Functional Analysis Of The Grx1 Gene In Testis Of Locusta Migratoria

Glutaredoxins?Grxs?,also known as thiol transferase,is a family of small molecular weight oxidoreductases that play an important role in maintaining the redox homeostasis in cells and protecting plants' and animals' cells from oxidative damage.In this paper,the full-length c DNA fragment encoding the Locusta migratoria Grx1 was amplified by RT-PCR.The sequence analysis showed that the open reading frame of the gene consists of 333 nucleotides,encoding 110 amino acid residues with a predicted molecular mass of 11.96 k Da and an isoelectric point of 6.56.Protein analysis revealed that the protein has a conserved domain:GRX_GRXh_12like.The amino acid sequence identity of Grx1 of Locusta migratoria and the Grx1 orthologs from other insects is between 60% and80%.The homology study shows that the locust Grx1 shares a common active site CPYC with other insect Grxs.Thus the locust Grx1 belongs to the glutaredoxion family and is named as Lm Grx1.The transcriptional level of LmGrx1 gene in various developmental stages and different tissues of Locusta migratoria was examined by real-time PCR.The results showed that Lm Grx1 was expressed from egg stage to adult,and it was highly expressed in adult.Lm Grx1 is expressed in insect antennae,intestine,gastric caecum,malpighian tube,fat body,testis,especially strongly expressed in testis.However,the expression levels in the ovaries,muscles,hemolymph,epidermis,and brain are extremely low.Following the developmental stages of the testis in migratory locust,the expression level of Lm Grx1 showed a trend of increasing first and then decreasing,and peaked on the fourth day of adult.To investigate the function of LmGrx1 in testis development,we injected the locust at the first day of fourth instar nymph with ds RNA,the Lm Grx1 was successfully silenced.It was found that during the whole developmental process from the fourth instar to adult,there was no significant difference between the treated group and the control group in both the overall morphology and the sperm cell development process.In order to further explore the role of Lm Grx1 in the developmental of the testis,the ds Lm Grx1 was injected into the nymphs on the first day of the fourth instar.After 24 hours,the testis of the treated and control groups?with ds GFP?were taken and the total RNA was extracted for RNA sequencing analysis.A total of7670 Unigenes were discovered,of which 3995 were functionally annotated;46 differentially expressed genes?DEGs?were obtained,of which 30 were up-regulated and 16 were down-regulated;further analysis revealed that interference with Lm Grx1 would affect intracellular transcription,the expression of genes in the translation process,as well as genes related to post-translational modification of proteins and some long non-coding RNAs.Eleven genes were further selected and the expression patterns of these DEGs were confirmed by RT-q PCR.In summary,the developmental stages and expression patterns of the Lm Grx1 in the migratory locust were detected.The function of the gene in the locust was explored by RNA interference technology.The transcriptome database of the testis in locust was constructed and analyzed by bioinformatics.The real-time quantitative detections of some genes were carried out to reveal its expression trends.The results of this study will provide basic data for the screening of molecular targets for the control of locusts.