Homology Cloning Research Articles

Molecular cloning and expression mechanism of Mnp65 in Megalobrama amblycephala response to Aeromonas hydrophilia challenge

p65 is one of the important subunits of the inflammation-related transcription factor NF-κB. In the present study, we cloned and identified the p65 from Megalobrama amblycephala (Mnp65) by homologous cloning and RACE technique. The full-length Mnp65 cDNA consisted of 2331 bp, and included one open reading frame encoding a 604-amino acid putative protein. The protein sequence included a DNA binding motif, a well conserved N-terminal Rel-homology domain (RHD), and a C-terminal IG-like plexins transcription (IPT). Mnp65 was closely related with the other p65 proteins of Cypriniformes and clearly distinct from that of Perciformes and Salmoniformes in terms of sequence homology. Mnp65 homodimer may interact with IκBα in the IPT domain based on the predicted 3D structure of IκBα/Mnp65 complex. Mnp65 was ubiquitously expressed in M. amblycephala tissues, and the highest levels were detected in muscle and liver. Intragastric infection with Aeromonas hydrophila caused respiratory burst and cytokine storm from 8 h to 48 h, showing significantly higher level of respiratory burst activities and significantly high cytokines levels, such as TNF-α, IL-1β, IL-6, IL-8 etc., compared to 0 h. In addition, the bacterial challenge downregulated the IkBα, and upregulated Mnp65 and TNF-α in the liver. IkBα-Mnp65 was regulated by the negative feedback of cytokine storm, to increase IkBα and decrease Mnp65. Then cytokine storm was relieved at 96 h. Finally, severe intestinal inflammation was observed from 24 h to 48 h after infection, characterized by extensive villous necrosis, epithelial hyperplasia and lymphocyte infiltration, all of which were relieved at 96 h. Taken together, Mnp65 plays a crucial role in the physiological response of teleost fish to bacterial infection.

Inhibitory effect of the Nth gene on drug resistance in Mycobacterium tuberculosis

To clarify the role of Nth gene in drug resistance of Mycobacterium tuberculosis (MTB), the clinical strain MTB were isolated and cultured from the sputum of pulmonary tuberculosis patients, and grouped according to the results of drug sensitivity tests. The expression levels of Nth mRNA and protein were detected by Polymerase chain reaction and Western blot, respectively. The bacteriophage-mediated homologous recombination gene knockout method and seamless cloning and ligation technology were used to knockout Nth gene and perform backfill overexpression experiments on MTB standard strain H37Rv. The resistance of the obtained strains was induced, and its growth was compared. In the results, the resistance rate of 193 clinical isolates to first-line drugs was 37.37%. The expression levels of Nth mRNA and protein in sensitive strains were higher than those in drug-resistant strains (P < 0.05). Strains without Nth were more likely to grow, while the growth of the Nthoverexpressing strain was inhibited. When two drugs are used in combination, the bacteria developed drug resistance later than those induced by single drug. In conclusion, the Nth gene has an inhibitory effect on the drug resistance of MTB.

Cloning and temporal-spatial expression analysis of dfr gene from Scutellaria baicalensis with different colors

Dihydroflavanol-4-reductase (Dfr) is a key enzyme that regulates the synthesis of anthocyanin and proanthocyanidin in the flavonoid biosynthesis pathway. To investigate the difference of dfr gene in Scutellaria baicalensis Georgi with different colors in the same ecological environment, three complete full-length sequences of dfr gene were cloned from the cDNA of S. baicalensis with white, purple-red and purple colors using homologous cloning and RACE techniques. The three genes were named Sbdfr1, Sbdfr2 and Sbdfr3, respectively, and their corresponding structures were analyzed. The results showed that all three Dfr proteins have highly conserved NADPH binding sites and substrate-specific binding sites. Phylogenetic analysis showed that they are closely related to that of the known S. viscidula (ACV49882.1). Analysis of key structural domains and 3D models revealed differences in the catalytically active regions on the surface of all three Dfr proteins, and their unique structural characteristics may provide favorable conditions for studying the substrate specificity of different Dfr proteins. qRT-PCR analysis shows that dfr was expressed at different level in all tissues except the roots of S. baicalensis in full-bloom. During floral development, the expression level of dfr in white and purple-flowered Scutellaria showed an overall upward trend. In purple-red-flowered Scutellaria, the expression first slowly increased, followed by a decrease, and then rapidly increased to the maximum. This research provides a theoretical basis for further exploring the mechanism and function of Dfr substrate selectivity, and are of great scientific value for elucidating the molecular mechanism of floral color variation in S. baicalensis.

