Comparative genomic analyses reveal the genetic basis of the yellow-seed trait in Brassica napus.

Yellow-seed trait is a desirable breeding characteristic of rapeseed (Brassica napus) that could greatly improve seed oil yield and quality. However, the underlying mechanisms controlling this phenotype in B. napus plants are difficult to discern because of their complexity. Here, we assemble high-quality genomes of yellow-seeded (GH06) and black-seeded (ZY821). Combining in-depth fine mapping of a quantitative trait locus (QTL) for seed color with other omics data reveal BnA09MYB47a, encoding an R2R3-MYB-type transcription factor, as the causal gene of a major QTL controlling the yellow-seed trait. Functional studies show that sequence variation of BnA09MYB47a underlies the functional divergence between the yellow- and black-seeded B. napus. The black-seed allele BnA09MYB47aZY821, but not the yellow-seed allele BnA09MYB47aGH06, promotes flavonoid biosynthesis by directly activating the expression of BnTT18. Our discovery suggests a possible approach to breeding B. napus for improved commercial value and facilitates flavonoid biosynthesis studies in Brassica crops.

Prevalence and influencing factors of malocclusion in adolescents in Shanghai, China.

BACKGROUND: The main purpose of the study was to investigate the prevalence and related risk factors of malocclusion in permanent dentition among adolescents in Shanghai, and provide basic data for government's preventive strategies and intervention plans. METHODS: 1799 adolescents aged 11-15 years old from 18 middle schools in 6 districts of Shanghai were recruited to investigate oral health status and related risk factors using cluster random sampling method in 2021. Malocclusion and caries were examined by on-site inspection. The investigation criteria referred to Bjoerk and the recommendation of the WHO. The malocclusion inspection items included molars relationship, canine relationship, overbite, overjet, midline displacement, anterior crossbite, posterior crossbite, scissors bite, crowding and spacing. The subjects were asked to fill in a questionnaire including parents' education level, oral health behaviors and dietary habits. The chi-square test and logistic regression analysis were used to analyze the relationship between malocclusion and risk factors. RESULTS: 1799 adolescents were included in the study and the prevalence of malocclusion in adolescents in Shanghai was 83.5%, and the proportion of molar relationship class I, class II, and class III was 48.9%, 14.7%, and 19.0%, respectively. The most common occlusal characteristic of malocclusion was anterior crowding, followed by midline irregularities and deep overbite, with prevalence rates of 44.8%, 39.0% and 38.6%, respectively. The prevalence rate of adolescents with caries was 34.3%. Those who had dental caries and preferred soft food were more likely to have abnormal occlusal characteristics (p < 0.05). CONCLUSION: The prevalence of malocclusion in adolescents in Shanghai is high, so it is of great significance to strengthen oral health education, allocate proper preventive strategies and carry out the early correction if necessary.

Multifunctional Nanoparticles and Nanopesticides in Agricultural Application.

The unscientific application of pesticides can easily cause a series of ecological environmental safety issues, which seriously restrict the sustainable development of modern agriculture. The great progress in nanotechnology has allowed the continuous development of plant protection strategies. The nanonization and delivery of pesticides offer many advantages, including their greater absorption and conduction by plants, improved efficacy, reduced dosage, delayed resistance, reduced residues, and protection from natural enemies and beneficial insects. In this review, we focus on the recent advances in multifunctional nanoparticles and nanopesticides. The definition of nanopesticides, the types of nanoparticles used in agriculture and their specific synergistic mechanisms are introduced, their safety is evaluated, and their future application prospects, about which the public is concerned, are examined.

Downregulation of Brassica napus MYB69 (BnMYB69) increases biomass growth and disease susceptibility via remodeling phytohormone, chlorophyll, shikimate and lignin levels.

MYB transcription factors are major actors regulating plant development and adaptability. Brassica napus is a staple oil crop and is hampered by lodging and diseases. Here, four B. napus MYB69 (BnMYB69s) genes were cloned and functionally characterized. They were dominantly expressed in stems during lignification. BnMYB69 RNA interference (BnMYB69i) plants showed considerable changes in morphology, anatomy, metabolism and gene expression. Stem diameter, leaves, roots and total biomass were distinctly larger, but plant height was significantly reduced. Contents of lignin, cellulose and protopectin in stems were significantly reduced, accompanied with decrease in bending resistance and Sclerotinia sclerotiorum resistance. Anatomical detection observed perturbation in vascular and fiber differentiation in stems, but promotion in parenchyma growth, accompanied with changes in cell size and cell number. In shoots, contents of IAA, shikimates and proanthocyanidin were reduced, while contents of ABA, BL and leaf chlorophyll were increased. qRT-PCR revealed changes in multiple pathways of primary and secondary metabolisms. IAA treatment could recover many phenotypes and metabolisms of BnMYB69i plants. However, roots showed trends opposite to shoots in most cases, and BnMYB69i phenotypes were light-sensitive. Conclusively, BnMYB69s might be light-regulated positive regulators of shikimates-related metabolisms, and exert profound influences on various internal and external plant traits.

