Phylogeny of Trigonotis in China-with a special reference to its nutlet morphology and plastid genome

The genus Trigonotis comprises nearly 60 species mainly distributed in East and Southeast Asia.China has the largest number of Trigonotis species in the world,with a total of 44 species,of which 38 are endemic.Nutlet morphology is useful for the taxonomic delimitation of Trigonotis.However,there are still controversial circumscriptions of nutlet shape in some species.In previous studies,interspecies phylogenetic relationships were inferred using few DNA markers and very few taxa,which possibly led to erroneous or incomplete conclusions.In this study,the nutlet morphology of 39 Trigonotis taxa and the characteristics of 34 complete chloroplast genomes(29 taxa)were investigated and analyzed.Then,the phylogenetic relationships were discussed within this genus based on complete chloroplast genomes.To the best of our knowledge,this study is the first comprehensive analysis of nutlet morphology and complete chloroplast genome of Trigonotis.Based on nutlet morphology,Trigonotis can be divided into two groups:Group 1,hemispherical or oblique tetrahedron with carpopodiums,and Group 2,inverted tetrahedron without carpopodiums.The chloroplast genome of Trigonotis exhibited a typical quadripartite structure,including 84-86 protein-coding,37 transfer RNA,and 8 ribosomal RNA genes,with a total length of 147,247-148,986 bp.Genes in the junctions were well conserved in Trigonotis,similar to those in other Boraginaceae s.str.species.Furthermore,Trigonotis chloroplast genomes showed relatively high diversity,with more conserved genic regions than intergenic regions;in addition,we detected 14 hot spots(Pi>0.005)in non-coding regions.Phylogenetic analyses based on chloroplast genome data identified highly resolved relationships between Trigonotis species.Specifically,Trigonotis was divided into two clades with strong support:one clade included species with hemispherical or oblique tetrahedron nutlets with carpopodiums and bracts,whereas the other clade included species with inverted tetrahedron nutlets without carpopodiums or bracts.Our results may inform future taxonomic,phylogenetic,and evolutionary studies on Boraginaceae.

木薯MeMinD的互作蛋白筛选

MinD参与质体分裂的精细调控,在质体形态维持中起着关键作用。实验室前期研究发现MeMinD蛋白参与了木薯质体的分裂,然而与MeMinD蛋白协同调控木薯质体分裂的相关蛋白尚未明确。本研究构建了pGBKT7-MeMinD载体,通过酵母双杂交文库筛选,共获得MeMinD蛋白的8个候选互作蛋白,点对点验证后发现烟酸磷酸核糖转移酶2(MeNAPRT2)与MeMinD存在互作关系。该研究结果为进一步解析MeMinD参与调控木薯质体分裂的相关机制提供新的信息。

栎属麻栎亚组与冬青栎组的质体捕获历史研究

栎属(Quercus)麻栎亚组(subsect.Campylolepides)包括麻栎(Q.acutissima)、栓皮栎(Q.variabilis)及小叶栎(Q.chenii)3个物种,是栎属土耳其栎组(section Cerris)的东亚分支,前人已经对麻栎亚组或组内物种的物种形成和谱系地理做过深入研究,也发现了土耳其栎组(section Cerris)曾与冬青栎组(section Ilex)发生过古基因渐渗并导致质体捕获,但目前麻栎亚组与冬青栎组质体的具体进化历史仍不清楚。该研究对15个冬青栎组的样品进行了基因组浅层测序,并整合先前发表的麻栎亚组及其近缘类群共计325个重测序数据,其中麻栎亚组3物种19居群276个体,利用这些数据进行质体基因组组装和分析。结果表明:(1)麻栎亚组3个物种间存在共享单倍型,但整个麻栎亚组的质体单倍型基本构成一个单系分支,嵌套在华中至四川凉山州一带冬青栎组物种组成的分支中。(2)麻栎亚组物种中一个来自辽东半岛的孑遗麻栎单倍型与冬青栎组物种万山栎(Q.pseudosetulosa)聚为一个进化枝。(3)两次质体捕获事件均发生在中新世中期,在此之后麻栎亚组与冬青栎组未发生过质体捕获,推测麻栎亚组跟冬青栎组物种目前已经形成近乎完全的生殖隔离。该研究表明冬青栎组和麻栎亚组在质体基因组的演化历史中经历了古老的质体捕获事件,并最终逐步形成了相对独立的演化路径。

Effects of Tobacco Plastid Division Genes NtFtsZ1 and NtFtsZ2 on the Division and Morphology of Chloroplasts

