d of stress,that of dhy gene was significantly higher in RT2 and RT4 than WT,and that of P5CS gene was significantly higher in RT2 than WT.The Cu/Zn-SOD gene expression level was higher in RT2 strain than WT at 6 d,while significantly higher in RT4 strain than WT at 9 d.Obviously,the changes of physiological and biochemical indexes were correlated with the expression of several genes in response to drought stress in the T2 generation of SoACLA-1 transgenic sugarcane.5.The genetic stability and functions of SoACLA-1 gene in the T1 and T2 generations of SoACLA-1 transgenic sugarcane were comprehensively analyzed.It is concluded that SoACLA-1 gene in the transgenic sugarcane could be kept in T1 and T2 generations and tended to be stable with the generation increase although loss phenomenon was observed.Efficient expression of bar and SoACLA-1 genes were found in the transgenic sugarcane,and the target traits can be expressed in the T1 and T2 generations,which reflected that the transgene could be inherited stably.It is concluded that the transgenic sugarcane has stronger drought resistance than non-transgenic sugarcane proved that the SoACLA-1 gene has the function of improving drought resistance of sugarcane.