Design, Synthesis, and Action Mechanism of 1,3-Benzodioxole Derivatives as Potent Auxin Receptor Agonists and Root Growth Promoters.

Deeper and longer roots allow crops to survive and flourish, but our understanding of the plant growth regulators promoting root system establishment is limited. Here, we report that, a novel auxin receptor agonist, named K-10, had a remarkable promotive effect on root growth in both Arabidopsis thaliana and Oryza sativa through the enhancement of root-related signaling responses. Using computer-aided drug discovery approaches, we developed potent lead compound by screening artificial chemicals on the basis of the auxin receptor TIR1 (Transport Inhibitor Response 1), and a series of N-(benzo[d] [1,3] dioxol-5-yl)-2-(one-benzylthio) acetamides, K-1 to K-22, were designed and synthesized. The results of bioassay showed that K-10 exhibited an excellent root growth-promoting activity far exceeding that of NAA (1-naphthylacetic acid). A further morphological investigation of the auxin related mutants (yucQ, tir1) revealed that K-10 had auxin-like physiological functions and was recognized by TIR1, and K-10 significantly enhanced auxin response reporter's (DR5:GUS) transcriptional activity. Consistently, transcriptome analysis showed that K-10 induced a common transcriptional response with auxin and down-regulated the expression of root growth-inhibiting genes. Further molecular docking analysis revealed that K-10 had a stronger binding ability with TIR1 than NAA. These results indicated that this class of derivatives could be a promising scaffold for the discovery and development of novel auxin receptor agonists, and the employment of K-10 may be effective for enhancing root growth and crop production.

Genetic mapping and physiological analysis of chlorophyll-deficient mutant in Brassica napus L.

BACKGROUND: Leaf color mutants have reduced photosynthetic efficiency, which has severely negative impacts on crop growth and economic product yield. There are different chlorophyll mutants in Arabidopsis and crops that can be used for genetic control and molecular mechanism studies of chlorophyll biosynthesis, chloroplast development and photoefficiency. Chlorophyll mutants in Brassica napus are mostly used for mapping and location research but are rarely used for physiological research. The chlorophyll-deficient mutant in this experiment were both genetically mapped and physiologically analyzed. RESULTS: In this study, yellow leaf mutant of Brassica napus L. mutated by ethyl methyl sulfone (EMS) had significantly lower chlorophyll a, b and carotenoid contents than the wild type, and the net photosynthetic efficiency, stomatal conductance and transpiration rate were all significantly reduced. The mutant had sparse chloroplast distribution and weak autofluorescence. The granule stacks were reduced, and the shape was extremely irregular, with more broken stromal lamella. Transcriptome data analysis enriched the differentially expressed genes mainly in phenylpropane and sugar metabolism. The mutant was mapped to a 2.72 Mb region on A01 by using BSA-Seq, and the region was validated by SSR markers. CONCLUSIONS: The mutant chlorophyll content and photosynthetic efficiency were significantly reduced compared with those of the wild type. Abnormal chloroplasts and thylakoids less connected to the stroma lamella appeared in the mutant. This work on the mutant will facilitate the process of cloning the BnaA01.cd gene and provide more genetic and physiological information concerning chloroplast development in Brassica napus.

Development of an efficient marker-free soybean transformation method using the novel bacterium Ochrobactrum haywardense H1.