As an important group of plant cellular organelles, the molecular mechanism of plastid division is poorly understood. Recent studies have revealed that the homologs of ftsZ gene, an essential prokaryotic cell division gene, are involved in plastid division process of plant cells. Antisense and sense expression constructions were employed to investigate the functions of the two ftsZ genes, NtFtsZ1 and NtFtsZ2, in transgenic Nicotiana tabacum L. plants. Although antisense expression of,NtFtsZs reduced the native protein level obviously, the size and number of chloroplasts in transgenic tobacco plants had no effect. In contrast, overexpression of NtFtsZs in transgenic plants strikingly changed the number and morphology of chloroplasts. Even only 1 - 2 huge chloroplasts could be seen in the mesophyll cells of some overexpression transgenic plants. Analyses of chloroplast ultrastructures and chlorophyll content of different transgenic plants suggested that NtFtsZs gene have no direct influence on the normal development and function of chloroplasts. ne changes in chloroplast morphology must be a compensation for the change in chloroplast number. The different phenotypes of chloroplasts in antisense and sense transgenic plants implied that different members from the same ftsZ gene family may have similar function in controlling plastid division. Meanwhile, the changes of chloroplast morphology in sense transgenic plants represented the possible plastoskeleton function of ftsZ in higher plant.

PATERNAL INHERITANCE OF PLASTID DNA IN GENUS PHARBITIS

The inheritance of plastid DNA in Pharbitis was studied by the method of restriction fragment length polymorphisms (RFLP).Experimental results showed that plastid DNA from Pharbitis was paternally inherited in reciprocal crosses,P. nil ×P. limbata and P. limbata×P. nil hybrids.But,in the cross of P. limbata×P. nil,the possibility of biparental inheritance of plastid DNA could not be roled out in our preliminary experiment.Thus Pharbitis became the third genus among angiosperms characterized with male plastid transmission.The mechanisms of paternal plastids DNA inheritance in Pharbitis is unclear.The authors proposed that dilution,exclusion and/or degeneration of maternal plastid,including their DNA,after fertilization should be considered.

Plastid Inheritance in Sweet Potato as Revealed by DNA Restriction Fingerprinting

The inheritance of chloroplast DNA (cpDNA) in sweet potato (Ipomoea batatas Lain.) was analyzed using DNA restriction fingerprinting. The cpDNA fingerprints of hybrids from reciprocal crosses between Xushu18 and AB78-1 were found to be identical to those of their female parents, which reveals that cpDNA of sweet potato is maternally inherited in this intervarietal crossing. This maternal cpDNA transmission pattern does not accord with the putative one based on former cytological studies. The plastid inheritance in Convolvulaceae has been briefly reviewed in this study, and the utility of DNA restriction fingerprinting analysis in the study of plastid inheritance is also discussed.

植物对重金属的吸收和转运途径及调控机制研究进展

论述了重金属进入植物根表皮层的途径,通过共质体与质外体水平运输的短程运输途径,以及木质部与韧皮部进行装卸的长程运输途径。共质体途径对重金属离子的转运速度慢,而质外体途径转运重金属离子的迁移阻力小、速率快。细胞壁吸附解析作用是影响重金属离子通过细胞质外体途径传入根木质部的内因,但根系皮层内凯氏带及软木脂发育,构成质外体屏障,能阻断重金属离子向木质部迁移的质外体途径。植物蒸腾作用及根压为木质部重金属离子的长距离运输提供驱动力,和植物的蒸腾速度有关。植物对重金属解毒与耐性的机理主要是诱导植物产生金属硫蛋白、胁迫植物形成植物螯合肽(PCs)和液泡对重金属的区域化作用。

Localization of Two GFP_tagged Tobacco Plastid Division Protein NtFtsZs in Escherichia coli

Two plastid division genes, NtFtsZ1 and NtFtsZ2 isolated from Nicotiana tabacum L. were fused with gfp and expressed in Escherichia coli . The regular localizations of full length NtFtsZs∶GFP along the filamentous bacteria indicated that the NtFtsZs could recognize the potential division sites in E. coli and be polymerized with heterogeneous FtsZ from bacteria. The overexpression of NtFtsZs ∶ gfp inhibited the division of host strain cells and resulted in the long filamentous bacterial morphology. These results suggested that eukaryotic ftsZs have similar function to their prokaryotic homologs. Meanwhile, the different deletions of motifs of NtFtsZs are also employed to investigate the functions of these proteins in E. coli . The results showed that the C_terminal domains of NtFtsZs were related to the correct localization of NtFtsZs in E. coli and the N_terminal domains of NtFtsZs were responsible for the polymerization of homogeneous and heterogeneous FtsZ proteins. The significance of these results in understanding the functions of NtFtsZs in plastid division were discussed.

