Recombinase-mediated gene stacking in cotton.

Site-specific gene stacking could reduce the number of segregating loci and expedite the introgression of transgenes from experimental lines to field lines. Recombinase-mediated site-specific gene stacking provides a flexible and efficient solution, but this approach requires a recombinase recognition site in the genome. Here, we describe several cotton (Gossypium hirsutum cv. Coker 312) target lines suitable for Mycobacteriophage Bxb1 recombinase-mediated gene stacking. Obtained through the empirical screening of random insertion events, each of these target lines contains a single intact copy of the target construct with precise sequences of RS2, lox, and attP sites that is not inserted within or close to a known gene or near a centromere and shows good expression of the reporter gene gfp. Gene stacking was tested with insertion of different combinations of three candidate genes for resistance to verticillium wilt into three cotton target lines: CTS1, CTS3, and CTS4. Nine site-specific integration events were recovered from 95 independently transformed embryogenic calluses. Southern and DNA sequence analyses of regenerated plants confirmed precise site-specific integration, and resistance to verticillium wilt was observed for plant CTS1i3, which has a single precise copy of site-specifically integrated DNA. These cotton target lines can serve as foundation lines for recombinase-mediated gene stacking to facilitate precise DNA integration and introgression to field cultivars.

Target lines for recombinase-mediated gene stacking in soybean.

KEY MESSAGE: Five soybean target lines with recombinase sites at suitable genomic positions were obtained and tested for site-specific gene stacking. For introgression of new transgenic traits to field cultivars, adding new DNA to an existing transgene locus would reduce the number of segregating loci to reassemble back into a breeding line. We described previously an in planta transgene stacking system using the Bxb1 integrase to direct new DNA into a genomic target, but for this system to operate, the target locus must have a preexisting recombination site for Bxb1-mediated integration. Here, we describe 5 soybean target lines from the screening of 118 Agrobacterium-mediated transgenic plants that were positive for gus expression. Each of the 5 target lines has a single copy of the transgenic DNA with precise DNA sequences of the recombinase recognition sites, located at least 1 kb away from the nearest coding region, not close to the centromere, and showed good expression of the reporter gene. We tested Bxb1 integrase-mediated integration of a gfp-containing plasmid into each of these lines and showed precise site-specific integration in bombarded calluses. For plant regeneration, we used embryonic axes of mature soybean seeds to conduct a new set of biolistic transformation with a DsRed-containing plasmid. Three integration events were regenerated into whole plants, demonstrating the principle that target lines can serve as foundation lines for the stacking of DNA to predefined locations in the soybean genome.

Target Lines for in Planta Gene Stacking in Japonica Rice.

The clustering of transgenes at a chromosome location minimizes the number of segregating loci that needs to be introgressed to field cultivars. Transgenes could be efficiently stacked through site-specific recombination and a recombinase-mediated in planta gene stacking process was described previously in tobacco based on the Mycobacteriophage Bxb1 site-specific integration system. Since this process requires a recombination site in the genome, this work describes the generation of target sites in the Japonica rice genome. Agrobacterium-mediated gene transfer yielded ~4000 random-insertion lines. Seven lines met the criteria of being single copy, not close to a centromere, not inserted within or close to a known gene or repetitive DNA, having precise recombination site sequences on both ends, and able to express the reporter gene. Each target line tested was able to accept the site-specific integration of a new gfp-containing plasmid and in three of those lines, we regenerated fertile plants. These target lines could be used as foundation lines for stacking new traits into Japonica rice.

Optimal guidance law design for three-dimensional trajectory tracking of missiles based on fixed-gain disturbance observer.

To address the trajectory tracking problem of missiles, an optimal disturbance rejection guidance law (ODRGL) in three dimensions is proposed under the premise of uncontrollable speed. Error models of the actual and the reference trajectory are built in channels of pitch and yaw, respectively. In the yaw channel, a modeling error is introduced as the model cannot be built directly. According to the separation theorem, the controller and the observer are designed respectively. A fixed-gain disturbance observer (FGDO) is proposed to estimate the disturbance such as wind disturbances and modeling errors, and the disturbance is compensated for feed-forward. The nominal error models of pitch and yaw channels are linearized by the differential geometric linearization theory, and then linear quadratic regulator (LQR) controllers are designed for the linearized models. The simulation results show that in the presence of wind disturbances and modeling errors, the optimal guidance law proposed in this paper can stabilize the original nonlinear system and ensure energy optimization.