We have discovered a novel bacterium, Ochrobactrum haywardense H1 (Oh H1), which is capable of efficient plant transformation. Ochrobactrum is a new host for Agrobacterium-derived vir and T-DNA-mediated transformation. Oh H1 is a unique, non-phytopathogenic species, categorized as a BSL-1 organism. We engineered Oh H1 with repurposed Agrobacterium virulence machinery and demonstrated Oh H1 can transform numerous dicot species and at least one monocot, sorghum. We generated a cysteine auxotrophic Oh H1-8 strain containing a binary vector system. Oh H1-8 produced transgenic soybean plants with an efficiency 1.6 times that of Agrobacterium strain AGL1 and 2.9 times that of LBA4404Thy-. Oh H1-8 successfully transformed several elite Corteva soybean varieties with T0 transformation frequency up to 35%. In addition to higher transformation efficiencies, Oh H1-8 generated high-quality, transgenic events with single-copy, plasmid backbone-free insertion at frequencies higher than AGL1. The SpcN selectable marker gene is excised using a heat shock-inducible excision system resulting in marker-free transgenic events. Approximately, 24.5% of the regenerated plants contained only a single copy of the transgene and contained no vector backbone. There were no statistically significant differences in yield comparing T3 null-segregant lines to wild-type controls. We have demonstrated that Oh H1-8, combined with spectinomycin selection, is an efficient, rapid, marker-free and yield-neutral transformation system for elite soybean.

Design, synthesis and mode of action of novel 3-chloro-6-pyrazolyl picolinate derivatives as herbicide candidates.

BACKGROUND: Picolinate/picolinic acid compounds are an important class of synthetic auxin herbicides. To explore the herbicidal activity of 6-pyrazolyl picolinate compounds, a series of 3-chloro-6-pyrazolyl-picolinate derivatives was designed and synthesized. RESULTS: Twenty-five 3-chloro-6-pyrazolyl-picolinate derivatives synthesized were tested for herbicidal activity and the IC50 value of compound c5 to the growth of Arabidopsis thaliana root was 27 times lower than that of the commercial herbicide clopyralid. Compound c5 displayed better post-emergence herbicidal activity and broader (Picloram, Clopyralid, Aminopyralid) herbicidal spectrum at a dosage of 400 g ha-1 in comparison with clopyralid; it also was safe to wheat and maize at this dosage. Arabidopsis thaliana phenotypes and expression of auxin-response genes demonstrated that compound c5 might be a novel auxin-type herbicide. Molecular docking analyses revealed that compound c5 had stronger binding ability to receptor AFB5 (auxin signaling F-box protein 5) than clopyralid. CONCLUSION: These 6-pyrazolyl picolinate compounds could be used as potential lead structures for the discovery of a novel synthetic auxin herbicide. © 2021 Society of Chemical Industry.

Author Correction: Characterization of DvSSJ1 transcripts targeting the smooth septate junction (SSJ) of western corn rootworm (Diabrotica virgifera virgifera).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characterization of DvSSJ1 transcripts targeting the smooth septate junction (SSJ) of western corn rootworm (Diabrotica virgifera virgifera).

Transgenic maize plants expressing dsRNA targeting western corn rootworm (WCR, Diabrotica virgifera virgifera) DvSSJ1 mRNA, a Drosophila snakeskin (ssk) ortholog, show insecticidal activity and significant plant protection from WCR damage. The gene encodes a membrane protein associated with the smooth sepate junction (SSJ) which is required for intestinal barrier function. To understand the active RNA form that leads to the mortality of WCR larvae by DvSSJ1 RNA interference (RNAi), we characterized transgenic plants expressing DvSSJ1 RNA transcripts targeting WCR DvSSJ1 mRNA. The expression of the silencing cassette results in the full-length transcript of 901 nucleotides containing a 210 bp inverted fragment of the DvSSJ1 gene, the formation of a double-stranded RNA (dsRNA) transcript and siRNAs in transgenic plants. Our artificial diet-feeding study indicates that dsRNAs greater than or equal to approximately 60 base-pairs (bp) are required for DvSSJ1 insecticidal activity. Impact of specificity of dsRNA targeting DvSSJ1 mRNA on insecticidal activities was also evaluated in diet bioassay, which showed a single nucleotide mutation can have a significant impact or abolish diet activities against WCR. These results provide insights as to the functional forms of plant-delivered dsRNA for the protection of transgenic maize from WCR feeding damage and information contributing to the risk assessment of transgenic maize expressing insecticidal dsRNA.

Engineering Lignin Nanomicroparticles for the Antiphotolysis and Controlled Release of the Plant Growth Regulator Abscisic Acid.