北京丁香质体基因组研究

为了解北京丁香的质体基因组特征,本研究分析了北京丁香的质体基因组结构和基因构成,并联合其它35个木犀科物种和2个泡桐科物种进行了系统发育分析。结果表明:(1)北京丁香的质体基因组共编码131个基因,包含36个tRNA基因、8个rRNA基因和87个mRNA基因,分别占所有注释基因的27.48%、6.11%和66.41%;(2)在该质体基因组的密码子组成中,RSCU值大于1的密码子有30个,31个密码子的RSCU值小于1,且密码子有以G和C结尾的使用偏好性;(3)北京丁香质体基因组中共识别出178个正向重复序列和177个回文重复序列;(4)NJ系统发育树表明北京丁香与丁香属植物暴马丁香亲缘关系较为密切,丁香属植物与女贞属植物亲缘关系密切。

β-Amylase Is Predominantly Localized to Plastids in the Developing Tuberous Root of Sweet Potato

Starch degradation in cells is closely associated with cereal seed germination, photosynthesis in leaves, carbohydrate storage in tuber and tuberous roots, and fleshy fruit development. Based on previously reported in vitro assays, β amylase is considered as one of the key enzymes catalyzing starch breakdown, but up to date its role in starch breakdown in living cells remains unclear because the enzyme was shown often extrachloroplastic in living cells. Recently we have shown for the first time that β_amylase is predominantly immuno_localized to plastids in living cells of developing apple fruit. But it remains to know whether this model of β_amylase compartmentation is more widespread in plant living cells. The present experiment, conducted in tuberous root of sweet potato ( Ipomea batatas Lam. cv. Xushu 18) and via immunogold electron_microscopy technique, showed that β amylase visualized by gold particles was predominantly localized in plastids especially at periphery of starch granules, but the gold particles were scarcely found in other subcellular compartments, indicating that the enzyme is subcellularly compartmented in the same zone as its starch substrates. The density of gold particles (β amylase) in plastids was increasing during growing season, but the predominantly plastid_distributed pattern of β amylase in cells was shown unchanged throughout the tuberous root development. These data prove that the enzyme is compartmented in its functional sites, and so provide evidence to support the possible widespread biological function of the enzyme in catalyzing starch breakdown in plant living cells or at least in living cells of plant storage organs.

A truncated hepatitis E virus ORF2 protein expressed in tobacco plastids is immunogenic in mice

AIM： To cost-effectively express the 23-ku pE2, the most promising subunit vaccine encoded by the E2 fragment comprising of the 3＇-portion of hepatitis E virus （HEV） open reading frame 2 （ORF2） in plastids of tobacco （Nicotiana tabacum cv. SR1）, to investigate the transgene expression and pE2 accumulation in plastids, and to evaluate the antigenic effect of the plastid-derived pE2 in mice. METHODS： Plastid-targeting vector pRB94-E2 containing the E2 fragment driven by rice psbA promoter was constructed. Upon delivery into tobacco plastids, this construct could initiate homologous recombination in psaB-trnfM and trnG-psbC fragments in plastid genome, and result in transgene inserted between the two fragments. The pRB94-E2 was delivered with a biolistic particle bombardment method, and the plastid-transformed plants were obtained following the regeneration of the bombarded leaf tissues on a spectinomycin-supplemented medium. Transplastomic status of the regenerated plants was confirmed by PCR and Southern blot analysis, transgene expression was investigated by Northern blot analysis, and accumulation of pE2 was measured by ELISA. Furthermore, protein extracts were used to immunize mice, and the presence of the pE2-reactive antibodies in serum samples of the immunized mice was studied by ELISA. RESULTS： Transplastomic lines confirmed by PCR and Southern blot analysis could actively transcribe the E2 mRNA. The pE2 polypeptide was accumulated to a level as high as 13.27 μg/g fresh leaves. The pE2 could stimulate the immunized mice to generate pE2-specific antibodies. CONCLUSION： HEV-E2 fragment can be inserted into the plastid genome and the recombinant pE2 antigen derived is antigenic in mice. Hence, plastids may be a novel source for cost-effective production of HEV vaccines.