T cell reactivity to regulatory factor X4 in type 1 narcolepsy

Type 1 narcolepsy is strongly (98%) associated with human leukocyte antigen (HLA) class II DQA1*01:02/DQB1*06:02 (DQ0602) and highly associated with T cell receptor (TCR) alpha locus polymorphism as well as other immune regulatory loci. Increased incidence of narcolepsy was detected following the 2009 H1N1 pandemic and linked to Pandemrix vaccination, strongly supporting that narcolepsy is an autoimmune disorder. Although recent results suggest CD4+ T cell reactivity to neuropeptide hypocretin/orexin and cross-reactive flu peptide is involved, identification of other autoantigens has remained elusive. Here we study whether autoimmunity directed against Regulatory Factor X4 (RFX4), a protein co-localized with hypocretin, is involved in some cases of narcolepsy. Studying human serum, we found that autoantibodies against RFX4 were rare. Using RFX4 peptides bound to DQ0602 tetramers, antigen RFX4-86, -95, and -60 specific human CD4+ T cells were detected in 4/10 patients and 2 unaffected siblings, but not in others. Following culture with each cognate peptide, enriched autoreactive TCRαβ clones were isolated by single-cell sorting and TCR sequenced. Homologous clones bearing TRBV4-2 and recognizing RFX4-86 in patients and one twin control of patient were identified. These results suggest the involvement of RFX4 CD4+ T cell autoreactivity in some cases of narcolepsy, but also in healthy donors.

English

Bletilla striata (Thunb.) Reihb.f., a traditional Chinese herbal medicine, has attracted increasing attention because of its wide range of applicability to the medical field and chemical industry. B. striata has been identified to be particularly sensitive to high temperatures. Thus, the study of temperature stress on gene transcription is of interest in the field. Use of reliable reference genes is essential for qRT-PCR analysis of genes. However, little information regarding suitable reference genes for the genus Bletilla has been published. In this study, the sequences of seven potential reference genes in B. striata were obtained via a homology cloning strategy. These genes were as follows: glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18S), elongation factor 1 alpha (EF1α), α-tubulin (TUA), β-tubulin (TUB), ubiquitin (UBI), and NAC domain protein (NAC). We then evaluated the stability levels of these transcripts in different tissues (root, tuber, and leaf) exposed to high temperature using three conventional software and comprehensive RefFinder algorithms. The results indicated that 18S and TUB were the best internal control genes among different periods of heat treatment and that a combination of 18S and UBI was the best in different tissues. Altogether, 18S and UBI were identified to be the best reference genes for all the samples, while NAC and TUA were the least stable reference genes. The results will be useful for studies on target gene expression in plants of the Bletilla genus. © 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers © 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers©

Cloning and functional characterization of auxin receptor TIR1 in Gossypium hirsutum

Transport inhibitor response 1 (TIR1), as an F-box family protein, is a nucleus receptor of auxin in plants. It is essential in the auxin signal transduction pathway and the expression of auxin-responsive genes. As the most important hormone in plants, auxin has a great influence on the ovule and fiber development of cotton (Gossypium hirsutum L.). Studies on the effect of auxin signal transduction pathway on cotton fiber development can fundamentally solve the problem of fiber yield and quality. The cDNA sequence of G. hirsutum TIR1 was obtained through homologous cloning and rapid-amplification of cDNA ends (RACE). The homology between GhTIR1 and that in Arabidopsis thaliana was 64.8%. To study the functions, subcellular localization, and the specific localization of GhTIR1 sequence, we constructed the fusion expression vector of GhTIR1 and its N-terminal or C-terminal deletion mutant genes with GFP. By transiently transforming Arabidopsis protoplasts and stably transforming Nicotiana tabacum BY-2 cell lines, GhTIR1 was localized in the nucleus through the N-terminal 1–197 amino acid sequence. The expression patterns of GhTIR1 in different organs and ovule and cotton fiber cells at different developmental stages showed that GhTIR1 was highly expressed in the initial and elongation stages of cotton fiber development. These results indicate that GhTIR1 plays an important role in the initiation and elongation of cotton fibers, especially the formation of primary fibers of cotton fiber cells.