[Diagnosis and treatment of otogenic sigmoid sinus thrombophlebitis].

Objective:To summarize and analyze the clinical data of sigmoid sinus thrombophlebitis and discuss its treatment strategy. Methods:The clinical manifestations, auxiliary examinations, surgical procedures and anticoagulant therapy of patients diagnosed with otosource sigmoid sinus thrombophlebitis in the Department of Otorhinolaryngology, People's Hospital of Xinjiang Uygur Autonomous Region from November 2014 to November 2021 were retrospectively analyzed. Results:Five patients had a history of otorrhea and hearing loss for more than 5 years. They had headache during the acute episode, and 4 patients had drosive fever. They had severe complications, including brain abscess, sepsis, septic shock, intracranial hemorrhage, and hemorrhagic disseminated pneumonia. HRCT of temporal bone showed defects in the sigmoid sinus wall in 4 cases, and gas accumulation around and inside the sigmoid sinus in 3 cases. T1WI showed low signal, isosignal, and high signal in the sigmoid sinus area on MRI, and T2WI showed high signal in the sigmoid sinus area. The transverse sinus, sigmoid sinus and internal jugular vein were not developed in 2 cases, and the transverse sinus and sigmoid sinus were not developed in 1 case, and the internal jugular vein was thin. All 5 cases underwent radical mastoidectomy and resection of sigmoid sinus wall granulation or peritosinusitis abscess. The patients were followed up for 4-12 months and recovered well. Conclusion:For the cases of sigmoid sinus bone wall destruction accompanied by headache and fever by HRCT, it is necessary to be alert to the occurrence of sigmoid sinus thrombophlegitis, early diagnosis and early surgery to prevent the progression of the disease. Radical mastoidectomy combined with anti-infection therapy is the main treatment, and anticoagulation is necessary to achieve a better prognosis.

A flow cytometer based protocol for quantitative analysis of bloom-forming cyanobacteria (Microcystis) in lake sediments.

A quantitative protocol for the rapid analysis of Microcystis cells and colonies in lake sediment was developed using a modified flow cytometer, the CytoSense. For cell enumeration, diluted sediment samples containing Microcystis were processed with sonication to disintegrate colonies into single cells. An optimized procedure suggested that 5 mg dw (dry weight)/mL dilution combined with 200 W x 2 min sonication yielded the highest counting efficiency. Under the optimized determination conditions, the quantification limit of this protocol was 3.3 x 10(4) cells/g dw. For colony analysis, Microcystis were isolated from the sediment by filtration. Colony lengths measured by flow cytometry were similar to those measured by microscopy for the size range of one single cell to almost 400 microm in length. Moreover, the relationship between colony size and cell number was determined for three Microcystis species, including Microcystis flos-aquae, M. aeruginosa and M. wessenbergii. Regression formulas were used to calculate the cell numbers in different-sized colonies. The developed protocol was applied to field sediment samples from Lake Taihu. The results indicated the potential and applicability of flow cytometry as a tool for the rapid analysis of benthic Microcystis. This study provided a new capability for the high frequency monitoring of benthic overwintering and population dynamics of this bloom-forming cyanobacterium.

Combining novel feature selection strategy and hyperspectral vegetation indices to predict crop yield.