Plastid phylogenomics and biogeography of the medicinal plant lineage Hyoscyameae(Solanaceae)

The cosmopolitan family Solanaceae,which originated and first diversified in South America,is economically important.The tribe Hyoscyameae is one of the three clades in Solanaceae that occurs outside of the New World;Hyoscyameae genera are distributed mainly in Europe and Asia,and have centers of species diversity in the Qinghai-Tibet Plateau and adjacent regions.Although many phylogenetic studies have focused on Solanaceae,the phylogenetic relationships within the tribe Hyoscyameae and its biogeographic history remain obscure.In this study,we reconstructed the phylogeny of Hyoscyameae based on whole chloroplast genome data,and estimated lineage divergence times according to the newly reported fruit fossil from the Eocene Patagonia,Physalis infinemundi,the earliest known fossil of Solanaceae.We reconstructed a robust phylogeny of Hyoscyameae that reveals the berry fruit-type Atropa is sister to the six capsule-bearing genera(Hyoscyameae sensu stricto),Atropanthe is sister to the clade(Scopolia,Physochlaina,Przewalskia),and together they are sister to the robustly supported AnisoduseHyoscyamus clade.The stem age of Hyoscyameae was inferred to be in the Eocene(47.11 Ma,95%HPD:36.75e57.86 Ma),and the crown ages of Hyoscyameae sensu stricto were estimated as the early Miocene(22.52 Ma,95%HPD:15.19e30.53 Ma),which shows a close correlation with the rapid uplift of the Qinghai-Tibet Plateau at the Paleogene/Neogene boundary.Our results provide insights into the phylogenetic relationships and the history of the biogeographic diversification of the tribe Hyoscyameae,as well as plant diversification on the Qinghai-Tibet Plateau.

Occurrence and light response of residual plastid genes in a Euglena gracilis bleached mutant strain OflB2

Euglena gracilis is a unicellular green eukaryotic microalga that features characteristics of both plants and animals.The photosynthetic function of its chloroplast is easily lost under stress resulting in bleached mutants,while the physiological role of their residual plastid DNAs remains unclear.In this study,we obtained five bleached mutants by ofloxacin(Ofl)treatment,identified 12 residual plastid genes in five bleached mutants,and determined the mRNA levels in the wild type E.gracilis(WT)and one bleached mutant(OflB2)under dark and light stimulation conditions by quantitative reverse transcribed PCR(qRTPCR).Results show that the expression of all selected plastid genes in both WT and OflB2 mutant did not change significantly in darkness,while their responses to light stimulation were different.Under the light stimulation conditions,half of the genes did not change significantly,while most of the other genes were down-regulated in OflB2 mutant and up-regulated in WT.Therefore,the bleached mutant retains part of the plastid genome and the plastid relic is responsive to light.Our research will help to understand the functions of residual plastid DNA and evolution of chloroplasts.

Construction of Lactuca sativa Plastid Transformation Vector and High-level Expression of gfp Gene in Escherichia coli

Using genomic DNA of bolting-tolerant lettuce as a template,flanking fragments of lettuce plastid rpo A gene were amplified and cloned by PCR. Targeting the sites of these two fragments,homologous recombinant fragments of exogenous gene were integrated to construct lettuce plastid expression vector p Brpo AGFP,which harbored the expression cassette Prrn-gfp-aad A-Tpsb A. The results showed that the amplified flanking fragments were 1.2 and 1.1 kb in size. After sequencing,restriction digestion,ligation and transformation,lettuce plastid expression vector containing expression cassette Prrn-gfp-aad A-Tpsb A was constructed and confirmed by SDS-PAGE electrophoresis. The results of SDS-PAGE electrophoresis indicated that gfp gene was efficiently expressed under the regulation of plasmid specific promoter Prrn and terminator Tpsb A. GFP accounted for 45. 6% of total soluble proteins; inclusion bodies accounted for 47.5 % of bacterial proteins,which reached relatively high expression levels. The construction of lettuce plastid expression vector p Brpo A-GFP laid a solid foundation for establishment of subsequent lettuce plastid transformation system and genetic improvement of lettuce using various functional genes.

