Analysis Of Drought Resistance Function Of Sugarcane ?~1-Pyrroline-5-carboxylate Synthase Gene(SoP5CS)

Sugarcane is an important sugar and energy crop,and also raw material of many industrial products.More than 80%of Chinese sugarcane is grown in upland areas that are lack of irrigation conditions.Drought has become an important restricting factor for the development of sugarcane and sugar industry in China.Based on the previous studies,sugarcane ?1-pyrroline-5-carboxylate synthase gene(SoP5CS)was cloned from the drought-resistant sugarcane variety GT21 that was bred by Guangxi province and characteristics of the gene sequence had been analyzed.The expression patterns of the SoP5CS gene in different organs under various stress treatments were analyzed by fluorescence quantitative PCR(qRT-PCR).The prokaryotic expression vector of SoP5CS gene was constructed and the SoP5CS protein was expressed in Escherichia coli.The expressed SoP5CS protein was purified and its monoclonal antibody was prepared.The plant expression vector of SoP5CS gene was constructed and transformated into sugarcane and tobacco by Agrobacterium mediated method.The aim of the present study is to provide a foundation for further study on the drought resistance of sugarcane and other crops by comprehensively exloring the drought resistance function of SoP5CS.The main results obtained in this study are as follows.1.The full length of SoP5CS gene was cloned from leaves of sugarcane variety GT21 by RT-PCR and registered in NCBI with the accession number KJ546350.1.SoP5CS was 2151 bp in length,encoding 716 amino acids with a predicted molecular weight of 77.73 kDa.It was predicted as a hydrophilic protein without transmembrane domains and located in the endoplasmic reticulum.The nucleotide sequence of SoP5CS showed high homology with other gramineous species and the function regions of the encoded protein was highly conservative with other higher plants.2.The expression of SoP5CS gene in different organs of sugarcane variety GT21 was analyzed by qRT-PCR.The results showed that the expression of SoP5CS was the highest in leaves,followed by the stems and the lowest in roots.In addition,the expression characteristics of SoP5CS in leaves of sugarcane variety GT21 seedlings under 0.1 mM ABA,4°C low temperature,15%PEG and 200 mM NaCl treatments were analyzed.The results showed that all treatments could induce the up-regulation of SoP5CS gene in varying degrees,but the expression patterns were different.It was speculated that SoP5CS gene might play an important role in sugarcane response to various stresses.3.The recombinant prokaryotic expression plasmid pET30a-SoP5CS was constructed by using PET-30a as a vector and the recombinant protein was expressed successfully.Then the recombinant protein was purified and used as the antigen to prepare monoclonal antibody by immunization mouse,cell fusion and culture.The titer of monoclonal antibody was high and the specificity was also quite good.The expression of SoP5CS protein in sugarcane leaves under different drought stress was also detected successfully by Western blotting.4.The plant expression vector of SoP5CS was constructed,which was driven by maize ubiquitin promoter and used herbicide resistance gene bar as resistance selection marker.SoP5CS was over-expressed using Agrobacterium tumefaciens-medidted transformation in sugarcane callus.A number of positive transformed plants were obtained,and then four transgenic lines were selected from the PCR positive plants for drought stress experiment.The relative expression levels of SoP5CS varied in different transgenic plants,the lines S3,S4 and S5 were significantly higher than those in control,which were 4.78-fold,4.82-fold and 2.23-fold,respectively,while S1 was lower and just 0.64-fold as compared to the control.The relative expression of SoP5CS protein was different from the expression of SoP5CS gene,which the S1,S4 and S5 lines were nearly the same as the control,while S3 was 2.53-fold.The accumulation of proline was consistent with the expression of SoP5CS levels in transgenic lines of S3,S4 and S5 whose proline contents were significantly higher than that of the control,while that of S1 was significantly lower.SOD activity in S3,S4 and S5 were also significantly higher than that of the control,while that in S1 showed no difference with the control.For MDA content,S3,S4 and S5 showed significantly lower than the control and S1.In addition,in the ABA content,S3,S4 and S5 were higher than the control,while S1 was slightly lower.S3,S4 and S5 transgenic lines were good drought resistant materials,which could be used for further research.5.The plant expression vector used in transformation of sugarcane was also used in the transformation of tobacco.Transgenic plants were identified by effective screening of herbicides,PCR detection and copy number detetmination.Four transgenic tobacco lines with low copy numbers were selected for drought stress experiment.The results showed that,under drought stress,both the relative expression levels of SoP5CS and its encoded protein were lowest in T9 transgenic tobacco lines,which the relative expression levels of SoP5CS in T1,T4 and T6 were 9.02-fold,3.93-fold and 8.51-fold as compared to T9,respectively,while the SoP5CS protein was 2.22-fold,3.91-fold and 1.36-fold,respectively.Further analysis showed that the contents of proline and ABA in four transgenic lines were all significantly higher than those of the control,and T1 and T6 were higher than the others.MDA content in the control was significantly higher than those in the transgenic plants,and the accumulation of MDA in T1 was the lowest.P5CR and P5CS activities were different between control and transgenic plants,the activities of both enzymes in T1 and T4 were nearly the same as those in the control,while those in T6 and T9 were significantly higher.CAT activity in T6 was the highest,and that in other transgenic lines was also significantly higher than that in the control.For SOD activity,all the transgenic lines showed significantly higher than the control,and T4 and T6 relatively higher.The relative water content in the leaves of the transgenic lines was significantly higher than that in the control,while the chlorophyll reduction was lower.A comprehensive analysis showed that the drought resistance of T6 line was the best,and the T1 line was also quite good.They could be used for further research.