BACKGROUND: Wheat is an important food crop globally, and timely prediction of wheat yield in breeding efforts can improve selection efficiency. Traditional yield prediction method based on secondary traits is time-consuming, costly, and destructive. It is urgent to develop innovative methods to improve selection efficiency and accelerate genetic gains in the breeding cycle. RESULTS: Crop yield prediction using remote sensing has gained popularity in recent years. This paper proposed a novel ensemble feature selection (EFS) method to improve yield prediction from hyperspectral data. For this, 207 wheat cultivars and breeding lines were grown under full and limited irrigation treatments respectively, and their canopy hyperspectral reflectance was measured at the flowering, early grain filling (EGF), mid grain filling (MGF), and late grain filling (LGF) stages. Then, 115 vegetation indices were extracted from the hyperspectral reflectance and combined with four feature selection methods, i.e., mean decrease impurity (MDI), Boruta, FeaLect, and RReliefF to train deep neural network (DNN) models for yield prediction. Next, a learning framework was developed by combining the predicted values of the selected and the full features using multiple linear regression (MLR). The results show that the selected features contributed to higher yield prediction accuracy than the full features, and the MDI method performed well across growth stages, with a mean R2 ranging from 0.634 to 0.666 (mean RMSE = 0.926-0.967 t ha-1). Also, the proposed EFS method outperformed all the individual feature selection methods across growth stages, with a mean R2 ranging from 0.648 to 0.679 (mean RMSE = 0.911-0.950 t ha-1). CONCLUSIONS: The proposed EFS method can improve grain yield prediction from hyperspectral data and can be used to assist wheat breeders in earlier decision-making.

Efficient genetic transformation and CRISPR/Cas9-mediated genome editing of watermelon assisted by genes encoding developmental regulators.

Cucurbitaceae is an important family of flowering plants containing multiple species of important food plants, such as melons, cucumbers, squashes, and pumpkins. However, a highly efficient genetic transformation system has not been established for most of these species (Nanasato and Tabei, 2020). Watermelon (Citrullus lanatus), an economically important and globally cultivated fruit crop, is a model species for fruit quality research due to its rich diversity of fruit size, shape, flavor, aroma, texture, peel and flesh color, and nutritional composition (Guo et al., 2019). Through pan-genome sequencing, many candidate loci associated with fruit quality traits have been identified (Guo et al., 2019). However, few of these loci have been validated. The major barrier is the low transformation efficiency of the species, with only few successful cases of genetic transformation reported so far (Tian et al., 2017; Feng et al., 2021; Wang JF et al., 2021; Wang YP et al., 2021). For example, Tian et al. (2017) obtained only 16 transgenic lines from about 960 cotyledon fragments, yielding a transformation efficiency of 1.67%. Therefore, efficient genetic transformation could not only facilitate the functional genomic studies in watermelon as well as other horticultural species, but also speed up the transgenic and genome-editing breeding.

Regulation of NAD(P)H dehydrogenase-dependent cyclic electron transport around PSI by NaHSO3 at low concentrations in tobacco chloroplasts.

Although bisulfite at low concentrations (L-NaHSO3) has been found to increase the cyclic electron transport around PSI (CET), its regulative mechanism remains unknown. In this work, the role of L-NaHSO3 (0.1-500 µM) in NAD(P)H dehydrogenase-dependent CET (the NDH pathway) was investigated. After treatment of tobacco leaves with L-NaHSO3, the NDH pathway, as reflected by a transient post-illumination increase in Chl fluorescence, the dark reduction of P700+ after far-red light and the amount of NDH, was increased after the light-dark-light transition, but was slightly lowered under continuous light. Meanwhile, the linear electron transport (LET) was accelerated by L-NaHSO3 under both the light regimes. Experiments in thylakoids further demonstrated that both LET, monitored by light-dependent oxygen uptake, and CET, as determined from the NADPH-dependent oxygen uptake and dark reduction of P700+, were enhanced by L-NaHSO3 and the enhancements were abolished by superoxide dismutase. Furthermore, L-NaHSO3-induced CET was partially impaired in thylakoids of the ΔndhCKJ mutant, while L-NaHSO3-induced LET was not affected. Based on these results, we propose that the photooxidation of L-NaHSO3 initiated by superoxide anions in PSI regulates NDH pathway to maintain efficient photosynthesis.

Wavelet Transform Artificial Intelligence Algorithm-Based Data Mining Technology for Norovirus Monitoring and Early Warning.

