Cloning And Functional Characterization Of Pxβtubulin0 And Its Promoter In Plutella Xylostella

The βTubulin plays an essential role in the formation of microtubules in animals.It is specifically expressed in the testis of Drosophila and silkworm,and plays an essential role in the formation of sperm and male reproductive development.In Drosophila,the mutation of βTubulin causes male sterility,and the promoter of the βTubulin has also been proved to be a testis-specific promoter in Drosophila and silkworm,which is important for the construction of male sterile transgenic lines.However,this gene and its promoter have not been studied in Plutella xylostella.The present study aims to identify and study the functions of PxβTubulin0 gene and the sequence characteristics of its promoter.The main research content and results are as follows:(1)The artificial diet strain G88 of P.xylostella was used in this study.Total RNA was isolated from the indiviudals and transcribed into the firststrand c DNA.Two Pxβtubulin genes(PxβTubulin0 and PxβTubulin2)were amplified by PCR.The stage-and sex-specific expression patterns of these two genes were identified through q RT-PCR.The results showed that Pxβtubulin0 is a male-specific gene,and it is specifically highly expressed in male pupae,followed by male adults.The tissue expression pattern of the gene was further identified by q RT-PCR,and the results showed that the gene was specifically and highly expressed in the testis.Therefore,the PxβTubulin0 of P.xylostella is determined to be a testis-specific gene.The gene consists of two exons and one intron.The full length of CDS is 1344 bp,encoding 447 proteins.Phylogenetic tree results showed that the PxβTubulin0 of P.xylostella had the closest relationship with citrus swallowtail and silkworm.CRISPR/Cas9 technology was used to clarify the role of PxβTubulin0 in the male reproductive development of P.xylostella.PxβTubulin0 gene of P.xylostella was knocked-out through CRISPR/Cas9,and two homozygous strains of(-5 bp)and(-18+1 bp)were obtained.Bioassay results showed that the knockout of the PxβTubulin0 in P.xylostella significantly reduced the egg amount and hatching rate and also significantly inhibited the testis development.However,the formation of spermatophore in females of P.xylostella gland formation,mating response time and total mating time were not affected.(2)The dual-luciferase reporter assay system was used to verify the in-vitro driving activity of the PxβTubulin0 promoter.A total of 2006 bp sequence of putative was PCR amplified.TATA box,GATAd,BR,Abd-B,hth,and some other common transcription factor binding sites were identified from the PxβTubulin0P,as well as gonad-specific doublesex binding sites,β0UE1 binding sites similar to silkworm testis-specific promoter β2tubulin P and the β0UE2 binding site similar to Drosophila testis-specific promoter Bmβ4P.Next,eight different lengths of promoter sequences of 2006 bp,1733 bp,1576 bp,1391 bp,1065 bp,870 bp,577 bp and 256 bp were used to construct PGL transient expression vectors.The results showed that,the promoter sequences were active except for the promoter of 870 bp;the promoter of 1065 bp had the highest activity.In summary,this study identified the testis-specific PxβTubulin0 in P.xylostella,and clarified the role of this gene in male reproductive development,providing a foundation for the further study on function of Tubulin in P.xylostella.At the same time,the in vitro driving activity of the PxβTubulin0 promoter was successfully identified and analyzed,laying the foundation for the analysis of the expression pattern of PxβTubulin0P in different tissues and different developmental stages.It is also a foundation for the potential use of this promoter to develop transgenic P.xylostella to access the in-vivo functions.Furthermore,the PxβTubulin0promoter can be used to develop the male-lethal genetic control for P.xylostella.