Overexpression Of LmGST Gene Promotes The Application Of Duckweed Hyperenrichment Of Cadmium

Duckweed（Lemna minor）is a fast-growing,environmentally adaptable Cd hyperaccumulator.Glutathione S-transferases（GST,E.C.2.5.1.18）often play a detoxicating role in plants under abiotic stress,furthermore,the GST gene in duckweed was proved to respond to Cd stress.Further studies on the role of GST gene in duckweed hyperenrichment of cadmium are essential for the application of duckweed.Therefore,LmGST gene were cloned,bioinformatics analysis and function verification were conducted.Prokaryotic expression was used to verify the role of LmGST gene in E.coli BL21,and subcellular localization was used to analyze the expression position of LmGST gene in cells.The genetic transformation system of duckweed callus and fronds was established,optimized and compared.The LmGST overexpressing duckweed strains were obtained by genetic transformation.The growth rate,chlorophyll content,antioxidant enzyme activities,GST enzyme activity,GSH content and relative gene expression level were measured and compared between the LmGST overexpressing duckweed and wild type duckweed under Cd stress.The effect of Cd enrichment and Cd removal by LmGST overexpressing duckweed was further tested at laboratory level.The following conclusions were mainly obtained:1.LmGST gene cloning and bioinformatics analysis:the complete coding sequences of LmGSTF3,LmGSTF4 and LmGSTU1 of duckweed were cloned with open reading frames of 561,645 and 675 bp,encoding 186,214 and 224 amino acids,respectively.The encoded GST proteins are stable acidic hydrophilic proteins,not membrane-spreading proteins or secretory proteins,which is predicted to be localized in the cytoplasm.Expression of LmGSTU1,LmGSTF3 and LmGSTF4 genes was up-regulated under Cd stress as verified by quantitative PCR.2.Prokaryotic expression and subcellular localization of the LmGST gene:LmGSTF3 and LmGSTU1 were constructed into prokaryotic expression vector pET30a and transformed into E.coli BL21.The results showed that the growth of E.coli BL21 pET30a-LmGSTF3 and LmGSTU1 was better than that of BL21 pET30a under 400～800 mg·L^-1CdCl₂stress.The transgenic GST E.coli formed more colonies on 200 mg·L^-1CdCl₂-stressed plates;expressed GST protein and the expression was up-regulated under Cd stress;the removal rate of Cd was higher than that of BL21pET30a,and BL21 pET30a-LmGSTF3 could remove more than 80%of Cd.LmGSTU1 was constructed onto a GFP vector and transiently transformed into tobacco leaves,and it was observed that LmGSTU1 was localized to the cytoplasm and nuclear membrane.3.Establishment and optimization of genetic transformation system of duckweed:the genetic transformation system of duckweed callus and fronds was established and optimized.The efficiency of the genetic transformation system of duckweed callus was 75%,which was higher than that of fronds（55%）.Both of the two transformation systems could be stably expressed.The experimental period of fronds genetic transformation system was about 1～2 months,which was much shorter than that of callus（4～5 months）.4.Expression analysis of LmGST overexpressing duckweed:LmGSTF3、LmGSTF4 and LmGSTU1 overexpressing duckweed was obtained by genetic transformation.Overexpression of LmGSTF4 and LmGSTU1 genes attenuated the inhibitory effect of 10 mg·L^-1Cd on root length and reduced root abscission caused by Cd,and alleviated the effect of Cd on the reduction of chlorophyll content.Overexpression of LmGSTF3 and LmGSTU1 genes increased GST enzyme activity（0.8～15.5 fold）and GSH content（1.7～4.6 fold）and significantly increased the expression levels of GST gene（1.5～8.0 fold）,as well as GSH pathway-related genes GR,GPX,pep A and pep N,by 0.8～77.9,1.1～15.5,4.3～161.3 and 2.2～22.7 fold,respectively.5.Application of LmGST overexpressing duckweed:The LmGSTF4-14,LmGSTU1-1 and LmGSTU1-18 strains achieved 83%,83%and 84%Cd removal after treatment with 10 mg·L^-1Cd for 7 d,respectively,which were much higher than WT（41%）.The Cd enrichment reached 3197～4126 mg·kg^-1,which was 1.2～1.5 times higher than that of WT.The three strains were further used to treat simulated discharge effluent and the results of the pilot test showed that LmGSTU1-1 and LmGSTU1-18 strains treated simulated sewage for 2 days and purified from inferior ClassⅤwater to ClassⅤwater.After 4 days of treatment,Cd in simulated sewage was completely removed,1 day faster than WT.In conclusion,this study laid the foundation for further revealing the Cd tolerance and detoxification mechanism of GST gene,and provided the basis for further use of duckweed for water Cd pollution remediation,which is of great significance for the development and application of LmGST overexpressing duckweed.