Lignin is the most abundant aromatic biopolymer in nature and is a major byproduct from the paper industry. The unlocking of lignin's potential for high-value applications has gained increasing attention in recent years. In this study, alkali lignin (AL), with a rigid conjugated structure and amphiphilic property, was used as a sustainable and eco-friendly encapsulation material for the protection and controlled release of photosensitive abscisic acid (ABA), an important and widely used plant growth regulator. Cetyltrimethylammonium bromide (CTAB) was used to induce the formation of AL-CTAB nanomicroparticles by self-assembly. The size and morphology of AL-CTAB particles were modified by changing the AL concentration and the dispersion agent. AL (0.3 M) dissolved in tetrahydrofuran could form a uniform size (300 nm) of particles with a regular spherical structure. Subsequently, ABA was loaded on the prepared nanomicroparticles to synthesize the capsule formulation of ABA@AL-CTAB. The controlled-release behavior and the antiphotolysis performance as well as the thermal stability of ABA@AL-CTAB were proved to be superior. Lasting inhibition of Arabidopsis and rice seed germination by ABA@AL-CTAB under light irradiations implied protection of ABA from photolysis. In addition, ABA@AL-CTAB could effectively regulate plant stomata, thereby increasing plant drought resistance. Overall, lignin is suitable for the preparation of agrochemical formulations with excellent controlled release and antiphotolysis performances.

Identification and Characterization of Major Constituents in Different-Colored Rapeseed Petals by UPLC-HESI-MS/MS.

Oilseed rape (Brassica napus L.) is the second highest yielding oil crop worldwide. In addition to being used as an edible oil and a feed for livestock, rapeseed has high ornamental value. In this study, we identified and characterized the main floral major constituents, including phenolic acids and flavonoids components, in rapeseed accessions with different-colored petals. A total of 144 constituents were identified using ultrahigh-performance liquid chromatography-HESI-mass spectrometry (UPLC-HESI-MS/MS), 57 of which were confirmed and quantified using known standards and mainly contained phenolic acids, flavonoids, and glucosinolates compounds. Most of the epicatechin, quercetin, and isorhamnetin derivates were found in red and pink petals of B. napus, while kaempferol derivates were in yellow and pale white petals. Moreover, petal-specific compounds, including a putative hydroxycinnamic acid derivative, sinapoyl malate, 1-O-sinapoyl-ß-d-glucose, feruloyl glucose, naringenin-7-O-glucoside, cyanidin-3-glucoside, cyanidin-3,5-di-O-glucoside, petunidin-3-O-ß-glucopyranoside, isorhamnetin-3-O-glucoside, kaempferol-3-O-glucoside-7-O-glucoside, quercetin-3,4'-O-di-ß-glucopyranoside, quercetin-3-O-glucoside, and delphinidin-3-O-glucoside, might contribute to a variety of petal colors in B. napus. In addition, bound phenolics were tentatively identified and contained three abundant compounds (p-coumaric acid, ferulic acid, and 8-O-4'-diferulic acid). These results provide insight into the molecular mechanisms underlying petal color and suggest strategies for breeding rapeseed with a specific petal color in the future.

Small RNA changes in synthetic Brassica napus.

MAIN CONCLUSION: Small RNAs and microRNAs were found to vary extensively in synthetic Brassica napus and subsequent generations, accompanied by the activation of transposable elements in response to hybridization and polyploidization. Resynthesizing B. napus by hybridization and chromosome doubling provides an approach to create novel polyploids and increases the usable genetic variability in oilseed rape. Although many studies have shown that small RNAs (sRNAs) act as important factor during hybridization and polyploidization in plants, much less is known on how sRNAs change in synthetic B. napus, particularly in subsequent generations after formation. We performed high-throughput sequencing of sRNAs in S1-S4 generations of synthetic B. napus and in the homozygous B. oleracea and B. rapa parent lines. We found that the number of small RNAs (sRNAs) and microRNAs (miRNAs) doubled in synthetic B. napus relative to the parents. The proportions of common sRNAs detected varied from the S1 to S4 generations, suggesting sRNAs are unstable in synthetic B. napus. The majority of miRNAs (67.2 %) were non-additively expressed in the synthesized Brassica allotetraploid, and 33.3 % of miRNAs were novel in the resynthesized B. napus. The percentage of miRNAs derived from transposable elements (TEs) also increased, indicating transposon activation and increased transposon-associated miRNA production in response to hybridization and polyploidization. The number of target genes for each miRNA in the synthesized Brassica allotetraploid was doubled relative to the parents, enhancing the complexity of gene expression regulation. The potential roles of miRNAs and their targets are discussed. Our data demonstrate generational changes in sRNAs and miRNAs in synthesized B. napus.

