A highly efficient genetic transformation system for broccoli and subcellular localization.

Introduction: Agrobacterium-mediated genetic transformation has been widely used for the identification of functional genes and regulatory and developmental mechanisms in plants. However, there are still some problems of low genetic transformation efficiency and high genotype dependence in cruciferous crops. Methods: In this study, broccoli, a worldwide Brassica crop, was used to investigate the effects of genotype, explant type, concentration of hygromycin B used during seedling selection, overexpression vector type, RNAi and CRISPR/cas9 on the genetic transformation efficiency. At the same time, two vectors, PHG-031350 and PHG-CRa, were used for subcellular localization of the glucoraphanin synthesis-related gene FMOGS-OX5 and clubroot resistance gene by a PEG-Ca2+-mediated transient transformation system for broccoli protoplasts. Finally, the Agrobacterium-mediated genetic transformation system of broccoli was optimized and improved. Results and Discussion: This study showed that hypocotyl explants are more suitable for Agrobacterium-mediated transgene and CRISPR/Cas9 gene editing of broccoli. In contrast to previous studies, we found that 5 mg/L hygromycin B was more advantageous for the selection of resistant broccoli sprouts, and genotype 19B42 reached the highest transformation rate of 26.96%, which is higher than that in Brassica oleracea crops. In addition, the inbred line 19B42 successfully achieved high genetic transformation of overexpression, RNAi and CRISPR/Cas9 vectors; thus, it is powerful recipient material for the genetic transformation of broccoli. Subcellular localization proved that the glucoraphanin metabolism-related gene Bol031350 and clubroot resistance gene CRa were both expressed in the cytoplasm and nucleus, which provided a scientific basis for studying the regulation of glucosinolate metabolism and clubroot resistance in cruciferous crops. Therefore, these findings will provide new insight into the improvement of the genetic transformation and molecular breeding of Brassica oleracea crops.

A high-efficiency PEG-Ca2+-mediated transient transformation system for broccoli protoplasts.

Transient transformation of plant protoplasts is an important method for studying gene function, subcellular localization and plant morphological development. In this study, an efficient transient transformation system was established by optimizing the plasmid concentration, PEG4000 mass concentration and genotype selection, key factors that affect transformation efficiency. Meanwhile, an efficient and universal broccoli protoplast isolation system was established. Using 0.5% (w/v) cellulase R-10 and 0.1% (w/v) pectolyase Y-23 to hydrolyze broccoli cotyledons of three different genotypes for 3 h, the yield was more than 5×106/mL/g, and the viability was more than 95%, sufficient to meet the high standards for protoplasts to be used in various experiments. The average transformation efficiency of the two plasmid vectors PHG-eGFP and CP507-YFP in broccoli B1 protoplasts were 61.4% and 41.7%, respectively. Using this system, we successfully performed subcellular localization of the products of three target genes (the clubroot resistance gene CRa and two key genes regulated by glucosinolates, Bol029100 and Bol031350).The results showed that the products of all three genes were localized in the nucleus. The high-efficiency transient transformation system for broccoli protoplasts constructed in this study makes it possible to reliably acquire high-viability protoplasts in high yield. This research provides important technical support for international frontier research fields such as single-cell sequencing, spatial transcriptomics, plant somatic hybridization, gene function analysis and subcellular localization.

[Intervention of auricular point sticking on perioperative psychological stress in patients with anorectal diseases].

OBJECTIVE: To explore and quantify the intervention effect of auricular point sticking on perioperative psychological stress in patients with anorectal diseases. METHODS: Eighty patients who underwent anorectal surgery were randomly divided into an observation group (40 cases) and a control group (40 cases). The routine preoperative guidance, preoperative visits, and informed of the postoperative condition were received in the control group. On the basis of the treatment in the control group, auricular point sticking was immediately applied at Shenmen (TF4), Shen (CO10), Wei (CO4), Gan (CO12), Pi (CO13), Pizhixia (AT4), E (AT1), Nie (AT2) and Zhen (AT3) in the observation group.The patients were pressed by themselves, 3 to 5 min per point each time, 5 times a day, and the contralateral auricular points were replaced every 2 or 3 days until 1 week after surgery. The Hamilton anxiety scale (HAMA), Hamilton depression scale (HAMD), and Pittsburgh sleep quality index (PSQI) scores were compared between the two groups before and 7 days after surgery. RESULTS: There was no significant difference in the total HAMA scores between after and before surgery in the observation group (P>0.05). The total HAMA score in the control group was higher than that before surgery (P<0.05). The total HAMA score in the observation group after surgery was lower than that in the control group (P<0.05). There was no significant difference in the total HAMD scores between the two groups before and after surgery (P<0.05). There was no significant difference in the total HAMD scores between the two groups after the surgery (P>0.05). The scores of somatic anxiety factor in the two groups were higher than those before surgery (P<0.05). The scores of somatic anxiety factor in the observation group were lower than those in the control group (P<0.05). The scores of psychotic anxiety factors in the two groups were lower than those before surgery (P<0.05). There was no significant difference in the score of psychotic anxiety factors between the two groups (P>0.05). The total score of PSQI in the two groups was lower than that before surgery (P<0.05), and the total score of PSQI in the observation group was lower than that in the control group (P<0.05). CONCLUSION: Auricular point sticking can effectively improve some psychological stress problems during perioperative period in patients with anorectal diseases.

