Regulation of AtbZIP1 Gene Expression by T-DNA Insertion in Arabidopsis thaliana

bZIP transcription factor family is one of the largest groups of the plant transcription factor families and plays an important role in plant growth and adaption to the abiotic stresses. In this study, two AtbZIP1 mutant Arabidopsis （bzipl） were used with T-DNA inserted into two different sites, designated as SALK-556773 and SALK-660942, in order to identify different effects on AtbZIP1 gene expression by different T-DNA insertion sites. PCR and RT-PCR results revealed that T-DNA insertion in CDS region could effectively inhibit AtbZIP1 gene expression, while T-DNA insertion in 3＇-UTR couldn＇t. The phenotype analysis further confirmed the differences and showed that T-DNA insertion in CDS region decreased plants＇ drought resistance, while in 3＇-UTR couldn＇t. The phenotype assays also suggested that AtbZIP1 held pivotal roles in plant response to drought stress.

Studies on rice seed quality through analysis of a large- scale T-DNA insertion population

米饭(Oryza sativa ) T-DNA 插入人口，它包括了超过 63 000 根独立的转基因的线和 8 840 识别 flanking 定序被印射到大米染色体上的标签(FST ) ，被开发全身地学习大米种子质量控制。FST 分发的染色体宽的分析证明 T-DNA 插入断然与表示基因被相关，但是否定地与 transposable 元素和小 RNA。另外，恢复 T-DNAs 优先地在表示基因的 untranslated 区域被定位。超过 11 000 根通常认为的同型结合的线通过种的多产生被获得，包括淀粉，直链淀粉，蛋白质和脂肪的内容并且屏蔽的抵抗，和种子质量的测量在他们的一半附近与一个非破坏性的在红外线附近的光谱学方法，与种子质量的唯一或多重的改变的参数识别了 551 异种。相应 FST 的分析证明包括新陈代谢的进程和 transcriptional 规定，参予多样的函数的基因被包含，显示种子质量被一个复杂网络调整。

Construction of Mutant Populations by T-DNA Insertion for Functional Genomic Study in Cotton

The use of transfer DNA(T-DNA)as amutagen has been developed for tagging genes inmany crops,and results showed that T-DNAinsertion is a random event,and that theinserted genes are stable through multiplegenerations.Through sequencing PCR-amplifiedfragments adjacent to the inserted elements,wecan construct the T-DNA flanking database,which would be useful for cloning the genestagged by T-DNA.

An efficient method for constructing a random insertional mutant library for forward genetics in Nannochloropsis oceanica

Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-strand breaks of DNA,causing insertional mutation.The random insertional mutant library constructed using this method has become a method of forward genetics for gene cloning.However,the establishment of a random insertional mutant library requires a high transformation efficiency of exogenous genes.Many microalgal species show a low transformation efficiency,making constructing random insertional mutant libraries difficult.In this study,we established a highly efficient transformation method for constructing a random insertional mutant library of Nannochloropsis oceanica,and tentatively tried to isolate its genes to prove the feasibility of the method.A gene that may control the growth rate and cell size was identified.This method will facilitate the genetic studies of N.oceanica,which should also be a reference for other microalgal species.

Velamentous Cord Insertion: Interest of Antenatal Diagnosis and Review of the Literature

Velamentous insertion of the umbilical cord corresponds to the insertion of the cord directly on amniotic membranes. It is a rare situation whose frequency varies from 0.5% to 1.69% of single pregnancies. It must be diagnosed during the morphological ultrasound of the 2nd trimester, actively looking for the association with a vasa previa, due to the risk of fetal haemorrhagic threat. We report an antenatal diagnosis of velamentous cord insertion and its management with literature review.