Extensive tRNA gene changes in synthetic Brassica napus.

Allopolyploidization, where two species come together to form a new species, plays a major role in speciation and genome evolution. Transfer RNAs (abbreviated tRNA) are typically 73-94 nucleotides in length, and are indispensable in protein synthesis, transferring amino acids to the cell protein synthesis machinery (ribosome). To date, the regularity and function of tRNA gene sequence variation during the process of allopolyploidization have not been well understood. In this study, the inter-tRNA gene corresponding to tRNA amplification polymorphism method was used to detect changes in tRNA gene sequences in the progeny of interspecific hybrids between Brassica rapa and B. oleracea, mimicking the original B. napus (canola) species formation event. Cluster analysis showed that tRNA gene variation during allopolyploidization did not appear to have a genotypic basis. Significant variation occurred in the early generations of synthetic B. napus (F1 and F2 generations), but fewer alterations were observed in the later generation (F3). The variation-prone tRNA genes tended to be located in AT-rich regions. BlastN analysis of novel tRNA gene variants against a Brassica genome sequence database showed that the variation of these tRNA-gene-associated sequences in allopolyploidization might result in variation of gene structure and function, e.g., metabolic process and transport.

Characterization and evolution of 5' and 3' untranslated regions in eukaryotes.

Untranslated regions (UTRs) in eukaryotes play a significant role in the regulation of translation and mRNA half-life, as well as interacting with specific RNA-binding proteins. However, UTRs receive less attention than more crucial elements such as genes, and the basic structural and evolutionary characteristics of UTRs of different species, and the relationship between these UTRs and the genome size and species gene number is not well understood. To address these questions, we performed a comparative analysis of 5' and 3' untranslated regions of different species by analyzing the basic characteristics of 244,976 UTRs from three eukaryote kingdoms (Plantae, Fungi, and Protista). The results showed that the UTR lengths and SSR frequencies in UTRs increased significantly with increasing species gene number while the length and G+C content in 5' UTRs and different types of repetitive sequences in 3' UTRs increased with the increase of genome size. We also found that the sequence length of 5' UTRs was significantly positively correlated with the presence of transposons and SSRs while the sequence length of 3' UTRs was significantly positively correlated with the presence of tandem repeat sequences. These results suggested that evolution of species complexity from lower organisms to higher organisms is accompanied by an increase in the regulatory complexity of UTRs, mediated by increasing UTR length, increasing G+C content of 5' UTRs, and insertion and expansion of repetitive sequences.

Characterization and functional annotation of nested transposable elements in eukaryotic genomes.

The movement of transposable elements (TE) in eukaryotic genomes can often result in the occurrence of nested TEs (the insertion of TEs into pre-existing TEs). We performed a general TE assessment using available databases to detect nested TEs and analyze their characteristics and putative functions in eukaryote genomes. A total of 802 TEs were found to be inserted into 690 host TEs from a total number of 11,329 TEs. We reveal that repetitive sequences are associated with an increased occurrence of nested TEs and sequence biased of TE insertion. A high proportion of the genes which were associated with nested TEs are predicted to localize to organelles and participate in nucleic acid and protein binding. Many of these function in metabolic processes, and encode important enzymes for transposition and integration. Therefore, nested TEs in eukaryotic genomes may negatively influence genome expansion, and enrich the diversity of gene expression or regulation.

Characterization of transcriptional activation and inserted-into-gene preference of various transposable elements in the Brassica species.

Transposable elements (TEs) have attracted increasing attention because of their tremendous contributions to genome reorganization and gene variation through dramatic proliferation and excision via transposition. However, less known are the transcriptional activation of various TEs and the characteristics of TE insertion into genomes at the genome-wide level. In the present study, we focused on TE genes for transposition and gene disruption by insertion of TEs in expression sequences of Brassica, to investigate the transcriptional activation of TEs, the biased insertion of TEs into genes, and their salient characteristics. Long terminal repeat (LTR-retrotransposon) accounted for the majority of these active TE genes (70.8%), suggesting that transposition activation varied with TE type. 6.1% genes were interrupted by LTR-retrotransposons, which indicated their preference for insertion into genes. TEs were preferentially inserted into cellular component-specific genes acted as "binding" elements and involved in metabolic processes. TEs have a biased insertion into some host genes that were involved with important molecular functions and TE genes exhibited spatiotemporal expression. These results suggested that various types of transposons differentially contributed to gene variation and affected gene function.