Charge transport in photofunctional nanoparticles self-assembled from zinc 5,10,15,20-tetrakis(perylenediimide)porphyrin building blocks.

Molecules designed to carry out photochemical energy conversion typically employ several sequential electron transfers, as do photosynthetic proteins. Yet, these molecules typically do not achieve the extensive charge transport characteristic of semiconductor devices. We have prepared a large molecule in which four perylene-3,4:9,10-tetracarboxydiimide (PDI) molecules that both collect photons and accept electrons are attached to a central zinc 5,10,15,20-tetraphenylporphyrin (ZnTPP) electron donor. This molecule self-assembles into ordered nanoparticles both in solution and in the solid-state, driven by van der Waals stacking of the PDI molecules. Photoexcitation of the nanoparticles results in quantitative charge separation in 3.2 ps to form ZnTPP(+)PDI(-) radical ion pairs, in which the radical anion rapidly migrates to PDI molecules that are, on average, 21 A away, as evidenced by magnetic field effects on the yield of the PDI triplet state that results from radical ion pair recombination. These nanoparticles exhibit charge transport properties that combine important features from both photosynthetic and semiconductor photoconversion systems.

Equilibrium Acidities and Homolytic Bond Dissociation Enthalpies of the Acidic C-H Bonds in As-Substituted Triphenylarsonium and Related Cations(1).

Equilibrium acidities (pK(HA)s) of As-fluorenyltriphenylarsonium, As-phenacyltriphenylarsonium, six As-(para-substituted benzyl)triphenylarsonium [p-GC(6)H(4)CH(2)(+)AsPh(3)] (G = H, Me, CF(3), CO(2)Me, CN, and NO(2)), and six P-(para-substituted benzyl)tri(n-butyl)phosphonium [p-GC(6)H(4)CH(2)(+)P(n-Bu)(3)] (G = H, Me, CF(3), CO(2)Me, CN, and NO(2)) bromide salts, together with the oxidation potentials [E(ox)(A(-))] of their conjugate bases (ylides) have been determined in dimethyl sulfoxide (DMSO) solution. Introduction of an alpha-triphenylarsonium (alpha-Ph(3)As(+)) group was found to increase the adjacent C-H bond acidities by 13-20 pK units (18-27 kcal/mol). The equilibrium acidities for the two series p-GC(6)H(4)CH(2)(+)AsPh(3) and p-GC(6)H(4)CH(2)(+)P(n-Bu)(3) cations were found to be nicely correlated with the Hammett sigma(-) constants of the corresponding para-substituents (G) (Figures 1 and 2). The homolytic bond dissociation enthalpies (BDEs) of the acidic C-H bonds determined by using eq 1 reveal that an alpha-Ph(3)As(+) group increases the BDE value of the adjacent acidic C-H bond by 2-5 kcal/mol, whereas the substituent effects for an alpha-Ph(3)P(+) or alpha-(n-Bu)(3)P(+) group was found to be dependent on the nature of the substituents attached to the alpha-carbon atom. Good linear correlations were obtained for the equilibrium acidities of As-(para-substituted benzyl)triphenylarsonium and P-(para-substituted benzyl)tri(n-butyl)phosphonium cations with the oxidation potentials of their conjugate bases (ylides) as shown in Figures 3 and 4, respectively.

Bond Dissociation Free Energies (BDFEs) of the Acidic H-A Bonds in HA(*)(-) Radical Anions by Three Different Pathways.

Cleavage of radical anions, HA(*)(-), have been considered to give either H(*) + A(-) (path a) or H(-) + A(*) (path b), and factors determining the preferred mode of cleavage have been discussed. It is conceivable that cleavage to give a proton and a radical dianion, HA(*)(-) right harpoon over left harpoon H(+) + A(*)(2)(-) (path c), might also be feasible. A method, based on a thermodynamic cycle, to estimate the bond dissociation free energy (BDFE) by path c has been devised. Comparison of the BDFEs for cleavage of the radical anions derived from 24 nitroaromatic OH, SH, NH, and CH acids by paths a, b, c has shown that path c is favored thermodynamically.