Treatment of benign rectal stricture caused by repeated anal insertion by endoscopy and balloon dilation:A case report

BACKGROUND Benign rectal strictures can be categorized as primary(disease-related)and secondary(surgical anastomosis-related).Secondary strictures arise from surgical complications,whereas primary strictures have diverse etiologies,including various inflammatory conditions.Benign strictures are usually managed by surgery and endoscopy.We present an unusual etiology of benign rectal stricture caused by the repeated insertion of foreign objects into the rectum for sexual purposes,resulting in rectal injury and subsequent chronic inflammation.CASE SUMMARY A 53-year-old man presented to the outpatient clinic of the Colorectal Surgery Department with symptoms of chronic constipation and bloody stools.The patient previously experienced rectal injury due to foreign object insertion for sexual purposes.Colonoscopy revealed benign circumferential narrowing of the rectum.He underwent treatment by endoscopic argon plasma coagulation and balloon dilation and follow-up as an outpatient for 4 months.A colonoscopy at the end of the follow-up period revealed no evidence of rectal stricture relapse.CONCLUSION A history of rectal injury,followed by chronic inflammation,should be considered in patients with benign rectal strictures.Management with endoscopic argon plasma coagulation and balloon dilation can prevent the need for surgical resection of benign rectal strictures.

Induction of the AOX1D Isoform of Alternative Oxidase in A. thaliana T-DNA Insertion Lines Lacking Isoform AOX1A Is Insufficient to Optimize Photosynthesis when Treated with Antimycin A

Plant respiration is characterized by two pathways for electron transfer to O2, namely the cytochrome pathway （CP） that is linked to ATP production, and the alternative pathway （AP）, where electrons from ubiquinol are directly transferred to O2 via an alternative oxidase （AOX） without concomitant ATP production. This latter pathway is well suited to dispose of excess electrons in the light, leading to optimized photosynthetic performance. We have characterized T- DNA-insertion mutant lines of Arabidopsis thaliana that do not express the major isoform, AOXIA. In standard growth conditions, these plants did not show any phenotype, but restriction of electron flow through CP by antimycin A, which induces AOXIA expression in the wild-type, led to an increased expression of AOXID in leaves of the aoxla-knockout mutant. Despite the increased presence of the AOX1D isoform in the mutant, antimycin A caused inhibition of photosyn- thesis, increased ROS, and ultimately resulted in amplified membrane leakage and necrosis when compared to the wild- type, which was only marginally affected by the inhibitor. It thus appears that AOX1 D was unable to fully compensate for the loss of AOXIA when electron flow via the CP is restricted. A combination of inhibition studies, coupled to metabolite profiling and targeted expression analysis of the P-protein of glycine decarboxylase complex （GDC）, suggests that the aoxla mutants attempt to increase their capacity for photorespiration. However, given their deficiency, it is intriguing that increase in expression neither of AOX1D nor of GDC could fully compensate for the lack of AOXIA to optimize pho- tosynthesis when treated with antimycin A. We suggest that the aoxla mutants can further be used to substantiate the current models concerning the influence of mitochondrial redox on photosynthetic performance and gene expression.

The alteration in the architecture of a T-DNA insertion rice mutant osmtd1 is caused by up-regulation of Micro RNA156f

Plant architecture is an important factor for crop production. Some members of microRNA156 (miR156) and their target genes SQUAMOSA Promoter-Binding Protein-Like (SPL) were identified to play essential roles in the establishment of plant architecture. However, the roles and regulation of miR156 is not well understood yet. Here, we identified a T-DNA insertion mutant Osmtd1 (Oryza sativa multi-tillering and dwarf mutant). Osmtd1 produced more tillers and displayed short stature phenotype. We determined that the dramatic morphological changes were caused by a single T-DNA insertion in Osmtd1. Further analysis revealed that the T-DNA insertion was located in the gene Os08g34258 encoding a putative inhibitor I family protein. Os08g34258 was knocked out and OsmiR156f was significantly upregulated in Osmtd1. Overexpression of Os08g34258 in Osmtd1 complemented the defects of the mutant architecture, while overexpression of OsmiR156f in wild-type rice phenocopied Osmtd1. We showed that the expression of OsSPL3, OsSPL12, and OsSPL14 were significantly downregulated in Osmtd1 or OsmiR156f overexpressed lines, indicating that OsSPL3, OsSPL12, and OsSPL14 were possibly direct target genes of OsmiR156f. Our results suggested that OsmiR156f controlled plant architecture by mediating plant stature and tiller outgrowth and may be regulated by an unknown protease inhibitor I family protein.

