Identification and expression analysis of the Xyloglucan transglycosylase/hydrolase (XTH) gene family under abiotic stress in oilseed (Brassica napus L.).

XTH genes are key genes that regulate the hydrolysis and recombination of XG components and plays role in the structure and composition of plant cell walls. Therefore, clarifying the changes that occur in XTHs during plant defense against abiotic stresses is informative for the study of the plant stress regulatory mechanism mediated by plant cell wall signals. XTH proteins in Arabidopsis thaliana was selected as the seed sequences in combination with its protein structural domains, 80 members of the BnXTH gene family were jointly identified from the whole genome of the Brassica napus ZS11, and analyzed for their encoded protein physicochemical properties, phylogenetic relationships, covariance relationships, and interoperating miRNAs. Based on the transcriptome data, the expression patterns of BnXTHs were analyzed in response to different abiotic stress treatments. The relative expression levels of some BnXTH genes under Al, alkali, salt, and drought treatments after 0, 6, 12 and 24 h were analyzed by using qRT-PCR to explore their roles in abiotic stress tolerance in B. napus. BnXTHs showed different expression patterns in response to different abiotic stress signals, indicating that the response mechanisms of oilseed rape against different abiotic stresses are also different. This paper provides a theoretical basis for clarifying the function and molecular genetic mechanism of the BnXTH gene family in abiotic stress tolerance in rapeseed.

Overexpression of BnMYBL2-1 improves plant drought tolerance via the ABA-dependent pathway.

Drought stress is a major environmental challenge that poses considerable threats to crop survival and growth. Previous research has indicated anthocyanins play a crucial role in alleviating oxidative damage, photoprotection, membrane stabilization, and water retention under drought stress. However, the presence of MYBL2 (MYELOBBLASTOSIS LIKE 2), an R3-MYB transcription factor (TF) which known to suppress anthocyanin biosynthesis. In this study, four BnMYBL2 members were cloned from Brassica napus L, and BnMYBL2-1 was overexpressed in Triticum aestivum L (No BnMYBL2 homologous gene was detected in wheat). Subsequently, the transgenic wheat lines were treated with drought, ABA and anthocyanin. Results showed that transgenic lines exhibited greater drought tolerance compared to the wild-type (WT), characterized by improved leaf water content (LWC), elevated levels of soluble sugars and chlorophyll, and increased antioxidant enzyme activity. Notably, transgenic lines also exhibited significant upregulation in abscisic acid (ABA) content, along with the transcriptional levels of key enzymes involved in ABA signalling under drought. Results also demonstrated that BnMYBL2-1 promoted the accumulation of ABA and anthocyanins in wheat. Overall, the study highlights the positive role of BnMYBL2-1 in enhancing crop drought tolerance through ABA signalling and establishes its close association with anthocyanin biosynthesis. These findings offer valuable insights for the development of drought-resistant crop varieties and enhance the understanding of the molecular mechanisms underlying plant responses to drought stress.

Study on exogenous application of thidiazuron on seed size of Brassica napus L.

Thidiazuron (TDZ) is a novel and efficient cytokinin commonly used in tissue culture, and numerous studies have demonstrated that TDZ can increase berry size. However, no study to date has explored the effect of TDZ on seed size of Brassica napus and the mechanism. To shed light on the effect of TDZ on the seed size of B. napus, four different concentrations of TDZ were applied to B. napus. Results indicated that TDZ treatment could increase the seed diameter and silique length of B. napus to varying degrees and 100 and 200 µmol/L TDZ treatments were the most effective with a 3.6 and 4.6% increase in seed diameter, respectively. In addition, the yield of B. napus was also substantially increased under TDZ treatment. On the other hand, confocal micrographs of embryos and cotyledon cells suggested that embryos and their cotyledon epidermal cells treated with 200 µmol/L TDZ were obviously larger in size than the control. Furthermore, TDZ promoted the upregulation of some key maternal tissue growth-related genes, including two G-protein signaling genes (AGG3 and RGA1) and two transcriptional regulators (ANT and GS2). The expression analysis of genes related to the auxin metabolic pathways, G-protein signaling, endosperm growth and transcriptional regulators confirmed that treatment with TDZ negatively regulated the key genes ABI5, AGB1, AP2, ARF2, and ARF18 during bud development stage and florescence. The results strongly suggested that TDZ might regulate the transcriptional levels of key genes involved in auxin metabolic pathways, G-protein signaling, endosperm growth and transcriptional regulators, which resulted in bigger cotyledon epidermal cells and seed size in B. napus. This study explored the mechanism of TDZ treatment on the seed size of B. napus and provided an important reference for improving rapeseed yield.

