Biological Function Analysis Of LTR Retrotransposons In Cotton

Crops can bring us significant economic benefits,and cotton is one of them,especially in China.More than 50% of the cotton genomes are composed of TEs,and LTR retrotransposons are the most abundant transposable elements in cotton TEs.Among them,Copia-like and Gypsy-like elements account for the highest proportion.In plants,only a few structural genes of LTR retrotransposons can be fully expressed to produce functional proteins with transposable activity.Premature termination will be formed due to the base substitution in most of LTR retrotransposons,and old TEs within new TEs inserted may also be one of the reason.The early termination of translation causes the fragmentation of the transposable elements,which can not form all of the functional proteins used for transposition,producing the TEs without transposable activity.We first analyzed the proportion,insertion time,sequence and three-dimensional molecular structure of Copia and Gypsy TEs in G.arboreum(AA),G.raimondii(DD)and G.hirsutum(AADD)using bioinformatics technologies,speculating the possible biological function of Copia proteins in cotton.Then,we analyzed the binding activity and binding position of Copia functional proteins in G.arboreum and G.hirsutum using ChIP assay and high-throughput sequencing.Finally,we transformed Copia coding genes into pichia pastor,analyzing the expression products from the pichia pastor by MS,which can help us know the expression pattern and biological function of Copia coding genes in cotton.The main research results are as follows:(1)According to the analysis of cotton genome,it was found that the proportion of LTR retrotransposon(Copia+Gypsy)in cotton genome from high to low was:G.arboreum,G.hirsutum and G.raimondii,respectively.Only a few conserveddomains of Copia-like elements can be expressed in cotton leaves,ovules and germinated seeds,and only IN and RT domain coding transcript can be identified according to the RNA-seq data.The sequence identities of IN and RT domain from Copia-like elements within the G.arboreum and G.hirsutum genomes were more than90%.(2)Quantitative analysis of two conserved domains,RT and IN,by RT-qPCR showed that the transcriptional level of these two domains were higher in G.arboreum than in G.hirsutum.Immunoblot assay was performed using the specific antibody anti-copiaRT,and it was found that the two domains of IN and RT may be co-expressed together as a single protein.ChIP-qPCR analysis of various tissue in G.arboreum and G.hirsutum using the specific antibody anti-copiaRT,showed that the IN and RT proteins preferentially bind to the first exons of the functional genes.(3)The complete Copia structural gene was cloned in G.arboreum,which was3957 bp in length and encoded 1318 amino acids.The Copia structural gene was then transformed into Pichia pastoris and the target gene was successfully expressed.And the MS analysis of induced expression product in pichia pastor,showed that the expression level of self-proteins in pichia pastor has been changed after insertion of Copia.Above all,the expression of the coding genes of Copia-like elements is not only an important part of the transposition mechanism,but also regulates the expression of functional genes by other means besides the transposition.