Effort and Contribution of T-DNA Insertion Mutant Library for Rice Functional Genomics Research in China:Review and Perspective

With the completion of the rice （Oryza sativa L.） genome-sequencing project, the rice research community proposed to characterize the func- tion of every predicted gene in rice by 2020. One of the most effective and high-throughput strategies for studying gene function is to employ genetic mutations induced by insertion elements such as T-DNA or transposons. Since 1999, with support from the Ministry of Science and Technology of China for Rice Functional Genomics Programs, large-scale T-DNA insertion mutant populations have been generated in Huazhong Agricultural University, the Chinese Academy of Sciences and the Chinese Academy of Agricultural Sciences. Currently, a total of 372,346 mutant lines have been generated, and 58,226 T-DNA or Tos17 flanking sequence tags have been isolated. Using these mutant resources, more than 40 genes with potential applications in rice breeding have already been identified. These include genes involved in biotic or abiotic stress responses, nutrient metabolism, pollen development, and plant architecture. The functional analysis of these genes will not only deepen our understanding of the fundamental biological questions in rice, but will also offer valuable gene resources for developing Green Super Rice that is high-yielding with few inputs even under the poor growth conditions of many regions of Africa and Asia.

Atomic-scale insights of indium segregation and its suppression by GaAs insertion layer in InGaAs/AlGaAs multiple quantum wells

The In segregation and its suppression in InGaAs/AlGaAs quantum well are investigated by using high-resolution x-ray diffraction(XRD)and photoluminescence(PL),combined with the state-of-the-art aberration corrected scanning transmission electron microscopy(Cs-STEM)techniques.To facility our study,we grow two multiple quantum wells(MQWs)samples,which are almost identical except that in sample B a thin GaAs layer is inserted in each of the InGaAs well and AlGaAs barrier layer comparing to pristine InGaAs/AlGaAs MQWs(sample A).Our study indeed shows the direct evidences that In segregation occurs in the InGaAs/AlGaAs interface,and the effect of the Ga As insertion layer on suppressing the segregation of In atoms is also demonstrated on the atomic-scale.Therefore,the atomic-scale insights are provided to understand the segregation behavior of In atoms and to unravel the underlying mechanism of the effect of GaAs insertion layer on the improvement of crystallinity,interface roughness,and further an enhanced optical performance of InGaAs/AlGaAs QWs.

Complications Related to the Insertion Technique of Jadelle® Contraceptive Implants

Introduction: Contraceptive implants are one of the most effective methods of birth spacing. Jadelle® implants consist of two strands that are easy to insert and remove. Although their effectiveness is no longer in question, their use (insertion) requires a surgical procedure with the corollary possibility of complications. These are mainly insertions that are too deep (in the arm muscle), vascular and nerve damage. Material and Methods: Our study focused on complications related to implant insertion. It was a descriptive and retrospective study over thirty-four months, from October 2016 to July 2019, and concerned all patients seen in consultation and who presented a complication related to the insertion of contraceptive implants in the Department of Gynecology and Obstetrics of the National Hospital of Pikine. Results: We collected nine complications managed at the Gynecology and Obstetrics Department of the Centre Hospitalier National de Pikine from 2016 to 2019. These were insertions that were too deep with sometimes nerve damage, infection or incident during anesthesia. The operative procedures were based on the type of complication. Conclusion: Although Jadelle® has the advantage of having only 2 rods compared to its predecessor Norplant®, its use is also conditioned by insertion and removal procedures which may experience complications.