Deciphering the transcriptional regulatory networks that control size, color, and oil content in Brassica rapa seeds.

BACKGROUND: Brassica rapa is an important oilseed and vegetable crop species and is the A subgenome donor of two important oilseed Brassica crops, Brassica napus and Brassica juncea. Although seed size (SZ), seed color (SC), and oil content (OC) substantially affect seed yield and quality, the mechanisms regulating these traits in Brassica crops remain unclear. RESULTS: We collected seeds from a pair of B. rapa accessions with significantly different SZ, SC, and OC at seven seed developmental stages (every 7 days from 7 to 49 days after pollination), and identified 28,954 differentially expressed genes (DEGs) from seven pairwise comparisons between accessions at each developmental stage. K-means clustering identified a group of cell cycle-related genes closely connected to variation in SZ of B. rapa. A weighted correlation analysis using the WGCNA package in R revealed two important co-expression modules comprising genes whose expression was positively correlated with SZ increase and negatively correlated with seed yellowness, respectively. Upregulated expression of cell cycle-related genes in one module was important for the G2/M cell cycle transition, and the transcription factor Bra.A05TSO1 seemed to positively stimulate the expression of two CYCB1;2 genes to promote seed development. In the second module, a conserved complex regulated by the transcription factor TT8 appear to determine SC through downregulation of TT8 and its target genes TT3, TT18, and ANR. In the third module, WRI1 and FUS3 were conserved to increase the seed OC, and Bra.A03GRF5 was revealed as a key transcription factor on lipid biosynthesis. Further, upregulation of genes involved in triacylglycerol biosynthesis and storage in the seed oil body may increase OC. We further validated the accuracy of the transcriptome data by quantitative real-time PCR of 15 DEGs. Finally, we used our results to construct detailed models to clarify the regulatory mechanisms underlying variations in SZ, SC, and OC in B. rapa. CONCLUSIONS: This study provides insight into the regulatory mechanisms underlying the variations of SZ, SC, and OC in plants based on transcriptome comparison. The findings hold great promise for improving seed yield, quality and OC through genetic engineering of critical genes in future molecular breeding.

Graphene oxide and indole-3-acetic acid cotreatment regulates the root growth of Brassica napus L. via multiple phytohormone pathways.