Adaptability variations and differences in photoprotection of young leaves of two tree species of subtropical forest to the light environment

In the high-light environment, young leaves accumulate anthocyanins as a photoprotection strategy. However, anthocyanin biosynthesis-related enzymes gene sequence is still unknown in the leaves of subtropical forest plants. There are thus few reports on the relationship between the expression level of these genes and photoprotection. In this study, Machilus chinensis and Castanopsis chinensis were taken as plant material in a subtropical forest. Non-full-length nucleotide sequences of chalcone synthase, dihydroflavonol 4-reductase, anthocyanin synthase, and β-tubulin gene were obtained by homologous and electronic cloning. The expression of those genes was verified and analyzed, and some physiological indicators were measured. The anthocyanin content and anthocyanin synthesis-related gene expression in the young leaves of C. chinensis were significantly higher than that of M. chinensis. Although young leaves of M. chinensis did not accumulate anthocyanins, they showed higher antioxidants and nonphotochemical quenching (NPQ). This study indicates that anthocyanins, antioxidants, and NPQ together mediate the positive effects on photoadaptation in young leaves.

Cloning and Expression Analysis of <i>PAP1</i> in <i>Brassica juncea</i>

Anthocyanins are important substances accounting for the leaf color in Brassica juncea and PAP1  gene is one of the key transport factors in the anthocyanin synthesis pathway. In this study, homologous cloning technology was used to clone the PAP1  gene sequences of Brassica juncea  with different leaf colors. Specific primers were designed according to the gene sequences of Brassica rapa  with high homology for PCR amplification. The PAP1  gene of Brassica juncea  is 1 348 bp~1 669 bp long, and the coding region sequence is 744 bp~753 bp, including 3 exons and 2 introns. Two MYB blinding domains are found in PAP1 protein at the site of 9~59 and 62~110 amino acids. Phylogenetic analysis showed that the PAP1  gene of Brassica juncea  had high homology with the related genes of Brassica rapa  and Brassica rapa subsp. rapa, but had low homology with Arabidopsis thaliana . Compared gene sequences in Brassica juncea  with different leaf colors, there are no differences between the coding sequence of purple and red leaf Brassica juncea , but the encoded protein have 22 amino acid differences from green leaves. We also observed the lower expression level of PAP1  and its related target genes such as DFR , TT19  in green leaves, which may lead to the differences of leaf color in Brassica juncea . This study provides a reference for exploring the function of PAP1  gene and the formation mechanism of different leaf color of Brassica juncea .

Extraction, Identification and Gene Cloning of Mn-SOD from Pleurotus tuber-regium

The manganese superoxide dismutase of Pleurotus tuber-regium was extracted and identified. The manganese superoxide dismutase (Mn-SOD) gene of P. tuber-regium was cloned from the mycelium of P.tuber-regium. Mn-SOD of P.tuber-regium was extracted by liquid nitrogen grinding, ultrasound, ammonium sulfate precipitation and dialysis. Mn-SOD activity of P. tuber-regium was determined by WST-8 method. SOD type of P. tuber-regium was identified by H2O2. Mn-SOD gene was cloned from P. tuber-regium by homologous cloning, rapid amplification of cDNA ends, fusion primers and nested PCR. Results showed that the activity of Mn-SOD of P. tuber-regium was 1.66 U. There was no significant difference between the enzyme treated with H2O2 and untreated, indicating that the main SOD in P. tuber-regium was Mn-SOD. PtMn-SOD was cloned and obtained. Its DNA sequence was 1025 bp in total, 663 bp in open reading frame and 220 amino acids were encoded. Sequence analysis and phylogenetic tree showed that PtMn-SOD was closely related to Pleurotus ostreatus (accession number:MH645359.1). It was predicted that the molecular weight of the protein was 24.54 kDa and the isoelectric point of the protein was 7.86. PtMn-SOD protein located in mitochondria and played a role in mitochondria.

