Screening Of Cu²⁺ Resistance-related Genes And Preliminary Study On The Resistance Mechanisms Of Pomacea Canaliculata

Objective:Pomacea canaliculata is a typical invasive herbivore and an important vector for the transmission of tropical diseases.It plays a very important role in the occurrence and transmission of Angiostrongylus cantonensis and poses a great threat to the health of Chinese residents.At present,the control effect of Pomacea canaliculata in China is not satisfactory.This species is easy to invade and difficult in control.One of the important reasons is its strong environmental adaptability.The study on the environmental adaptability mechanism of Pomacea canaliculata will help us to understand the biological characteristics of the invasive vectors related to the transmission of this important tropical disease,and it will be of great significance to formulate and improve the future control strategies of Pomacea canaliculata in China.Field investigation shows that Pomacea canaliculata has strong adaptability to heavy metal polluted water.Compared with other heavy metals such as Fe and Cd,Cu has a wider application in electroplating metallurgy,mechanical manufacturing,disinfectant and pesticide production,and it is easier to be discharged into the water body to cause pollution.Cu in water mainly exists in the form of 2-valent ions.Its biological toxicity is remarkable and its content is getting higher and higher.The pollution situation is getting worse and worse.The previous results showed that the tolerance of Cu²⁺ of Pomacea canaliculata was significantly higher than that of other freshwater snails.However,the current study on the adaptability of Cu²⁺ f Pomacea canaliculata was limited to the self-avoidance behavior and pathophysiological changes of Pomacea canaliculata.The related functional genes have not been studied yet.Therefore,this study screened out the functional genes related to Cu²⁺ resistance of Pomacea canaliculata,and provided a number of valuable genetic research materials for further study of the molecular mechanism of Cu²⁺ tolerance of Pomacea canaliculata.In addition,the basic characteristics of metallothionein gene?PcMT?and glutathione sulfotransferase gene?PcGST1 and PcGST2?in candidate genes,the genetic evolution relationship with homologous genes of other species,the background level of tissue expression,the changes of expression level before and after Cu²⁺ stress and the function of Cu²⁺ resistance were analyzed,and prokaryotic expression vectors were constructed to obtain purified PcMT and PcGST2 protein,The possible mechanism of Cu²⁺ tolerance of Pomacea canaliculata were preliminarily explored.Methods:?Based on the results of blood transcriptome sequencing of Pomacea canaliculata under Cu²⁺ stress,a number of Cu²⁺ resistance-related functional genes were screened and their reliability was verified by real-time fluorescence quantitative PCR?qPCR?.?PcMT,PcGST1 and PcGST2 genes were selected to further study.The coding regions of these two genes were amplified by RT-PCR and cloned,and the basic characteristics of genes were preliminarily analyzed by bioinformatics.The evolutionary tree was constructed by MEGA 7.0 software to analyze the genetic and evolutionary relationship between the gene and the homologous genes of other species.qPCR was used to analyze the background expression level of the gene in tissues?liver,kidney,gill,blood and midgut?and changes of gene expression in tissues?liver,kidney and gill?under Cu²⁺?50,100,150 ? g/L?stress?1,7,14 days?.?Using pET28a as expression-vector,pET28a-PcMT and pET28a-PcGST2 plasmids were constructed.IPTG was used to induce the expression of PcMT and PcGST2 proteins in E.coli BL21?DE3?.PcMT and PcGST2 proteins were purified by Ni-NDA affinity chromatography.The change of OD600 value was analyzed and the growth of transformed and non-transformed strains under Cu²⁺ stress was compared,the Cu²⁺ resistance of PcMT and PcGST2 genes was preliminarily verified.Results:? Differential expression of metallothionein,heat shock protein,glutathione sulfotransferase,polymyosin and D-aspartate oxidase genes in Pomacea canaliculata was correlated with Cu²⁺ stress.?The coding region of PcMT sgene is 272 bp,encoding 87 amino acids.It does not contain aromatic and histidine.The terminal sequence CQCGKGCTGADSCKCDRKCSCK is identical with the conservative sequence CXCXXXCTGXXXCXXXCXCK of mollusk MT.It can be classified as metallothionein family 2,subfamily mog:gastropoda metallothionein.The higher Cys content?22,25.3%?and the more conservative structure?17?Cys-X?1-3?-Cys of PcMT suggest that the protein may have strong metal ion binding ability.Compared with amino acid sequence,PcMT had the highest homology with metallothionein of Pomacea b,ridgesi,which was 93%.?The background expression level of PcMT gene in the tissues of Pomacea canaliculata was as follows:gill?2.4± 0.4?>liver?1.0±0.1?>midgut?0.9±0.1?>kidney?0.7±0.1?