| Bt gene is widely studied and applied in transgenic plants for insect resistance.Its integration and expression not only make the receptor plants obtain insect resistance,but also may cause some unintended effects,such as affecting the growth and development of plants.In order to explore the molecular mechanism of the effect of Bt gene on the growth of receptor plants,obtain transgenic dual Bt poplar with high insect resistance and broad insect-resistance spectrum and their growth were unaffected,and to analyze the mechanism of Bt toxin on target insects.The single Bt gene transgenic poplar 741,dual Bt gene transgenic poplar 741 and non-transgenic control potted seedlings were used as the research objects.The growth physiological indexes of each line were observed,to analyze the growth differences of different types of Bt transgenic poplar 741.The integration characteristics of exogenous genes were identified by NGS sequencing technology.The differences of transcriptome and metabolome of different types of Bt transgenic poplar 741 leaves were analyzed by transcriptome sequencing and untargeted metabolomics technology,to explore the molecular mechanism of dual Bt gene integration and expression affecting the growth of poplar 741.The plant expression vector carrying dual Bt genes Cry1Ac and Cry3A were transformed into Populus×euramericana‘Neva’by Agrobacterium-mediated method.The integration and expression of exogenous genes and insect resistance of transgenic lines were identified by molecular detection and indoor insect feeding experiments.And the growth of transgenic lines was investigated.The differences in the transcriptomes of Anoplophora glabripennis larvae fed transgenic Populus×euramericana‘Neva’lines or non-transgenic control by RNA-seq analyses were determined,so as to reveal the mechanism of Bt toxin inhibiting the development of A.glabripennis larvae.The main results are as follows:（1）Compared with control poplar 741 and single Bt transgenic poplar 741,dual Bt transgenic poplar 741 showed obvious variation.The plant height,ground diameter,section spacing,biomass,photosynthetic pigment content and chlorophyll fluorescence parameters of dual Bt transgenic poplar 741 were significantly lower than those of the control,while the photosynthetic parameters Ci,Cond,and Tr,as well as the contents of IAA,IBA and ABA were higher than those of control.The contents of CTK and GA in some lines were also increased.（2）PCR results showed that the exogenous Bt gene was stably integrated into the poplar741 genome.Based on NGS sequencing technology,two insertion sites of the exogenous gene in CC84 were detected.Two and five insertion sites of Cry1Ac and Cry3A were detected in pc3,and two and four insertion sites for Cry1Ac and Cry3A in pc9,respectively.Five insertion sites in 3 samples were verified by PCR,and the results were basically consistent with those of NGS sequencing.The adjacent genes and their functions within 10 kb upstream and downstream of the insertion site were analyzed.The T-DNA of chromosome 3 was inserted in the 5’untranslated region of the endogenous gene in pc3 and pc9,while the other insertion sites were in the intergenic region.The expression of adjacent gene coding polycomb group protein EMF 2 was upregulated in CC84,while the expression of JGB was downregulated.The expression levels of actin7 and uncharacterized LOC7489800 increased in pc3,while that of lysine like histidine transporter 8 decreased.The expression levels of genes coding chaperone protein Dna J GFA2,catalase-2 and UDP-D-apiose/UDP-D-xylose synthase 2 in pc9 increased,while those of genes coding ubiquitin domain-containing protein DSK2b and BTB/POZ domain-containing protein At3g44820 decreased.（3）Comparative analysis of transcriptome in leaves of single and dual Bt genes transgenic poplar 741 and control poplar 741 showed that,compared with control and CC84,the number of differentially expressed genes（DEGs）in dual Bt transgenic poplar 741 was more,and the number of upregulated genes was higher than that of downregulated genes,while compared with pb29,the number of DEGs compared with pb29 was less.The genes that may be involved in response to the expression of Cry1Ac and Cry3A,secondary transformation and gene interaction were screened out by the analysis of the common DEGs among the different comparison groups.The DEGs were mainly enriched in the pathways of pentose and glucuronate interconversions,starch and sucrose metabolism,flavonoid biosynthesis,gap junction,phenylalanine metabolism,phenylpropanoid biosynthesis,amino sugar and nucleotide sugar metabolism and so on.Compared with control and CC84,WRKY,b HLH and other transcription factor families were differentially expressed,and WRKY was mainly downregulated,while b HLH was upregulated.Compared with pb29,b HLH and other transcription factor families were differentially expressed,and b HLH was mainly downregulated.In plant hormone signal transduction pathway,compared with control and single Bt transgenic poplar 741,the downstream regulatory genes or transcription factors,such as AUX1/LAX,AUX/IAA,SAUR,ARF,TIR1,ARR-B,Sn RK2,PIF,PP2C,EIN3,JAZ,NPR1,TGA and so on,were differentially expressed in dual Bt transgenic poplar 741,which can promote or inhibit the hormone signal transduction and affect the expression of downstream genes,thus affecting plant growth.