The Potential of DNA Barcode-Based Delineation Using Seven Putative Candidate Loci of the Plastid Region in Inferring Molecular Diversity of Cowpea at Sub-Species Level

The novelty and suitability of the mitochondrial gene CO1 in DNA barcoding as a reliable identification tool in animal species are undisputed. This is attributed to its standardized sequencing segment of the mitochondrial cytochrome c oxidase-1 gene (CO1) which has the necessary universality and variability making it a generally acceptable barcode region. CO1 is a haploid single locus that is uniparentally-inherited. Protein-coding regions are present in high-copy numbers making it an ideal barcode. The mitochondrial oxidase subunit I (COI) gene is a robust barcode with a suitable threshold for delineating animals and is not subject to drastic length variation, frequent mononucleotide repeats or microinversions. However, a low nucleotide substitution rate of plant mitochondrial genome [mtDNA] precludes the use of CO1 as a universal plant DNA barcode and makes the search for alternative barcode regions necessary. Currently, there exists no universal barcode for plants. The plastid region reveals leading candidate loci as appropriate DNA barcodes yet to be explored in biodiversity studies in Kenya. Four of these plastid regions are portions of coding genes (matK, rbcL, rpoB, and rpoC1), and three noncoding spacers (atpF-atpH, trnH-psbA, and psbK-psbL) which emerge as ideal candidate DNA loci. While different research groups propose various combinations of these loci, there exists no consensus;the lack thereof impedes progress in getting a suitable universal DNA barcode. Little research has attempted to investigate and document the applicability and extend of effectiveness of different DNA regions as barcodes to delineate cowpea at subspecies level. In this study we sought to test feasibility of the seven putative candidate DNA loci singly and in combination in order to establish a suitable single and multi-locus barcode regions that can have universal application in delineating diverse phylogeographic groups of closely related Kenyan cowpea variants. In this study, our focus was based on genetic parameters including analyses of intra- and infra-specific genetic divergence based on intra- and infra-specific K2P distances;calculation of Wilcoxon signed rank tests of intra-specific divergence among loci and coalescence analyses to delineate independent genetic clusters. Knowledge of DNA candidate loci that are informative will reveal the suitability of DNA barcoding as a tool in biodiversity studies. Results of this study indicate that: matK, trnH-psbA, psbK-psbL, and rbcL are good barcodes for delineating intra and infraspecific distances at single loci level. However, among the combinations, matK + trnH-psbA, rpoB + atpF-atpH + matK are the best barcodes in delineating cowpea subvariants. rbcL gene can be a suitable barcode marker at single locus level, but overall, multi locus approach appears more informative than single locus approach. The present study hopes to immensely contribute to the scanty body of knowledge on the novelty of DNA barcoding in cataloguing closely related cowpea variants at molecular level and hopes to open up future research on genomics and the possibility of use of conserved regions within DNA in inferring phylogenetic relationships among Kenyan cowpea variants.

国内外几种知名品牌雪茄化学及风格特征比较

【目的】筛选表征国内外雪茄质量特征的关键指标。【方法】检测了18种古巴、5种大卫杜夫和9种长城雪茄的常规化学成分、生物碱、多酚、质体色素、常规烟气成分及风格特征,对比分析了古巴、大卫杜夫和长城雪茄的化学和风格特征。【结果】(1)不同品牌雪茄化学成分的含量存在较大差异,长城雪茄淀粉、降烟碱、绿原酸等的含量显著高于古巴雪茄;总糖、β-胡萝卜素、新烟草碱等的含量显著低于古巴雪茄。长城雪茄植物碱、还原糖、烟碱等的含量显著高于大卫杜夫雪茄;钾含量显著低于大卫杜夫雪茄。(2)不同品牌雪茄常规烟气成分无显著差异。(3)长城雪茄和大卫杜夫雪茄风格特征相近;与长城雪茄相比,古巴雪茄香韵种类较为丰富,香气更富有变化。【结论】古巴、大卫杜夫和长城雪茄总糖、β-胡萝卜素、烟碱、新绿原酸等化学物质的含量存在显著差异;不同品牌雪茄的风格特征存在较大差异。

