Genome-Wide Identification and Abiotic Stress Expression Analysis of CKX and IPT Family Genes in Cucumber (Cucumis sativus L.).

Cytokinins (CKs) are among the hormones that regulate plants' growth and development, and the CKX and IPT genes, which are CK degradation and biosynthesis genes, respectively, play important roles in fine-tuning plants' cytokinin levels. However, the current research on the function of IPT and CKX in cucumber's growth, development, and response to abiotic stress is not specific enough, and their regulatory mechanisms are still unclear. In this study, we focused on the IPT and CKX genes in cucumber, analyzed the physiological and biochemical properties of their encoded proteins, and explored their expression patterns in different tissue parts and under low light, salt stress, and drought stress. Eight CsCKX and eight CsIPT genes were identified from the cucumber genome. We constructed a phylogenetic tree from the amino acid sequences and performed prediction analyses of the cis-acting elements of the CsCKX and CsIPT promoters to determine whether CsCKXs and CsIPTs are responsive to light, abiotic stress, and different hormones. We also performed expression analysis of these genes in different tissues, and we found that CsCKXs and CsIPTs were highly expressed in roots and male flowers. Thus, they are involved in the whole growth and development process of the plant. This paper provides a reference for further research on the biological functions of CsIPT and CsCKX in regulating the growth and development of cucumber and its response to abiotic stress.

Effect of Nickel Ions on the Physiological and Transcriptional Responses to Carbon and Nitrogen Metabolism in Tomato Roots under Low Nitrogen Levels.

Nickel (Ni) is an essential trace element for plant growth and a component of the plant body that has many different functions in plants. Although it has been confirmed that nickel ions (Ni2+) havea certain regulatory effect on nitrogen (N) metabolism, there are not enough data to prove whether exogenous Ni2+ can increase the carbon (C) and N metabolism in the roots of tomato seedlingsunder low-nitrogen (LN) conditions. Therefore, through the present experiment, we revealed the key mechanism of Ni2+-mediated tomato root tolerance to LN levels. Tomato plants were cultured at two different N levels (7.66 and 0.383 mmol L-1) and two different Ni2+ levels (0 and 0.1 mg L-1 NiSO4 6H2O) under hydroponic conditions. After nine days, we collected roots for physiological, biochemical, and transcriptome sequencing analyses and found that the activities of N assimilation-related enzymes decreased at LN levels. In contrast, Ni2+ significantly increased the activities of N assimilation-related enzymes and increased the contents of nitrate (NO3-), ammonium (NH4+), and total amino acids. Through root transcriptomic analysis, 3738 differentially expressed genes (DEGs) were identified. DEGs related to C and N metabolism were downregulated after LN application. However, after Ni2+ treatment, PK, PDHB, GAPDH, NR, NiR, GS, GOGAT, and other DEGs related to C and N metabolism were significantly upregulated. In conclusion, our results suggest that Ni2+ can regulate the C and N metabolism pathways in tomato roots to alleviate the impact of LN levels.

The reference genome and full-length transcriptome of pakchoi provide insights into cuticle formation and heat adaption.

Brassica rapa includes various vegetables with high economic value. Among them, green petiole type pakchoi (B. rapa ssp. chinensis) is one of the major vegetables grown in southern China. Compared with other B. rapa varieties, green petiole type pakchoi shows a higher level of heat resistance, which is partially derived from the rich epicuticular wax. Here we sequence a high-quality genome of green petiole type pakchoi, which has been widely used as the parent in breeding. Our results reveal that long terminal repeat retrotransposon insertion plays critical roles in promoting the genome expansion and transcriptional diversity of pakchoi genes through preferential insertions, particularly in cuticle biosynthetic genes. After whole-genome triplication, over-retained pakchoi genes escape stringent selection pressure, and among them a set of cuticle-related genes are retained. Using bulked-segregant analysis of a heat-resistant pakchoi cultivar, we identify a frame-shift deletion across the third exon and the subsequent intron of BrcCER1 in candidate regions. Using Nanopore long-read sequencing, we analyze the full-length transcriptome of two pakchoi cultivars with opposite sensitivity to high temperature. We find that the heat-resistant pakchoi cultivar can mitigate heat-caused leaf damage by activating an unfolded protein response, as well as by inhibiting chloroplast development and energy metabolism, which are presumably mediated by both transcriptional regulation and splicing factors. Our study provides valuable resources for Brassica functional genomics and breeding research, and deepens our understanding of plant stress resistance.

Piriformospora indica colonization increases the growth, development, and herbivory resistance of sweet potato (Ipomoea batatas L.).