Norovirus monitoring and early warning can be used for diagnosis without etiological testing, and the treatment of this disease does not require the antibiotics. It often occurs in preschool children and affects their growth and development, so the coping measures for this disease are more prevention than treatment. In this study, the clinical data of 2133 children with diarrhea were collected. Based on the artificial intelligence (AI) algorithm of wavelet transform, a related model for data mining and processing of children's intestinal ultrasound images and stool specimens was constructed. Then, the norovirus infection trend was warned based on the wavelet analysis algorithm model. The results showed that the intestinal ultrasound image processed by the wavelet transform algorithm was clearer. The positive detection rate of norovirus in children with clinical diarrhea was as high as 59%, and the children had different degrees of body damage, of which the probability of compensatory metabolic acidosis was the highest. The epidemiological analysis found that children with norovirus infection were mainly concentrated in the age group under 2 years old and over 5 years old and showed a peak of infection in December. In summary, the intelligent algorithm based on wavelet transform can realize the noise reduction of intestinal ultrasound, and it should protect children with susceptible age and susceptible seasons to reduce the clinical infection rate of norovirus.

Evaluation of the protective effects of Chinese herbs against biomolecule damage induced by peroxynitrite.

This work evaluates the protective effects of Chinese herbs against ONOO(-)-induced biomolecule damage. Thirty-two Chinese traditional herbs were preliminarily screened for their ONOO(-)-scavenging activity by the fluorometric method. The potency of scavenging activity was in the following order: Apis cerana Fabricius (Propolis) > Rosmarinus officinalis L (Rosemary) > Pseudolarix amabilis (Nelson) Rehd. (Pine Bark PE) > Echinacea Moenck. (Echinacea); the remaining twenty-eight herbs performed unsatisfactorily for their scavenging activity. The four extracts with high ONOO(-) scavenging activity were selected for further characterization of their inhibiting effects on DNA single breaks, tyrosine nitration and LDL oxidation. A further study of the origin of this scavenging activity on the major active components showed cichoric acid and rosemary acid to both have strong ability to scavenge ONOO(-). These scavengers might be developed as therapeutic drugs for preventing ONOO(-)-involved diseases.

A microsatellite-based, gene-rich linkage map reveals genome structure, function and evolution in Gossypium.

The mapping of functional genes plays an important role in studies of genome structure, function, and evolution, as well as allowing gene cloning and marker-assisted selection to improve agriculturally important traits. Simple sequence repeats (SSRs) developed from expressed sequence tags (ESTs), EST-SSR (eSSR), can be employed as putative functional marker loci to easily tag corresponding functional genes. In this paper, 2218 eSSRs, 1554 from G. raimondii-derived and 754 from G. hirsutum-derived ESTs, were developed and used to screen polymorphisms to enhance our backbone genetic map in allotetraploid cotton. Of the 1554 G. raimondii-derived eSSRs, 744 eSSRs were able to successfully amplify polymorphisms between our two mapping parents, TM-1 and Hai7124, presenting a polymorphic rate of 47.9%. However, only a 23.9% (159/754) polymorphic rate was produced from G. hirsutum-derived eSSRs. No relationship was observed between the level of polymorphism, motif type, and tissue origin, but the polymorphism appeared to be correlated with repeat type. After integrating these new eSSRs, our enhanced genetic map consists of 1790 loci in 26 linkage groups and covers 3425.8 cM with an average intermarker distance of 1.91 cM. This microsatellite-based, gene-rich linkage map contains 71.96% functional marker loci, of which 87.11% are eSSR loci. There were 132 duplicated loci bridging 13 homeologous At/Dt chromosome pairs. Two reciprocal translocations after polyploidization between A2 and A3, and between A4 and A5, chromosomes were further confirmed. A functional analysis of 975 ESTs producing 1122 eSSR loci tagged in the map revealed that 60% had clear BLASTX hits (<1e(-10)) to the Uniprot database and that 475 were associated mainly with genes belonging to the three major gene ontology categories of biological process, cellular component, and molecular function; many of the ESTs were associated with two or more category functions. The results presented here will provide new insights for future investigations of functional and evolutionary genomics, especially those associated with cotton fiber improvement.

Cranial nerve palsy secondary to cerebrospinal fluid diversion.