BACKGROUND: Studies have indicated that graphene oxide (GO) could regulated Brassica napus L. root growth via abscisic acid (ABA) and indole-3-acetic acid (IAA). To study the mechanism and interaction between GO and IAA further, B. napus L (Zhongshuang No. 9) seedlings were treated with GO and IAA accordance with a two factor completely randomized design. RESULTS: GO and IAA cotreatment significantly regulated the root length, number of adventitious roots, and contents of IAA, cytokinin (CTK) and ABA. Treatment with 25 mg/L GO alone or IAA (> 0.5 mg/L) inhibited root development. IAA cotreatment enhanced the inhibitory role of GO, and the inhibition was strengthened with increased in IAA concentration. GO treatments caused oxidative stress in the plants. The ABA and CTK contents decreased; however, the IAA and gibberellin (GA) contents first increased but then decreased with increasing IAA concentration when IAA was combined with GO compared with GO alone. The 9-cis-epoxycarotenoid dioxygenase (NCED) transcript level strongly increased when the plants were treated with GO. However, the NCED transcript level and ABA concentration gradually decreased with increasing IAA concentration under GO and IAA cotreatment. GO treatments decreased the transcript abundance of steroid 5-alpha-reductase (DET2) and isochorismate synthase 1 (ICS), which are associated with brassinolide (BR) and salicylic acid (SA) biosynthesis, but increased the transcript abundance of brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1), cam-binding protein 60-like G (CBP60) and calmodulin binding protein-like protein 1, which are associated with BR and SA biosynthesis. Last, GO treatment increased the transcript abundance of 1-aminocyclopropane-1-carboxylic acid synthase 2 (ACS2), which is associated with the ethylene (ETH) pathway. CONCLUSIONS: Treatment with 25 mg/L GO or IAA (> 0.5 mg/L) inhibited root development. However, IAA and GO cotreatment enhanced the inhibitory role of GO, and this inhibition was strengthened with increased IAA concentration. IAA is a key factor in the response of B. napus L to GO and the responses of B. napus to GO and IAA cotreatment involved in multiple pathways, including those involving ABA, IAA, GA, CTK, BR, SA. Specifically, GO and IAA cotreatment affected the GA content in the modulation of B. napus root growth.

Graphene oxide and ABA cotreatment regulates root growth of Brassica napus L. by regulating IAA/ABA.

Previous studies have proven that graphene oxide (GO) regulates abscisic acid (ABA) and indole-3-acetic acid (IAA) contents and modulates plant root growth. To better understand the mechanism of plant growth and development regulated by GO and crosstalk between ABA and GO, Zhongshuang No. 9 seedlings were treated with GO and ABA. The results indicated that GO and ABA significantly affected the morphological properties and endogenous phytohormone contents in seedlings, and there was significant crosstalk between GO and ABA. ABA treatments combined with GO led to a rapid decrease in triphenyltetrazolium chloride (TTC) reduction intensity, and the inhibitory effect was enhanced with increasing ABA concentration. The treatments significantly affected the transcriptional levels of some key genes involved in the ABA, IAA, cytokinin (CTK), salicylic acid (SA), and ethane (ETH) pathways and increased the ABA and gibberellin (GA) contents in rapeseed seedlings. The effects of the treatments on the IAA and CTK contents were complex, but, importantly, the treatments suppressed root elongation. Correlation analysis also indicated that the relationship between root length and IAA/ABA could be described by a polynomial function: y = 88.11x2 - 25.15x + 4.813(R²â€¯= 0.912). The treatments increased the ACS2 transcript abundance for ETH biosynthesis and the ICS1 transcriptional level of the key genes involved in salicylic acid (SA) biosynthesis, as well as the downstream signaling genes CBP60 and SARD1. This finding indicated that ABA is an important factor regulating the effects of GO on the growth and development of Brassica napus L., and that ETH and SA pathways may be potential pathways involved in the response of rape seedlings to GO treatment.

CRISPR-Cas13a Nanomachine Based Simple Technology for Avian Influenza A (H7N9) Virus On-Site Detection.

It is urgent to find an avian influenza A H7N9 detection simple method which is suitable for on-site detection. The Cas13a protein just likes a nanomachine, when specifically bound to target RNA by single-stranded RNA (crRNA), changes its protein structure and produces RNase activity, which degrades RNA non-specifically. Harnessing Cas13a, the paper aims to establish an underlying on-site H7N9 virus nucleic acid detection method. LwCas13a protein nanomachine was expressed in a prokaryotic expression system and purified by nickel column. In vitro transcribed RNA of H7N9 HA gene has been used as a target, to design a specific crRNA. The activity of Cas13a was verified with a single-stranded RNA-bound fluorescent group and a quenching fluorophore as signals. Using Cas13a, a room temperature H7N9 detection system was established. Detection of 1 nm of single-stranded RNA can be done within 5 min. When combined with the RT-RPA and T7 transcription system at room temperature, the detection limits of HA and NA are 1 fM and the reaction time is 50 min. Excellent specificity was achieved by comparison with subtype viruses such as H1N1 and H5N1. The rapid detection method based on CRISPR-Cas13a nanomachine H7N9 has been successfully established, which can detect H7N9 quickly and specifically. In the future, it can be quickly detected in the field with portable fluorescence detector.