Complicated expansion trajectories of insertion sequences and potential association with horizontal transfer of Wolbachia DNA

DEAR EDITOR,Insertion sequences(ISs) are the simplest structural transposable elements(TEs) in prokaryotes, consisting only of a transposase coding sequence and its bilateral short terminal inverted repeats. Due to their gradually streamlined genomic construction, TEs rarely exist in the genomes of obligate endosymbionts. However, TE content, especially ISs.

Promoter insertion leads to polyembryony in mango-a case of convergent evolution with citrus

Sexual reproduction in plants is the main pathway for creating new genetic combinations in modern agriculture.In heterozygous plants,after the identification of a plant with desired traits,vegetative propagation(cloning)is the primary path to create genetically uniform plants.Another natural plant mechanism that creates genetically uniform plants(clones)is apomixis.In fruit crops like citrus and mango,sporophytic apomixis results in polyembryony,where seeds contain multiple embryos,one of which is sexually originated and the others are vegetative clones of the parent mother tree.Utilizing the mango genome and genetic analysis of a diverse germplasm collection,we identified MiRWP as the gene that causes polyembryony in mango.There is a strong correlation between a specific insertion in the gene’s promoter region and altered expression in flowers and developing fruitlets,inducing multiple embryos.The MiRWP gene is an ortholog of CitRWP that causes polyembryony in citrus.Based on the data,we speculate that promoter insertion events,which occurred independently in citrus and mango,induced nucellar embryogenesis.The results suggest convergent evolution of polyembryony in the two species.Further work is required to demonstrate the utility of these genes(mango and citrus)in other biological systems as a tool for the clonal production of other crops.

The Structural Features of Thousands of T-DNA Insertion Sites Are Consistent with a Double- Strand Break Repair-Based Insertion Mechanism

Transformation by Agrobacterium tumefaciens, an important tool in modern plant research, involves the integration of T-DNA initially present on a plasmid in agrobacteria into the genome of plant cells. The process of attachment of the agrobacteria to plant cells and the transport of T-DNA into the cell and further to the nucleus has been well described. However, the exact mechanism of integration into the host＇s DNA is still unclear, although several models have been proposed. During confirmation of T-DNA insertion alleles from the GABI-Kat collection of Arabidopsis thaliana mutants, we have generated about 34 000 sequences from the junctions between inserted T-DNA and adjacent genome regions. Here, we describe the evaluation of this dataset with regard to existing models for T-DNA integration. The results suggest that integration into the plant genome is mainly mediated by the endogenous plant DNA repair machinery. The observed integration events showed characteristics highly similar to those of repair sites of double- strand breaks with respect to microhomology and deletion sizes. In addition, we describe unexpected integration events, such as large deletions and inversions at the integration site that are relevant for correct interpretation of results from T-DNA insertion mutants in reverse genetics experiments.

T-DNA transfer and integration as a tool for insertional mutagenesis in the taxol-producing fungus

Agrobacterium tumefaciens-mediated DNA transformation method was applied to transformNodulisporium sylviforme fusant HDF-68,a taxol-producing fungus.We constructed a binary vectorpBI121-43 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA.Optimal co-cultivation of N.sylviforme with A.turnefaciens containing pBI121-43 led to 110～130 hy-gromycin-resistant transformants per million conidia.Putative transformants were found to be mitoticallystable.The molecular analysis of transformants demonstrated the random integration of single copy of theT-DNA into the host genome.This transformation system serves as a basic tool for insertional mutagenesisin N.sylviforme fusant HDF-68,and the development of such system lays a solid foundation for con-structing high-yied gene engineering strain and clarifying taxol biosynthesis pathway in this fungus.