（4）The gene modules blue and brown,extremely significant positive correlation and negative correlation with plant height,ground diameter,section spacing and biomass,respectively,were selected based on the WGCNA analysis and the correlation analysis between gene modules and traits.The co-expression network of DEGs in the two modules was constructed,and candidate hub genes were screened,including genes coding CBL-interacting serine/threonine-protein kinase 11,protein disulfide-isomerase A1,disease resistance-like protein DSC1,glucomannan 4-beta-mannosyltransferase 2,CYP86A4S,putative cyclin-dependent serine/threonine-protein kinase,ABCG15 and so on.（5）Comparative analysis of leaf metabonomic of poplar 741 with single and dual Bt genes and control poplar 741 showed that,there were significant differences in metabonomic between poplar 741 with dual Bt genes and control,pb29,and CC84.The differentially expressed metabolites（DEMs）that may be involved in response to Bt gene expression,secondary transformation and gene interaction were screened out through the analysis of common DEMs among different comparative groups.The enriched metabolic pathways of DEMs include one carbon pool by folate,biosynthesis of unsaturated fatty acids,fatty acid metabolism,sphingolipid metabolism,fatty acid elongation in mitochondria,phenylpropanoid biosynthesis,glycerophospholipids metabolism,flavonoid biosynthesis,porphyrin and chlorophyll metabolism,zeatin biosynthesis and so on.Phenylpropanoid biosynthesis and flavonoid biosynthesis pathways were the common metabolic pathways enriched in most of the comparative groups.The enriched DEMs mainly included 4-coumaroylshikimate,N1,N5,N10-triferuloyl spermidine,feruloyl-Co A,chavicol,syringin,N1,N5,N10-tricaffeoyl spermidine,N1,N5,N10-tricoumaroyl spermidine,cyanidin,delphinidin,and so on.Zeatin biosynthesis and porphyrin and chlorophyll metabolism pathways were the metabolic pathways co-enriched in the six comparative groups of dual Bt transgenic poplar741 compared with pb29 and CC84.The enriched DEMs mainly include N6-（delta2-Isopentenyl）-adenosine 5’-monophosphate,isopentenyladenosine-5’-triphosphate,trans-zeatin riboside monophosphate,siroheme,magnesium protoporphyrin,magnesium protoporphyrin monomethyl ester,protoporphyrinogen IX,chlorophyllide b,coproporphyrin I,divinylprotochlorophyllide and so on.（6）The metabolic pathways involved together by DEGs and DEMs were analyzed.Compared with control,pb29 and CC84,the key enzyme genes such as 4CL,CYP98A3,HCT,CCo AOMT and bgl B were differentially expressed in the phenylpropanoid biosynthesis pathway,which affected the metabolism levels of feruloyl-Co A,N1,N5,N10-tricoumaroyl spermidine,N1,N5,N10-triferuloyl spermidine,and coumarine in the downstream.The differential expression of CHS,FLS,CYP98A3,CCo AOMT and other genes in flavonoid biosynthesis pathway further affected the metabolism of myricetin,2’,4,4’,6’-tetrahydroxychalcone 4’-O-beta-D-glucoside,feruloyl-Co A and aureusidin 6-O-beta-D-glucoside.In porphyrin and chlorophyll metabolism pathway,genes such as CAO,Hem H,CHLI and POR were downregulated,which affected the metabolism levels of heme O and chlorophyll b.（7）Sixteen transgenic lines were obtained by Agrobacterium-mediated transformation with dual Bt genes into Populus×euramericana‘Neva’genome.The exogenous genes were expressed at both transcriptional and translational levels,and Cry3A protein levels were much higher than those of Cry1Ac.There were some differences in insect resistance among transgenic lines.Some of the transgenic lines exhibited high resistance to the first instar larvae of Hyphantria cunea and Micromelalopha troglodyta,and the first and second instar larvae and adults of Plagiodera versicolora（Corrected mortality rates reached 100%）.Six transgenic lines inhibited the growth and development of A.glabripennis larvae.The average weight of larvae and cellulase activity in midgut were lower than the control.Most of the 1-year-old and 2-year-old seedlings of dual Bt transgenic Populus×euramericana‘Neva’had no significant differences in plant height and ground diameter compared with the control,indicating that the growth of dual Bt transgenic Populus×euramericana‘Neva’was not affected.（8）Transcriptome sequencing was carried out for A.glabripennis larvae fed on dual Bt genes transgenic Populus×euramericana‘Neva’or non-transgenic control,and the DEGs were identified.Hierarchical clustering,functional enrichment of GO and KEGG and analysis of transcription factors were carried out.The reliability of sequencing results was confirmed by RT-qPCR.Candidate genes involved in Bt toxin response were screened,including Bt prototoxin activation,digestive enzymes,binding receptors,detoxifications and and protective enzymes.The results showed that Bt toxin could inhibit the growth and development of A.glabripennis larvae by regulating the expression of genes related to the growth and development of A.glabripennis larvae.This study lay a theoretical foundation for developing resistance to A.glabripennis in poplar,and provide a foundation for exploring the mechanism of Bt toxin action on Cerambycidae insects. |