Graphene Oxide Affects Growth and Resistance to Sclerotinia sclerotiorum in Brassica napus L.

Researchers have shown that graphene oxide has a significant effect on plant and pathogen growth and development. To better understand the effect of graphene oxide on the resistance of Brassica napus L. to Sclerotinia sclerotiorum, Zhongshuang 11 was used to evaluate changes in the morphology and physiology after graphene oxide treatment. Detached leaf inoculation was used to detect S. sclerotiorum infection in rapeseed. The results indicated that treatment with a low concentration of graphene oxide had no significant effect on the growth of B. napus "Zhongshuang 11." Graphene oxide inhibited S. sclerotiorum in PDA medium. Treatment with 15 mg/L graphene oxide for 8-24 h in seeds and 8-16 h in seedlings suppressed S. sclerotiorum growth compared to the control samples. These results demonstrate that a low concentration of graphene oxide did not harm the growth of B. napus but did enhance its resistance to S. sclerotiorum.

Preparation of Graphene Oxide and Its Mechanism in Promoting Tomato Roots Growth.

Graphene oxide is a new kind of nanomaterial. The graphene oxide was prepared and its quality detected by atomic force microscopy (AFM) and transmission electron microscopy (TEM), for better understanding of effects of the nanomaterial on plants. Wild type. (WT) tomato (Solanum lycopersicum) germplasm 'New Yorker' and corresponding transgenic plants (Prd29A::LeNCED1) were treated with prepared graphene oxide. 9-cis-epoxycarotenoid dioxygenase (NCED) is a key gene for ABA biosynthesis and overexpression of the NCED resulted in ABA accumulation and higher drought tolerance. Seminal root length in the WT tomato was longer than that in the control samples when the seedlings were treated with 20 mg/L graphene oxide for 15 days. In contrast, the same treatment resulted in shorter seminal root length in the transgenic plants compared with control samples. The graphene oxide treatments led to lower Superoxide Dismutase (SOD), Peroxidase (POD), Catalase (CAT) activity and Malondialdehyde (MDA) content in the WT and transgenic plants. 20 mg/L graphene oxide treatment also affected the transcript levels of IAA7, IAA4 and IAA10 but the effect on the wild type and corresponding transgenic plants was different. IAA4 transcription level decreased both in the WT and Prd29A::LeNCED1 transgenic plants while the IAA7 transcription level decreased in the transgenic plants and increased in the WT tomato. The IAA10 transcription level decreased in the WT tomato and increased in the Prd29A::LeNCED1 transgenic plants. Graphene oxide treatments resulted in higher transcription level of ABCG25 and ABCG40 in the WT plants but had no significant effect on transgenic plants. The transcription level of NCED in the WT and Prd29A::LeNCED1 transgenic plants treated with graphene oxide increased significantly, however, it was higher in the transgenic plants than in the WT tomato after 15 d treatment, indicating that the graphene oxide activated the rd29A promoter as does drought and salt. The HD-ZIP transcription level only decreased significantly in the treated Prd29A::LeNCED1 transgenic plants. All these results suggested that there was a crosstalk between ABA and graphene oxide and the graphene oxide affected plant growth through the ABA and IAA pathway.

Graphene oxide modulates root growth of Brassica napus L. and regulates ABA and IAA concentration.