保水剂对干旱胁迫下雪茄烟叶次生代谢物质及化学成分含量的影响

为研究干旱胁迫下保水剂种类及用量对雪茄烟叶次生代谢物质及化学成分含量的影响,以德雪3号为试材,采用盆栽试验研究了保水剂种类(SAP保水剂、沃特保水剂和自制TS-PAA保水剂)与用量(2、4和6 g·株^(-1))对雪茄烟生长过程中质体色素、多酚、有机酸含量的消长规律及其调制后化学成分含量变化的影响。结果表明,(1)干旱胁迫下施用3种保水剂均能显著增加烟叶中叶绿素、叶绿素a、叶绿素b和类胡萝卜素的含量,且有利于烟株前中期叶绿素的快速积累和后期叶绿素的充分降解,以确保烟株适时落黄;(2)生长发育期间绿原酸、芸香苷、莨菪亭含量呈先增加后缓慢降低的趋势,且显著高于对照,说明施加3种保水剂均有利于多酚类物质的积累,能间接提升调制后烟叶的香吃味;(3)在移栽后75 d时,3种保水剂处理下苹果酸、草酸、丙二酸含量均有不同程度的提高,而柠檬酸含量与对照无显著差异;(4)施加3种保水剂均能显著提高总糖、还原糖和钾含量,并降低总氮、烟碱、淀粉和氯含量,进而增加两糖比、糖碱比、钾氯比和氮碱比,使烟叶中化学成分更加均衡协调。不同处理相比较,TS-PAA保水剂和SAP保水剂以施用量为4 g·株~(^(-1)),沃特保水剂以施用量为6 g·株^(-1)时对次生代谢物质积累和烟叶质量改善作用效果最佳。

植物质体基因工程调控元件研究进展

随着植物合成生物学的发展,质体逐渐成为许多具有商业价值的次生代谢产物和治疗性蛋白异源生产的理想平台。与核基因工程相比,质体基因工程在外源基因高效表达和生物安全性等方面具有其独特优势。然而,外源基因在质体系统中的组成型表达或对植物生长不利,因此需进一步挖掘、设计调控元件实现对外源基因的精准调控。本文概述了质体基因工程调控元件的研究进展,内容包括操纵子设计与优化思路、多基因共表达调控策略及新型表达调控元件的挖掘等,为植物合成生物学的发展提供参考。

Insights into cryptic speciation of quillworts in China

Cryptic species are commonly misidentified because of high morphological similarities to other species.One group of plants that may harbor large numbers of cryptic species is the quillworts(Isoetes spp.),an ancient aquatic plant lineage.Although over 350 species of Isoetes have been reported globally,only ten species have been recorded in China.The aim of this study is to better understand Isoetes species diversity in China.For this purpose,we systematically explored the phylogeny and evolution of Isoetes using complete chloroplast genome(plastome)data,spore morphology,chromosome number,genetic structure,and haplotypes of almost all Chinese Isoetes populations.We identified three ploidy levels of Isoetes in Chinaddiploid(2n=22),tetraploid(2n=44),and hexaploid(2n=66).We also found four megaspore and microspore ornamentation types in diploids,six in tetraploids,and three in hexaploids.Phylogenetic analyses confirmed that I.hypsophila as the ancestral group of the genus and revealed that Isoetes diploids,tetraploids,and hexaploids do not form monophyletic clades.Most individual species possess a single genetic structure;however,several samples have conflicting positions on the phylogenetic tree based on SNPs and the tree based on plastome data.All 36 samples shared 22 haplotypes.Divergence time analysis showed that I.hypsophila diverged in the early Eocene(~48.05 Ma),and most other Isoetes species diverged 3-20 Ma.Additionally,different species of Isoetes were found to inhabit different water systems and environments along the Yangtze River.These findings provide new insights into the relationships among Isoetes species in China,where highly similar morphologic populations may harbor many cryptic species.

A faster killing effect of plastid-mediated RNA interference on a leaf beetle through induced dysbiosis of the gut bacteria

The expression of double-stranded RNAs(dsRNAs)from the plastid genome has been proven to be an effective method for controlling herbivorous pests by targeting essential insect genes.However,there are limitations to the efficiency of plastid-mediated RNA interference(PM-RNAi)due to the initial damage caused by the insects and their slow response to RNA interference.In this study,we developed transplastomic poplar plants that express dsRNAs targeting the b-Actin(dsACT)and Srp54k(dsSRP54K)genes of Plagiodera versicolora.Feeding experiments showed that transplastomic poplar plants can cause significantly higher mortality in P.versicolora larvae compared with nuclear transgenic or wild-type poplar plants.The efficient killing effect of PM-RNAi on P.versicolora larvae was found to be dependent on the presence of gut bacteria.Importantly,foliar application of a gut bacterial strain,Pseudomonas putida,will induce dysbiosis in the gut bacteria of P.versicolora larvae,leading to a significant acceleration in the speed of killing by PM-RNAi.Overall,our findings suggest that interfering with gut bacteria could be a promising strategy to enhance the effectiveness of PM-RNAi for insect pest control,offering a novel and effective approach for crop protection based on RNAi technology.