| Dendrolimus punctatus(Lepdoptera, Lasiocampidae) is the most important defoliator in China and Hyphantria cunea is a worldwide quarantine pest. These two pests not only lead to huge losses of forestry production and ecological environment, but also seriously affect the stability of forest ecosystem in China. So it’s urgent to develop new methods on long-term and continuous forest pests control. Population genetic manipulation method has great potential on pest control for overcoming the drawback of traditional methods which could not control pests persistently. We carried out researches on selecting sex-related genes from transcriptome data,cloning and functional studying of predicted sex determination genes, constructing genetic transformation and genome editing platform and so on to develop new methods on persistently pests control and obtained the following results:First, Transcriptome analysis of D. punctatus To get a comprehensive understanding of genetics characteristics of D. punctatus, we employed Illumina high-throughput pyrosequencing to generate EST databases of a total 11 samples: 4 developmental stages(egg, larva, pupa,adult), 5 organs of 5 instars larvae(head, midgut, fatbody, ovary, testis) and 2 adult antenna samples(female and male). Totally 510 M reads were obtained, with an average read length was 950.1051 bp. These reads were assembled into 50 M transcripts and 54102 unigenes.BlastX search against the NCBI-NR database revealed that 17985(33.2%) of these unigenes match significantly.In the four developmental stages of D. punctatus, larval stage showed the greatest number change of genes. In the five organs of D. punctatus, testis and ovary have a greater difference than the non-reproductive organs; Compared testis with ovary, there were 894 high expression genes in ovary while 1344 genes in testis; The adult antennae are the major organs of sexual dimorphisms, in comparison with female and male antennae, there were 589 highly expressed genes in male antennae and 227 genes in female antennae.Compared with the reported sex-determining genes in Drosophila and Bombyx, we obtained 21 homologous genes in D.punctatus. In the initial signal X: A, Da, Emc and Gro homologous genes were found; In metering compensation way, Msl3, Mle and Mof showed homologous genes; In somatic cell sex determination ways, Sxl, Tra2, Dsx, Ix, Doa, Snf, Vir and Fl(2)d had homologous genes; In the mating behavior and species differentiation pathway,Fru, Dsf, Out and Ovo had homologous genes; Other genes reported in silkworm, Imp, Hrp28,Psi and Piwi were also found in D. punctatus. Of top20 genes in testis, 11 genes without fun-ctional annotation may be involved in sex determination pathway which remains to be further investigated.Second, Cloning and functional studies of Dsx, Tra2 and Ix in D. punctatus We first cloned Dsx, Tra2 and Ix genes and knockdown of these genes’ expression by RNAi. The complete ORFs of three genes’ were amplified by RACE. Six-specifically splice pre-mRNAs of DpDsx coding for one male- and two female-specific proteins. There were five female-specific DpDsx, comprised of 884 bp, 990 bp, 1148 bp, 1269 bp and 1962 bp,respectively, and coding for two proteins with 243 and 252 amino acids; one male-specific DpDsx, of which total length of mRNA was 1612 bp encoding protein with 275 amino acids.There were four alternative splicing isoforms of DpTra-2, which length of CDS were 765 bp,768 bp, 843 bp and 858 bp, coding for four proteins with 254, 255, 255 and 285 amino acids,respectively; In this study, two splicing forms of DpIx were detected in D. punctatus, named DpIx-a and DpIx-b, respectively. The nucleotide length of DpIx-b was 576 bp encoding a protein of 192 amino acids; While DpIx-a was shorter in the deduced second exon and contained termination codon at the deduced third exon, it was only about 222 bp encoding 74 amino acids. RNAi results indicated that DpDsx, DpTra2 and DpIx had a connection between each other, however, no phenotype was obtained for more effective gene editing tools need to be used.Third, genetic analyses in D. punctatus and H. cunea In order to test the feasibility of transgenosis, pBac[A3-3*P3/EGFP] and pBac[IE1/dsRed] plasmids were injected into the embryos of D. punctatus and H. cunea. Fluorescence detection and excision assay resultsshowed Piggybac transposon system could be used to transgenosis construct in D. punctatus and H. cunea.Instantaneous expression experiments indicated that pBac [IE1/ds Red] could work in D.punctatus and H. cunea. On this basis, we coinjected pBac [IE1/dsRed] with pBac[A3/hepler]and transposase mRNA into these two pests at embryonic stages. We finally got G1 generation in D. punctatus, but the red fluorescence disappeared gradually during the development stages.PCR and inverse PCR results suggested that Piggybac transposons might not be able to stably expressed in D. punctatus.Forth, genome editing in D. punctatus and H. cunea using the CRISPR/Cas9 system To achieve the genome editing in D. punctatus and H. cunea, Cas9 mRNA and sgRNAs were co-injected into embryos. CRISPR/Cas9 could mediated Abdominal-a and Wnt-1 mutagenesis in D. punctatus with the 70.4% and 77.5% mortality rate, respectively. Of mutations, 47.5%and 55% have deletions, 17.5% and 32.9% showed observable phenotypes in G0 gametes,respectively. Knocking out Abd-a and Wnt-1 induced same phenotype of abnormal posterior segments. Besides this, variable phenotypes were observed in Wnt-1 mutations with a loss of limbs and deformation of heads, indicating that Wnt-1 signaling pathway is also necessary for the anterior segments development and appendages development. In H. cunea,CRISPR/Cas9-based mutagenesis of HcWnt-1 led to a high mortality rate(99.8%) and a mutation efficiency of 62.5% at embryonic stages following injection of Cas9 mRNA and sgRNAs with 1000 eggs. Segments fusion and appendages deficiency were obtained from HcWnt-1 mutants. These results demonstrated that CRISPR/Cas9 system is efficient in inducing mutations at specific genome locus of D. punctatus and H. cunea, which is suiltable for functional research in non-traditional species. RT-PCR and Immunohistochemistry were used to explore the expression pattern of HcWnt-1, indicating that the segmentation model of H.cunea belongs to short and intermediate germ band. Overall, these results suggested that Wnt-1involve in segmentation and A-P axis formation in D. punctatus and H. cunea at early embryonic stages, while Abd-a plays a role in posterior segemental identify form from second to seven abdominal segments at later embryonic stages.Genetic manipulation is a new way for sustainable pest control strategy. We found that Piggybac carrier is suitable for genetic operation in D. punctatus and H. cunea. In addition, the CRISPR/Cas9 system could work effectively in D. punctatus and H. cunea, providing a new tool for functional research in non-model organisms. The transgenosis and genome editing is the basis of genetic control of pests, which would provide theoretical support for sustainable control of pests. |
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