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MAIN CONCLUSION: We performed genome-wide and heterologous expression analysis of the safflower cysteine protease family and found that inhibition of CtCP1 expression enhanced plant cold resistance. Cysteine protease (CP) is mainly involved in plant senescence and stress responses. However, the molecular mechanism of endogenous cysteine protease inhibition in plant stress tolerance is yet unknown. Here, we report the discovery and functional characterization of a candidate CP1 gene from safflower. The conserved structural topology of CtCPs revealed important insights into their possible roles in plant growth and stress responses. The qRT-PCR results implied that most of CtCP genes were highly expressed at fading stage suggesting that they are most likely involved in senescence process. The CtCP1 expression was significantly induced at different time points under cold, NaCl, H2O2 and PEG stress, respectively. The in-vitro activity of heterologously expressed CtCP1 protein showed highest protease activity for casein and azocasein substrates. The expression and phenotypic data together with antioxidant activity and physiological indicators revealed that transgenic plants inhibited by CtCP1-anti showed higher tolerance to low temperature than WT and CtCP1-OE plants. Our findings demonstrated the discovery of a new Cysteine protease 1 gene that exerted a detrimental effect on transgenic Arabidopsis under low-temperature stress.

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The MYB transcription factors comprise one of the largest superfamilies in plants that have been implicated in the regulation of plant-specific metabolites and responses to biotic and abiotic stresses. Here, we present the first comprehensive genome-wide analysis and functional characterization of the CtMYB family in Carthamus tinctorius. A total of 272 CtMYBs were identified and classified into 12 subgroups using comparative phylogenetic analysis with Arabidopsis and rice orthologs. The overview of conserved motifs, gene structures, and cis elements as well as the expression pattern of CtMYB genes indicated the diverse roles of these transcription factors during plant growth, regulation of secondary metabolites, and various abiotic stress responses. The subcellular localization and transactivation analysis of four CtMYB proteins indicated predominant localization in the nuclei with enhanced transcriptional activation in yeast. The expression of CtMYB63 induced with various abiotic stress conditions showed upregulation in its transcription level. In addition, the expression analysis of the core structural genes of anthocyanin biosynthetic pathway under drought and cold stress in CtMYB63 overexpressed transgenic lines also supports the notion of CtMYB63 transcriptional reprogramming in response to abiotic stress by upregulating the anthocyanin biosynthesis. Together, our findings revealed the underlying regulatory mechanism of CtMYB TF network involving enhanced cold and drought stress tolerance through activating the rapid biosynthesis of anthocyanin in C. tinctorius. This study also presents useful insights towards the establishment of new strategies for crop improvements.

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Objective:To explore the accuracy of array comparative genomic hybridization(aCGH) in the unexpected detection of Duchenne muscular dystrophy ( DMD) gene duplication/deletion in prenatal diagnosis. Methods:A retrospective analysis was performed on 31 cases with DMD gene duplication/deletion detected by aCGH among 5 025 prenatal diagnosis samples without family history of DMD in Henan Provincial People's Hospital from July 2018 to December 2019. The multiplex ligation-dependent probe amplification (MLPA) method was used to verify the above results. The American College of Medical Genetics and Genomics (ACMG) guideline was referred for pathogenicity analysis of the detected duplicates/deletions. Descriptive analysis was adopted in analysis. Results:The total unexpected DMD gene duplication/deletion rate was 0.62% (31/5 025), among which 25 cases were with microduplication/microdeletion ≤ 200 kb and six were >200 kb; there were 24 cases of deletion, seven cases of duplication; exon or intron duplication/deletion were accounted for 19 and 12 cases, respectively. According to the five classification standards of ACMG guideline, there were 17 cases with pathogenic variants and 14 cases with uncertain pathogenicity/likely benign variants. Of the 19 with exon mutations, 17 cases were DMD intragenic variants, and two cases involved variants in and outside DMD gene, which were verified by MLPA whose results were all positive. Conclusions:The duplication/deletion of exon region of DMD gene detected by aCGH technique is accurate and reliable, which plays an important role in the diagnosis of DMD. For these cases involved both internal and external regions of DMD gene, aCGH can identify the upstream and downstream breaking points of DMD gene, thus providing the basis for ACMG grading.

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Flavonoids are mainly associated with growth, development, and responses to diverse abiotic stresses in plants. A growing amount of data have demonstrated the biosynthesis of flavonoids through multienzyme complexes of which the membrane-bounded cytochrome P450 supergene family shares a crucial part. However, the explicit regulation mechanism of Cytochrome P450s related to flavonoid biosynthesis largely remains elusive. In the present study, we reported the identification of a stress-tolerant flavonoid biosynthetic CtCYP82G24 gene from Carthamus tinctorius. The transient transformation of CtCYP82G24 determined the subcellular localization to the cytosol. Heterologously expressed CtCYP82G24 was effective to catalyze the substrate-specific conversion, promoting the de novo biosynthesis of flavonoids in vitro. Furthermore, a qRT-PCR assay and the accumulation of metabolites demonstrated that the expression of CtCYP82G24 was effectively induced by Polyethylene glycol stress in transgenic Arabidopsis. In addition, the overexpression of CtCYP82G24 could also trigger expression levels of several other flavonoid biosynthetic genes in transgenic plants. Taken together, our findings suggest that CtCYP82G24 overexpression plays a decisive regulatory role in PEG-induced osmotic stress tolerance and alleviates flavonoid accumulation in transgenic Arabidopsis.