OBJECTIVE: Cranial nerve palsy (CNP) secondary to cerebrospinal fluid (CSF) diversion is less familiar to us as a result of its rarity in incidence and insidiousness in presentation. This study aims to further expound the pathophysiological mechanism, clinical presentation, diagnosis, management and prognosis of CNP. METHODS: From June 2012 to February 2015, 5 of 347 consecutive patients with CNPs secondary to different CSF diversion procedures were treated at our institution. A systematic PubMed search of published studies written in English for patients developing CNPs after CSF diversion procedures from January 1950 to June 2015 was conducted. RESULTS: Overall, 29 studies and 5 patients of the current series totaling 53 CNPs met the inclusion criteria. CN II, III, IV, V, VI, VII and VIII were got involved in 2 (3.8%), 2 (3.8%), 5 (9.4%), 1 (1.9%), 44 (83.0%), 4 (7.5%) and 1 (1.9%) patients respectively. Thirty-eight patients (71.7%) developed CNPs following inadvertent lumbar puncture, 8 (15.1%) following lumbar drainage, and 7 (13.2%) following ventriculoperitoneal shunt. Forty-eight (90.6%) patients got resolved completely. CONCLUSIONS: The proposed mechanism of CNP after CSF diversion procedure is CSF hypovolemia and subsequent downward displacement of the brain and traction and distortion of the vascular and peripheral neural structures. As a result of its distinct anatomic characteristics rather than long intracranial course, CN VI is most commonly affected. With early recognition and timely conservative management, most patients could get favorable recovery.

Method for Biolistic Site-Specific Integration in Plants Catalyzed by Bxb1 Integrase.

Crop improvement is a never ending process. With a transgenesis approach, it is not inconceivable to envision a continuous addition of new transgenes to existing cultivars. Previously, we described a recombinase-directed gene stacking method in tobacco (Hou et al., Mol Plant 7:1756-1765, 2014). Being able to stack DNA to a previous location ensures that the number of genetic loci does not increase with each new round of transgene addition. Whereas the previous demonstration was conducted through polyethylene glycol to mediate uptake of DNA into tobacco protoplasts, we now describe protocols for using biolistic transformation to stack DNA in tobacco and rice.

Mcl-1 downregulation sensitizes glioma to bortezomib-induced apoptosis.

Glioma is the most aggressive form of primary brain tumor, with dismal patient outcome and no effective therapeutic approaches available. Targeting the ubiquitin-proteasome pathway has recently emerged as a potent rational anticancer strategy. Bortezomib, a specific proteasome inhibitor, has been approved for the treatment of relapsed or refractory multiple myeloma and other hematological malignancies as a single agent or as part of a combination therapy. However, bortezomib alone or in combination showed only minimal effects in the treatment of solid tumors. Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein which protects tumor cells against spontaneous and chemotherapy-induced apoptosis. In multiple myeloma, specific downregulation of Mcl-1 induces apoptosis. Furthermore, previous studies demonstrated that proteasome inhibitors induce Mcl-1 accumulation that, in turn, slows down their pro-apoptotic effects, and the cell survival in multiple myeloma is highly dependent on Mcl-1. In the present study, we investigated the role of Mcl-1 downregulation in bortezomib-induced apoptosis in gliomas. We observed that bortezomib triggers caspase-3 and PARP activation, upregulates cytochrome c expression and induces apoptosis. Furthermore, we demonstrated that effective targeting of Mcl-1 in glioma cells by gene silencing technology augments the glioma cell sensitivity to bortezomib-induced apoptosis. In conclusion, the present study demonstrates that Mcl-1 plays a critical role in bortezomib-induced apoptosis. Mcl-1 inhibitor in combination with bortezomib present a promising novel strategy to trigger cell death pathways in the treatment of gliomas.

An open-source system for in planta gene stacking by Bxb1 and Cre recombinases.

The rapid development of crops with multiple transgenic traits arouses the need for an efficient system for creating stacked cultivars. Most major crops rely on classical breeding to introgress the transgene from a laboratory variety to the numerous cultivars adapted to different growing regions. Even with vegetative propagated crops, genetic crosses are conducted during varietal improvement prior to vegetative cloning. The probability to assort the 'x' number of transgenic loci into a single genome may seem trivial, (») (x) for a diploid species, but given the 'y' number of other nontransgenic traits that breeders also need to assemble into the same genome, the (») (x+y) probability for a 'breeding stack' could quickly make the line conversion process unmanageable. Adding new transgenes onto existing transgenic varieties without creating a new segregating locus would require site-specific integration of new DNA at the existing transgenic locus. Here, we tested a recombinase-mediated gene-stacking scheme in tobacco. Sequential site-specific integration was mediated by the mycobacteriophage Bxb1 integrase-catalyzed recombination between attP and attB sites. Transgenic DNA no longer needed after integration was excised by Cre recombinase-mediated recombination of lox sites. Site-specific integration occurred in ~10% of the integration events, with half of those events usable as substrates for a next round of gene stacking. Among the site-specific integrants, however, a third experienced gene silencing. Overall, precise structure and reproducible expression of the sequentially added triple traits were obtained at an overall rate of ~3% of the transformed clones--a workable frequency for the development of commercial cultivars. Moreover, since neither the Bxb1-att nor the Cre-lox system is under patent, there is freedom to operate.