A Gγ subunit promoter T-DNA insertion mutant——A1-412 of Magnaporthe grisea is defective in appressorium formation,penetration and pathogenicity

In our previous studies,a nonpathogenic mutant of Magnaporthe grisea,A1-412,which was defective in appressorium formation,penetration and infectious growth,was obtained by T-DNA insertional mutagenesis. Here we reported the identification and characterization of the corresponding gene. Se- quence analysis of the genomic DNA franking T-DNA isolated by TAIL-PCR technique showed that T-DNA was inserted into the promoter region of the predicted G protein γ-subunit gene MGG1 (for Magnaporthe grisea G protein Gamma subunit). MGG1 is predicted to encode a 93-aa protein with a typical G-protein gamma like domain (GGL) and C-terminal CAAX box. The amino-acid sequence of MGG1 is highly identical to the Gγ subunits of other filamentous fungi. Further phenotypic investigation of A1-412 showed that ex- ogenous cAMP could induce appressorium formation,although the formed appressoria were abnormal in shape and unable to penetrate onion epidermis or rice leaves. Moreover,few perithecia were observed when A1-412 was crossed with the appropriate mat- ing-type strain. The above phenotypes in A1-412 were partially complemented by reintroduction of the gene MGG1. Our results indicate that the G-protein gamma subunit MGG1 may be involved in regulating morphogenesis,mating and pathogenicity in M. grisea.

Novel GaN-based double-channel p-heterostructure field-effect transistors with a p-GaN insertion layer

GaN-based p-channel heterostructure field-effect transistors(p-HFETs)face significant constraints on on-state currents compared with n-channel high electron mobility transistors.In this work,we propose a novel double heterostructure which introduces an additional p-GaN insertion layer into traditional p-HFETs.The impact of the device structure on the hole densities and valence band energies of both the upper and lower channels is analyzed by using Silvaco TACD simulations,including the thickness of the upper AlGaN layer and the doping impurities and concentration in the GaN buffer layer,as well as the thickness and Mg-doping concentration in the p-GaN insertion layer.With the help of the p-GaN insertion layer,the C-doping concentration in the GaN buffer layer can be reduced,while the density of the two-dimensional hole gas in the lower channel is enhanced at the same time.This work suggests that a double heterostructure with a p-GaN insertion layer is a better approach to improve p-HFETs compared with those devices with C-doped buffer layer alone.

Illumina Sequencing Technology as a Method of Identifying T-DNA Insertion Loci in Activation- Tagged Arabidopsis thaliana Plants

Dear Editor, Forward genetic screens are commonly used as unbiased tools to isolate genes responsible for a phenotype of interest. In Arabidopsis thaliana, especially T-DNA activation tagging pop- ulations are frequently employed. These populations are gener- ated using vectors containing multiple copies of the constitutive 35S promoters derived from cau and often result in isolation i flower mosaic virus （35S CaMV） of dominant gain-of-function alleles （Weigel et al., 2000; Nakazawa et al., 2003）. This allows the study of members of large gene families that are often func- tionally redundant and, therefore, hard to identify in loss-of- function screens. Moreover, due to the dominant nature,

Researchers Develop New Tools for Precise Large DNA Insertions

GAO Caixia’s group from the Institute of Genetics and Developmental Biology(IGDB)of the Chinese Academy of Sciences(CAS)has developed a new genome editing technology that achieves efficient and precise targeted insertion of large DNA segments in plants.The new technology,called prime editing-mediated recombination of opportune targets(PrimeRoot),combines an optimized dual-ePPE editor protein previously published by the group with a highly efficient tyrosine site-specific recombinase,Cre.It can achieve one-step,precise targeted insertion of large DNA segments in rice and maize with an efficiency up to 6%and has been used to successfully insert DNA segments up to 11.1 kb.

Esophageal inserted subclavian catheter:a rare complication

Central venous catheterization(CVC) is an invasive procedure for administering fluids,nutrients,and drugs;monitoring central venous pressure;performing pulmonary artery catheterization;and placing transvenous pacemakers in intensive care units and all specialties,from anesthesia to emergency medicine,for the treatment of trauma and hemodynamically unstable pediatric and adult patients.[1,2]Complications have been observed in more than 15% of patients who underwent CVC.Mechanical,infectious,and thrombotic complications have been reported in 5%–19%,5%–26%,and 2%–26% of patients,respectively.[3] Malposition,on the other hand,is common in subclavian catheter insertion and is usually associated with an initially misplaced guidewire.[4]