>blood?0.5±0.09?.?The results of the first experiment of corresponding tissue samples of Pomacea canaliculata without Cu²⁺ stress were used as control?1.0±0·0?.It was found that PcMT gene was sensitive to Cu²⁺ and could be induced to express in liver,gill and kidney tissues at 50 ? g/L.The effect of inducing expression was positively correlated with the stress time.The expression level changed to 2.03±0.3,3.4±0.4 and 2.8±0.6 at 14d,respectively.Under 100 ? g/L Cu²⁺ stress,the expression of PcMT gene in the liver of Pomacea canaliculata continued to increase with the increase of stress duration.At 14d,the expression of PcMT gene was 2.3 ± 0.3.The expression of PcMT gene in kidney and gill tissues showed an inverted "U" pattern with the stress duration.The peak values were 5.2 ± 0.6 and 8.5 ± 1.1 on 7d,respectively.The expression of PcMT sene in hepatopancreas,gills and kidney tissues of Pomacea canaliculata under 150 ? g/L Cu²⁺ stress showed an inverted "U" pattern,with peaks of 3.3± 0.8?7.001 ±0.4 and 9.1 ± 0.8 at 7d and 1d,respectively.? The C-terminal domains of PcGST1 and PcGST2 genes studied were GST-C-3 and GST-C-?superfamily domains respectively,which could bind to harmful electrophilic substrates produced by heavy metals.Both of them were GST-N-? domain superfamily and could bind to reduced glutathione.PcGSTl and PcGST2 are closely related to Aplysia californica of the same Gastropoda class and Biomphalaria-glabrata,and clustered into one branch.However,they-are far related to ?-type GST proteins of the bivalve mollusks,such as Mizuhopecten yessoensis,Azumapecten farreri and Argopecten irradians,and have longer branches.?-type GST proteins may differentiate to some extent during the formation of Gastropoda and bivalves in mollusks.?The background expression level of GSTs gene was not identical among different subtypes.The background expression level of PcGST1 gene was as follows:blood?8.5±0.3?>gill?1.4±0.2?>liver?1.1±0.2?>midgut?1.03±0.1?>kidney?0.4 ± 0.08?· The background expression level of PcGST2 gene was as follows:bloodcelis?5.8±0.9?>midgut?1.6±0.4?>liver?0.8±0.1?>gill?0.2±0.02?>kidney?0.2±0.01?.The expression patterns of Cu²⁺stress were not identical among GSTs genes of different subtypes.The first experimental results of corresponding tissue samples of Pomacea canaliculata without Cu²⁺ tress were used as control?1.0±0?.The effects of Cu²⁺ stress on the expression of PcGST1 gene were as follows:in liver tissue,the expression of PcGSTl gene increased continuously for 0-14 days under 50?g/L and 100 ? g/LCu²⁺ stress,respectively,at 14 days,2.1±0.1 and 5.2±0.7.Under 150 ? g/L Cu²⁺ stress,the peak value reached at 1 day,11.3 ± 1.8.In gill tissue,there was no significant change in gene expression under 50 ? g/L concentration of Cu²⁺ stress,but the peak expression was 15.8±8.3 at 150 g/L concentration of Cu²⁺ stress on 1 day.50 ? g/L,100 ? g/L and 150 ?g/L could induce expression in kidney tissue.The induction period of 50 ? g/L was shorter than 7 days,and the expression peak was lower 3.1 ± 0.3,while the induction period of high concentration was longer 14 days,but the expression peak was higher 5.6±2.6.?The first experimental results of corresponding tissue samples of Pomacea canaliculata without Cu²⁺ stress were used as control?1.0±0.0?.The effects of Cu²⁺ Stress on the expression of PcGST2 gene were as follows:in liver tissue,the expression of PcGST2 gene increased continuously from 0 to 14 days under 50 u g/LCu²⁺ stress,4.9±0.5 at 14 days,Under 100 ? g/L and 150 ? g/L Cu²⁺ stress,the expression reached its peak at 7 day,which was 13.01 ± 3.03 and 12.21 ± 2.2,respectively.Under 50 and 100 ? g/L Cu²⁺ stress in gill tissues,the relative expression of PcGST2 gene peaked at 7 days,5.3 ± 0.7.?23.3 ± 16.5,respectively.Under 150 g/L Cu²⁺ stress,the relative expression of PeGST2 gene peaked at 1 day,9.8±3.3.50 g/L Cu²⁺ was not very sensitive to renal tissue stress,and no significant change of PcGST2 gene expression was detected.The expression level continued to rise at 150 ? g/L concentration,and reached its peak at 14 days,which was 18.02±0.8.?coli BL211?DE3?containing pET28a-PcMT and pET28a-PcGST2 plasmids was successfully constructed.Under the induction of 1 mmol/L IPTG and 20 ?,17000 PcMT protein and 27 000 PcGST2 protein were expressed respectively.After purification by Ni-NDA affinity chromatography,PcMT and PcGST2 protein were finally obtained.? The growth curves of pET28a-PcMT,pET28a-PcGST2 transformed strains and non-transformed strains containing pET28a empty vector were similar in LB liquid medium,there was no significant difference.In the LB liquid medium containing 0.2 mmol/L CuS04.the transformed strains were more resistant to Cue+,and the growth curve was better than that of non-transformed strains.The difference was statistically significantConclusion:?The molecular mechanism of Cu²⁺ adaptation of Pomacea canaliculata is very complex,involving the coordinated participation of many genes.The 17 differentially expressed genes obtained by qRT-PCR are highly reliable.They can be used as candidate gene materials for further study on the molecular mechanism of Cu²⁺ stress adaptation in Pomacea canaliculata.?Cu²⁺ stress can significantly induce the expression of metallothionein gene and glutathione sulfur transferase genes in Pomacea canaliculata.Metallothionein gene and glutathione sulfotransferase gene may play a role in adaptation to Cu²⁺ stress in Pomacea canaliculata.?Metallothionein and glutathione sulfur transferase genes can endow the transformed strains with certain resistance to Cu²⁺ and have the function of resistance to Cu²⁺ This laid the foundation for obtaining the direct evidence that metallothionein and glutathione sulfur transferase genes genes can improve the tolerance of Cu²⁺ in Pomacea canaliculata.