[Nitrogen balance in dairy farm: research progress].

Large dairy farm with intensive management has high stocking density, but generally does not have enough space and normative feces disposal system, resulting in the discharged nitrogen surpassed the environmental carrying capacity of unit area land. Dairy farm is one of the major emission sources of nitrogen discharges in agriculture, where the nitrogen balance has being aroused attention by the experts abroad. The research on the nitrogen flow and nitrogen balance in dairy farm is the basis of the dairy farm nitrogen cycling and management study, as well as the basis for the construction of environmental laws, regulations and policies. The most reliable indicators to evaluate the nitrogen flow and nitrogen balance in dairy farm are nitrogen surplus and nitrogen use efficiency. This paper introduced the concept of nitrogen balance on farm-scale and the nitrogen flow within farm, compared the application scope of nitrogen surplus and nitrogen use efficiency, analyzed the factors affecting the nitrogen balance in dairy farm, and summarized the effective strategies to reduce the nitrogen discharges from dairy farm, aimed to provide references for the nitrogen management of dairy farm in China.

Characteristics, development and mapping of Gossypium hirsutum derived EST-SSRs in allotetraploid cotton.

In order to construct a saturated genetic map and facilitate marker-assisted selection (MAS) breeding, it is necessary to enhance the current reservoir of known molecular markers in Gossypium. Microsatellites or simple sequence repeats (SSRs) occur in expressed sequence tags (EST) in plants. Many ESTs are publicly available now and represent a good tool in developing EST-SSRs. From 13,505 ESTs developed from our two cotton fiber/ovule cDNA libraries constructed for Upland cotton, 966 (7.15%) contained one or more SSRs and from them, 489 EST-SSR primer pairs were developed. Among the EST-SSRs, 59.1% are trinucleotides, followed by dinucleotides (30%), tetranucleotides (6.4%), pentanucleotides (1.8%), and hexanucleotides (2.7%). AT/TA (18.4%) is the most frequent repeat, followed by CTT/GAA (5.3%), AG/TC (5.1%), AGA/TCT (4.9%), AGT/TCA (4.5%), and AAG/TTC (4.5%). One hundred and thirty EST-SSR loci were produced from 114 informative EST-SSR primer pairs, which generated polymorphism between our two mapping parents. Of these, 123 were integrated on our allotetraploid cotton genetic map, based on the cross [(TM-1xHai7124)TM-1]. EST-SSR markers were distributed over 20 chromosomes and 6 linkage groups in the map. These EST-SSR markers can be used in genetic mapping, identification of quantitative trait loci (QTLs), and comparative genomics studies of cotton.

Complete assignment of the chromosomes of Gossypium hirsutum L. by translocation and fluorescence in situ hybridization mapping.

Significant progress has been made in the construction of genetic maps in the tetraploid cotton Gossypium hirsutum. However, six linkage groups (LGs) have still not been assigned to specific chromosomes, which is a hindrance for integrated genetic map construction. In the present research, specific bacterial artificial chromosome (BAC) clones constructed in G. hirsutum acc. TM-1 for these six LGs were identified by screening the BAC library using linkage group-specific simple-sequence repeats markers. These BAC clones were hybridized to ten translocation heterozygotes of G. hirsutum. L as BAC-fluorescence in situ hybridization probes, which allowed us to assign these six LGs A01, A02, A03, D02, D03, and D08 to chromosomes 13, 8, 11, 21, 24, and 19, respectively. Therefore, the 13 homeologous chromosome pairs have been established, and we have proposed a new chromosome nomenclature for tetraploid cotton.