Analysis On The Roles Of KIF3A And KIF18A During Spermiogenesis Of Gampsocleis Gratiosa

Gampsocleis gratiosa belongs to Tettigoniidae in Orthoptera,which is commonly known as katydid.G.gratiosa is easily reared and observed with large size,unique arrow shape of sperm,complicated acrosome complex and clear nucleus under the optical microscope.So it is considered to be an ideal material for studying spermiogenesis of Orthoptera.Firstly,KIF3A(kif3a)and KIF18A(kif18a)are identified by gene cloning and bioinformatics.Secondly,RNA interference,real-time fluorescence quantitative PCR,frozen section and immunofluorescence,paraffin section and PAS staining are used to analyze the effects of RNA silencing during spermiogenesis of Gampsocleis gratiosa.This study lay a foundation for the research of the role of kinesins in spermiogenesis of Orthoptera insects.The four transcriptome of G.gratiosa were compared to select one sequence of KIF3 A and KIF18 A respectively.Kif3 a and kif18 a gene are cloned in G.gratiosa transcriptome and analyzed of their bioinformatics.Kif3 a cDNA length is 3590 bp,including an open reading frame of 2130 bp encoding a protein of 709 amino acids residues with molecular mass of about 80.33 KD and isoelectric point of approximately 5.35.Kif18 a cDNA length is 6766 bp,including an open reading frame of 4674 bp encoding a protein of 1557 amino acids residues with molecular mass of about 171.25 KD and isoelectric point of approximately 8.62.The sequence alignment of KIF3 A and KIF18 A in different species showed that their ATP binding sites and microtubule binding sites were highly conserved.The neighbor-joining tree using possion model with KIF3 A and KIF18 A acid sequence analysis showed that KIF3 A and KIF18 A were conservative in evolution.Based on RNA interference,we inject dskif3 a and dskif18 a into the second or third abdominal sternum of G.gratiosa respectively.Compared to the control group,the treatment groups of dskif3 a and dskif18 a showed changes at the fourth,fifth,sisth,seventh(the deform period of spermatid).At the rhombic phase,the overall morphology of sperm cells was basically diamond-shaped in the control group.The microfilaments are three dots in acrosome complex and the fluorescence intensity is the weakest in the middle.Furthermore,the fluorescence intensity at both ends is stronger along with the acrosome proper.The acrosome proper begin to form,and the fluorescence intensity of the microtubules on both sides of the acrosome proper increase,which may be the formation of manchette-like structure.In the dskif3 a treatment group,the fluorescence intensity of the dotted microfilaments in the middle was enhanced.While in the dskif18 a treatment group,the morphology of sperm cells changed significantly.There were no bright microtubule fluorescence signals on both sides of acrosome proper.The dot-like structure in the middle disappeared,and the morphology of nucleus changed obviously,showing a semicircle.Compared with the control group,the microtubule structure of the front of acrosome complex in dskif3 a group at the cylindrical phase is disappeared.In the dskif18 a group,the brighter microtubule structure on both sides of acrosome complex is changed.There is no significant change about nuclear morphology in both groups.At the critical period of sperm morphogenesis,the microtubules around acrosome complex and nucleus in treatment groups are breaked,and the apical capsule microtubules is disappeared.In addition,the nucleus become heterogeneous.At the pre-mature period,the microtubules around nucleus appear several small circles,and the flagella is broken.But the morphology of the nucleus is not changed significantly in the treatment of dskif3 a.The microtubules and microfilaments around nucleus also disappear partially,as well as,the nucleus larges obviously in the treatment group of dskif18 a.In PAS staining of the two groups,the nuclei became uneven and granular substances could be observed clearly.By comprehensive analysis of the results,the conclusions are as follows:(1)Kif3a and kif18 a genes are identified and analyzed in this paper.Real-time Quantitative PCR Detecting System shows that kif3 a and kif18 a genes are highest expressed in the testis of the male adult of Gampsocleis gratiosa.(2)After injection of dskif3 a and dskif18 a respectively,the expression quantity of kif3a mRNA and kif18 a mRNA are the lowest on the fourth day,that is,when the interference time is 96 hours,the interference effect is the most significant.(3)After RNAi,the sperm development of Gampsocleis gratiosa is disordered and the microtubule structure changes obviously.It is speculated that KIF3 A and KIF18 A may tether microtubule during spermiogenesis of Gampsocleis gratiosa.