KEY MESSAGE: Piriformospora indica symbiosis promoted the growth and photosynthesis, and simultaneously enhanced the resistance against insect herbivory by regulating sporamin-dependent defense in sweet potato. Piriformospora indica (P. indica), a versatile endophytic fungus, promotes the growth and confers resistance against multiple stresses by root colonization in plant hosts. In this study, the effects of P. indica colonization on the growth, physiological change, and herbivore resistance of leaf-vegetable sweet potato cultivar were investigated. P. indica symbiosis significantly improved the biomass in both above- and under-ground parts of sweet potato plants. In comparison with the non-colonized plants, the content of photosynthetic pigments and the efficiency of photosynthesis were increased in P. indica-colonized sweet potato plants. Further investigation showed that the activity of catalase was enhanced in both leaves and roots of sweet potato plants after colonization, but ascorbate peroxidase, peroxidase, and superoxide dismutase were not enhanced. Furthermore, the interaction between P. indica and sweet potato plants also showed the biological function in jasmonic acid (JA)-mediated defense. The plants colonized by P. indica had greatly increased JA accumulation and defense gene expressions, including IbNAC1, IbbHLH3, IbpreproHypSys, and sporamin, leading to elevated trypsin inhibitory activity, which was consistent with a reduced Spodoptera litura performance when larvae fed on the leaves of P. indica-colonized sweet potato plants. The root symbiosis of P. indica is helpful for the plant promoting growth and development and has a strong function as resistance inducers against herbivore attack in sweet potato cultivation by regulating sporamin-dependent defense.

[Cloning, sequencing of G3PD gene from Brugia malayi and prediction of B cell epitopes in its amino acid sequence].

Specific primers were designed and synthesized based on the reported glyceraldehyde-3-phosphate dehydrogenase (BmG3PD) gene of Brugia malayi (GenBank Accession No. U18137). Total RNA was extracted from Brugia malayi and its BmG3PD gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The PCR product was purified and cloned into plasmid pGEM-T, then transformed into Escherichia coli DH5alpha. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification. The positive recombinant plasmid pGEM-T-BmG3PD was confirmed by sequencing and homology comparison. Five parameters and methods were used to predict B-cell epitopes in amino acid sequence of BmG3PD. The amplified DNA fragment (1,020 bp) had a high identity of 99% with the BmG3PD gene sequence of Brugia malayi. B-cell epitopes of BmG3PD were probably at or adjacent to 22-36, 242-255, 303-318 and 326-336 in its amino acid sequence.

[Cloning and eukaryotic expression of the gene encoding myosin from Brugia malayi].

Total RNA was extracted from periodic microfilariae of Brugia malayi and its myosin partial gene (Bm-M55) was amplified by RT-PCR. The PCR product was cloned and then subcloned into pcDNA3.1 (+)vector. The recombinant eukaryotic plasmids were screened and identified by digestion with restriction enzyme and PCR amplification, and was transfected into COS-7 cells subsequently. The expressed protein was identified by SDS-PAGE. Bm-M55 mRNA was highly expressed in transfected COS-7 cells. The deduced amino acid sequence showed to be identical with that of Bm-M55, and the recombinant protein was about Mr 55000.

[Construction of eukaryotic expression plasmids with paramyosin gene of periodic Brugia malayi].

Total RNA was extracted from periodic Brugia malayi Specific primers were designed on the basis of known sequences of paramyosin gene from B. malayi (BmPmy). The desired gene was amplified by PCR technique from cDNA. The PCR products were purified and cloned into plasmid pGEM-T by T-A cloning method, transformed into Escherichia coli (E. coli) strain DH5alpha. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification. The right gene fragments encoding BmPmy in positive clones for prokaryotic and eukaryotic expression plasmids were digested with restrictive endonuclease, and were subcloned into pcDNA3.1(+). The recombinant eukaryotic plasmid (pcDNA3.1-BmPmy) was then transfected into COS-7 cells. The transient expression of BmPmy was examined with RT-PCR. BmPmy mRNA was highly expressed in transfected COS-7 cells.

Enhanced transfection efficiency of a systemically delivered tumor-targeting immunolipoplex by inclusion of a pH-sensitive histidylated oligolysine peptide.

Successful cancer gene therapy depends on the development of non-toxic, efficient, tumor cell- specific systemic gene delivery systems. Our laboratory has developed a systemically administered, ligand-liposome complex that can effectively and preferentially deliver its therapeutic payload to both primary and metastatic tumors. To further improve the transfection efficiency of this targeting complex, a synthetic pH-sensitive histidylated oligolysine K[K(H)KKK]5-K(H)KKC (HoKC), designed to aid in endosomal escape and condensation of DNA, was included in the complex. The presence of HoKC increased the in vitro transfection efficiency over that of the original complex. Moreover, no increase in cytotoxicity was observed due to the presence of the HoKC peptide. In a DU145 human prostate cancer xenograft tumor model in athymic nude mice, inclusion of the HoKC peptide did not interfere with the tumor targeting specificity of the i.v. administered ligand/liposome/DNA complex. Most importantly, the level of transgene expression was significantly elevated in the tumors, but not in the normal tissue in those animals receiving the complex incorporating HoKC. The in vivo enhancement of transfection efficiency by this modified gene delivery vehicle could lead to a reduction in the number of administrations required for antitumor efficacy.