Characterization and comparison of gene-based simple sequence repeats across Brassica species.

Simple sequence repeats (SSRs) are important components of eukaryotic genomes and may play an important role in regulating gene expression. However, the characteristics of genic SSRs and the effect of interspecific hybridization and polyploidization on genic SSRs seem not to have received desired attention in terms of scientific investigations. To determine the features of genic SSRs and elucidate their role in polyploidization process of the Brassica family, we identified SSRs in Plant Genome Database-assembled unique transcripts (PUTs) of Brassica species. A higher density of SSRs and a greater number of compound motif SSRs and mononucleotide motif types with large average number of repeats were detected in allotetraploid Brassica napus than in the diploid parental species (Brassica rapa and Brassica oleracea). In addition, a greater proportion of SSR-PUTs were found to be associated with the stress response and developmental processes in B. napus than in the parents. A negative correlation between the repeat number and the motif type and the total length, and a positive correlation between the repeat number and the total length of SSRs were observed. PUT-SSR might be generated from A/T-rich regions. The successful development of 123 pairs of SSR primers for Brassica PUTs showed that SSR-PUTs could be exploited as gene-based SSR functional markers for application in Brassica breeding. These results indicate that interspecific hybridization and polyploidization could trigger the amplification of SSRs, and long SSRs might become shorter to enable the plant to adapt to environmental and artificial selection.

TRANSPARENT TESTA 12 genes from Brassica napus and parental species: cloning, evolution, and differential involvement in yellow seed trait.

Molecular dissection of the Brassica yellow seed trait has been the subject of intense investigation. Arabidopsis thaliana TRANSPARENT TESTA 12 (AtTT12) encodes a multidrug and toxic compound extrusion (MATE) transporter involved in seed coat pigmentation. Two, one, and one full-length TT12 genes were isolated from B. napus, B. oleracea, and B. rapa, respectively, and Southern hybridization confirmed these gene numbers, implying loss of some of the triplicated TT12 genes in Brassica. BnTT12-1, BnTT12-2, BoTT12, and BrTT12 are 2,714, 3,062, 4,760, and 2,716 bp, with the longest mRNAs of 1,749, 1,711, 1,739, and 1,752 bp, respectively. All genes contained alternative transcriptional start and polyadenylation sites. BrTT12 and BoTT12 are the progenitors of BnTT12-1 and BnTT12-2, respectively, validating B. napus as an amphidiploid. All Brassica TT12 proteins displayed high levels of identity (>99%) to each other and to AtTT12 (>92%). Brassica TT12 genes resembled AtTT12 in such basic features as MatE/NorM CDs, subcellular localization, transmembrane helices, and phosphorylation sites. Plant TT12 orthologs differ from other MATE proteins by two specific motifs. Like AtTT12, all Brassica TT12 genes are most highly expressed in developing seeds. However, a range of organ specificity was observed with BnTT12 genes being less organ-specific. TT12 expression is absent in B. rapa yellow-seeded line 06K124, but not downregulated in B. oleracea yellow-seeded line 06K165. In B. napus yellow-seeded line L2, BnTT12-2 expression is absent, whereas BnTT12-1 is expressed normally. Among Brassica species, TT12 genes are differentially related to the yellow seed trait. The molecular basis for the yellow seed trait, in Brassica, and the theoretical and practical implications of the highly variable intron 1 of these TT12 genes are discussed.

Identification of the RNA binding regions of SRP68/72 and SRP72 by systematic mutagenesis of human SRP RNA.

Within the large domain of the human signal recognition particle (SRP), 18 mutant SRP RNAs were constructed to disrupt Watson-Crick and G-U basepairs in helices 5, 6 and 8. Using a double-filter assay, the competitive binding of the mutant RNAs to purified human SRP68/72 or to a 7.4 kDa RNA-binding fragment of SRP72 (72frg) was measured. Binding of SRP68/72 was impaired by several mutations in the large domain with the most pronounced effects caused by changes in helix 5 (residues 222-231) and helix 8 (residues 176-191 and 202-214). Binding of the 72frg was diminished prominently by altering helix 5, in particular residues 120-128, and was unaffected by deleting helices 6 and 8. Deleting helix 8 diminished binding of SRP68/72 to a greater extent than deleting helix 6. The data suggest that nucleotide residues throughout most of the large SRP domain are directly and/or indirectly engaged in the binding of SRP68. In contrast, SRP72 binds only to a portion of the 5ef region.