Researchers have proven that nanomaterials have a significant effect on plant growth and development. To better understand the effects of nanomaterials on plants, Zhongshuang 11 was treated with different concentrations of graphene oxide. The results indicated that 25-100mg/l graphene oxide treatment resulted in shorter seminal root length compared with the control samples. The fresh root weight decreased when treated with 50-100mg/l graphene oxide. The graphene oxide treatment had no significant effect on the Malondialdehyde (MDA) content. Treatment with 50mg/l graphene oxide increased the transcript abundance of genes involved in ABA biosynthesis (NCED, AAO, and ZEP) and some genes involved in IAA biosynthesis (ARF2, ARF8, IAA2, and IAA3), but inhibited the transcript levels of IAA4 and IAA7. The graphene oxide treatment also resulted in a higher ABA content, but a lower IAA content compared with the control samples. The results indicated that graphene oxide modulated the root growth of Brassica napus L. and affected ABA and IAA biosynthesis and concentration.

[Screening of high-yield PUFAs Mortierella isabellina strain].

The original strain Mortierella isabellina As3.3410 was treated by microwave and ultraviolet. Mutated strains were screened by acetyl salicylic acid and low temperature (15°C). A high-yield strain named as A35-4 was successfully selected. The biomass of this strain was 17.9 g/L, oil content was 67.8%, oil production was 12.12 g/L, polyunsaturated fatty acids (PUFAs) content was 20.2%, and production of PUFAs was 2.46 g/L, which increased 32.6%, 49.8%, 98.69%, 14.0%, and 125.7% compared with the original A0 stain, respectively. The continuous slope transmission experiments confirmed that the strain had a good genetic stability. The study is beneficial for cloning high efficiency genes for PUFAs and producing PUFAs in this stain, and lays the ground work for creation of transgenic plants containing high levels of PUFAs.

Cloning and molecular characterization of a functional flavonoid 3'-hydroxylase gene from Brassica napus.

A flavonoid 3'-hydroxylase (F3'H) gene, denoted BnF3'H-1, was cloned from oilseed rape (Brassica napus). The gene of 3038 base pairs (bp) contains 3 introns. The complementary DNA (cDNA) consists of 1820bp and has an open reading frame of 1536bp encoding a polypeptide of 511 amino acids with a molecular weight of 56.62kDa and an isoelectric point of 7.08. BnF3'H-1 shows high homology to known F3'H genes, especially F3'H from Arabidopsis thaliana. Untranslated regions (UTRs) may play important roles in regulating the expression of BnF3'H-1. Besides containing a Kozak sequence, the first 77-bp region is C-rich but G-poor, and the 26-bp 5'-UTR contains 3 sites of ACCACT-like sequences. Alternative polyadenylation in the 3'-UTR is adopted by this gene to generate heterogeneous transcripts. Conserved domain search and motif characterization identified BnF3'H-1 as a cytochrome P450. All F3'H-featured motifs, VVVAAS, GGEK and VDVKG, are unchanged in BnF3'H-1. The N-terminal signal peptide/anchor and 3 transmembrane helices were predicted in BnF3'H-1, and its subcellular localization is most probably at the endoplasmic reticulum. Since 16 phosphorylation sites could be predicted, phosphorylation may be a necessary post-translational modification of BnF3'H-1. The secondary structure is dominated by alpha-helices and random coils. Most helices are located in the middle region, while extended strands mainly intersperse in terminal regions. DNA gel blot analysis indicated that 2 different F3'H genes might exist in B. napus. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and RNA gel blot analysis showed that flowers have the highest F3'H expression, followed by pericarp and seed, and lower levels in some other organs. This species-featured expression pattern is in obedience to multiple functional roles that F3'H gene(s) play(s) in various organs of B. napus. The BnF3'H-1 coding region was expressed in Escherichia coli, and enzyme activity of the His-tagged protein was demonstrated by monitoring the conversion of the substrate naringenin using high-performance liquid chromatography (HPLC), suggesting that BnF3'H-1 is catalytically functional. RT-PCR analysis suggests that transcription level of the F3'H gene(s) is not the reason for the different seed colorations found in near-isogenic lines (black-seeded L1 and yellow-seeded L2) of B. napus.