Systemic tumor-targeted gene delivery by anti-transferrin receptor scFv-immunoliposomes.

An ideal therapeutic for cancer would be one that selectively targets to tumor cells, is nontoxic to normal cells, and that could be systemically delivered, thereby reaching metastases as well as primary tumor. Immunoliposomes directed by monoclonal antibody or its fragments are promising vehicles for tumor-targeted drug delivery. However, there is currently very limited data on gene delivery using these vehicles. We have recently described a cationic immunoliposome system directed by a lipid-tagged, single-chain antibody Fv fragment (scFv) against the human transferrin receptor (TfR) that shows promising efficacy for systemic p53 tumor suppressor gene therapy in a human breast cancer metastasis model. However, the extremely low yield of this lipid-tagged scFv limited further downstream development and studies. Here we report a different expression strategy for the anti-TfR scFv, which produces high levels of protein without any tags, and a different approach for complexing the targeting scFv to the liposomes. This approach entails covalently conjugating the scFv to the liposome via a cysteine at the 3'-end of the protein and a maleimide group on the liposome. Our results show that this conjugation does not impair the immunological activity or targeting ability of the scFv. The scFv-cys targets the cationic liposome-DNA complex (lipoplex) to tumor cells and enhances the transfection efficiencies both in vitro and in vivo in a variety of human tumor models. This scFv-immunoliposome can deliver the complexed gene systemically to tumors in vivo, where it is efficiently expressed. In comparison with the whole antibody or transferrin molecule itself, the scFv has a much smaller size for better penetration into solid tumors. It is also a recombinant protein rather than a blood product; thus, large scale production and strict quality control are feasible. This new approach provides a promising system for tumor-targeted gene delivery that may have potential for systemic gene therapy of various human cancers.

A novel secreted splice variant of vascular endothelial cell growth inhibitor.

Vascular endothelial cell growth inhibitor (VEGI), a member of the tumor necrosis factor (TNF) family, is an endothelial cell-specific inhibitor of angiogenesis. Overexpression by cancer cells of a secretable VEGI fusion protein resulted in abrogation of xenograft tumor progression, but overexpression of full-length VEGI was completely without effect. This finding indicates that secretion is essential for VEGI action. Here we report the identification of two new VEGI isoforms consisting of 251 and 192 amino acid residues. Both isoforms show endothelial cell-specific expression and share a C-terminal 151-residue segment with the previously described VEGI, which comprises 174 residues. The isoforms are generated from a 17 kb human gene by alternative splicing. Their expression is regulated in parallel by inflammatory cytokines TNF-alpha and interferon-gamma. VEGI-251, the most abundant isoform, contains a putative secretion signal. VEGI protein is detected in conditioned media of endothelial cells and VEGI-251-transfected mammalian cells. Overexpression of VEGI-251 in endothelial cells causes dose-dependent cell death. VEGI-251-transfected cancer cells form xenograft tumors of reduced growth rate and microvessel density compared with tumors of empty vector transfectants. These findings support the view that endothelial cell-secreted VEGI may function as an autocrine inhibitor of angiogenesis and a naturally existing modulator of vascular homeostasis.

Self-assembly of a virus-mimicking nanostructure system for efficient tumor-targeted gene delivery.

Molecular therapy, including gene therapy, is a promising strategy for the treatment of human disease. However, delivery of molecular therapeutics efficiently and specifically to the target tissue remains a significant challenge. A human transferrin (Tf)-targeted cationic liposome-DNA complex, Tf-lipoplex, has shown high gene transfer efficiency and efficacy with human head and neck cancer in vitro and in vivo (Xu, L., Pirollo, K.F., Tang, W.H., Rait, A., and Chang, E.H. Hum. Gene Ther. 1999;10:2941-2952). Here we explore the structure, size, formation process, and structure-function relationships of Tf-lipoplex. We have observed Tf-lipoplex to have a highly compact structure, with a relatively uniform size of 50-90 nm. This nanostructure is novel in that it resembles a virus particle with a dense core enveloped by a membrane coated with Tf molecules spiking the surface. More importantly, compared with unliganded lipoplex, Tf-lipoplex shows enhanced stability, improved in vivo gene transfer efficiency, and long-term efficacy for systemic p53 gene therapy of human prostate cancer when used in combination with conventional radiotherapy. On the basis of our observations, we propose a multistep self-assembly process and Tf-facilitated DNA cocondensation model that may provide an explanation for the resultant small size and effectiveness of our nanostructural Tf-lipoplex system.