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The basic helix-loop-helix (bHLH) family is the second largest superfamily of transcription factors that belongs to all three eukaryotic kingdoms. The key function of this superfamily is the regulation of growth and developmental mechanisms in plants. However, the bHLH gene family in Carthamus tinctorius has not yet been studied. Here, we identified 41 bHLH genes in Carthamus tinctorius that were classified into 23 subgroups. Further, we conducted a phylogenetic analysis and identified 10 conserved protein motifs found in the safflower bHLH family. We comprehensively analyzed a group of bHLH genes that could be associated with flavonoid biosynthesis in safflower by gene expression analysis, gene ontology annotation, protein interaction network prediction, subcellular localization of the candidate CtbHLH40 gene, and real-time quantitative expression analysis. This study provides genome-wide identification of the genes related to biochemical and physiological processes in safflower.

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Objective To study the clinical application effect of Xuebijing Injection in the treatment of capillary leak syndrome.Methods One hundred patients with capillary leak syndrome in our hospital from January 2013 to April 2016 were selected and randomly divided into the control group and observation group according to the ID number of admission.The two groups were given the Western medicine routine therapy of capillary leak syndrome.The observation group was simultaneously given Xuebijing Injection.The APACHE II score,Mashall score and hemodynamics were compared between the two groups.The disease condition change was dynamically observed.Results The APACHE II score and Mashall score before and after treatment in the observation group were significantly suprior to those in the control group(P<0.05);the peripheral blood circulating endothelial cells (CEC) number at admission in the two groups were higher than the normal level,the CEC content after taking the treatment was gradually decreased (P<0.05),but the CEC content decrease in the observation group was less than that of the control group (P<0.01);were observed before and after the treatment group APACHE II and Mashall score was significantly better than the control group (P<0.05);but the CEC content in the observation group was significnatly superior to the control group(P<0.01);the expressions of vascular endothelial growth factor (VEGF),TNF alpha and interleukin-6 (IL-6) in the observation group were better than those in the control group (P<0.05).Conclusion Using Xuebijing Injection in treating capillary leak syndrome can effectively protect the vascular endothelial cells,improves the living quality of patients and can be popularized and applied in clinical treatment.

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<p><b>OBJECTIVE</b>To establish genetic transformation system of active fibroblast growth factor (aFGF) in Carthamus tinctorius.</p><p><b>METHOD</b>The culture condition was optimized by orthogonal experiment design with cotyledon of C. tinctorius as the explant. The aFGF was transferred into safflower through Agrobacterium-mediated transformation and screened under different concentrations of antibiotics, and then PCR was identified.</p><p><b>RESULT</b>It confirmed the optimal differentiation medium: MS + BA 1.0 mg x L(-1) + NAA 0.2 mg x L(-1), the optimal root medium: 1/4 MS + NAA 2.0 mg x L(-1) + IAA0.5 mg x L(-1). The bacteriostatic effect of the three antibiotics showed slight difference. From them Tim was selected with the concentration of 400 mg x L(-1). It showed the bacteriostatic effect and promoted also differentiation. The selective concentration of hyg was confirmed to be 6 mg x L(-1). The eight transformed plants were identified, the positive rate was 25%.</p><p><b>CONCLUSION</b>It was determined the best hormones and the ratios for the differentiation and rooting of the safflower by organogenesis. It was identified the optimal concentration of inhibitory antibiotics and selection antibiotics. The aFGF gene was cloned in a part of plant by PCR analysis. It is shown that the aFGF gene has been integrated into safflower genome.</p>

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Recently, more research about the plant bioreactor expressing genes encoding human proteins was reported. In the present study, the cDNA of the human gene keratinocyte growth factor 2 (KGF2) was replaced with plant preferred codons by PCR, and the modified full-length cDNA was cloned into the plant expression vector pCAMBIA-YO containing the oil-body promoter. The fusion construct pCAMBIA-YO-KGF2 was transformed into Brassica napus by Agrobacterium tumefacien-mediated cotyledon transformation method. The transgenic seedlings were identified by PCR, Southern and western blot analysis all showed that KGF2 gene was successfully expressed in in transgenic Brassica napus.

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Marker-free plants have been public concern. Co-transformation and site-specific recombination system are more important methods in self-gene excision. We reviewed the Cre/lox site-specific system and its applications in plants, also, we discussed perspectives of the system in according with our experience.

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