A 3-ketoacyl-CoA synthase 11 (KCS11) homolog from Malania oleifera synthesizes nervonic acid in plants rich in 11Z-eicosenoic acid.

Nervonic acid (24:1) is a major component in nerve and brain tissues and it has important applications in food and pharmaceutical industries. Malania oleifera seeds contain about 40% nervonic acid. However, the mechanism of nervonic acid biosynthesis and accumulation in seeds of this endangered tree species remains unknown. In this study, developmental changes in fatty acid composition within embryos and their pericarps were investigated. Nervonic acid proportions steadily increased in developing embryos but 24:1 was not detected in pericarps at any stage. Two 3-ketoacyl-CoA synthase (KCS) homologs have been isolated from M. oleifera developing seeds by homologous cloning methods. Both KCSs are expressed in developing embryos but not detected in pericarps. Based on a phylogenetic analysis, these two KCSs were named as MoKCS4 and MoKCS11. Seed-specific expression of the MoKCS11 in Arabidopsis thaliana led to about 5% nervonic acid accumulation, while expression of the MoKCS4 did not show an obvious change in fatty acid composition. It is noteworthy that the transformation of the same MoKCS11 construct into two Brassica napus cultivars with high erucic acid did not produce the expected accumulation of nervonic acid, although expression of MoKCS11 was detected in the developing embryos of transgenic lines. In contrast, overexpression of MoKCS11 results in similar level of nervonic acid accumulation in camelina, a species which contains a similar level of 11Z-eicosenoic acid as does Arabidopsis thaliana. Taken together, the MoKCS11 may have a substrate preference for 11Z-eicosenoic acid, but not for erucic acid, in planta.

Molecular cloning, expression HSP70 and its response to bacterial challenge and heat stress in Microptenus salmoides.

The gene encoding HSP70 was isolated from Microptenus salmoides by homologous cloning and rapid amplification of cDNA ends (RACE). The HSP70 transcripts were 2116bp long and contained 1953 open reading frames encoding proteins of 650 amino acids with a molecular mass of 71.2kDa and theoretical isoelectric point of 5.22. The qRT-PCR analysis showed that the HSP70 gene was differentially expressed in various tissues under normal conditions, and the highest HSP70 level was observed in the spleen and the lowest levels in the muscle and heart. The clear time-dependent expression level of HSP70 was observed after bacterial challenge and heat stress. A significant increase in HSP70 expression level was detected and reached a maximum at 3h and 6h in liver, spleens and gill tissues after Aeromonas hydrophila infection and heat stress, respectively (P < 0.05). As time progressed, the expression of HSP70 transcript was downregulated and mostly dropped back to the original level at 48h. The concentration of cortisol, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increased as the time of stress progressed, with the highest level found on 3h and later declined rapidly and reached to the control levels at the 48h. Those results suggested that HSP70 was involved in the immune response to bacterial challenge and heat stress. The cloning and expression analysis of the HSP70 provide theoretical basis to further study the mechanism of anti-adverseness in Microptenus salmoides.

Genome-Wide Identification and Expression Analysis of the Strawberry FvbZIP Gene Family and the Role of Key Gene FabZIP46 in Fruit Resistance to Gray Mold.

A total of 54 FvbZIP genes were identified from the strawberry genome. These genes were found to be unevenly distributed on seven different chromosomes, and two of the genes had no matching chromosomal localization. FvbZIP genes were divided into 10 subfamilies according to protein sequence, and the structures of these genes were found to be highly conserved. Based on the bioinformatics analysis of FvbZIP genes, the expression of FabZIP genes changed during different stages of its growth and of its infection with gray mold disease. FabZIP46 was substantially upregulated, and its expression remained relatively high. FabZIP46 was cloned from cultivated strawberries by homologous cloning. The results of a transient transgenic assay revealed that the damage to the fruit tissue was markedly alleviated in strawberries overexpressing FabZIP46, with the incidence rate being substantially lower than that in the control group. By contrast, a brief silencing of FabZIP46 had the opposite effect. The results revealed that FabZIP46 played a positive role in the resistance of strawberries to Botrytis cinerea. The study findings provide valuable insights into the role of bZIP transcription factors as well as a theoretical reference for the regulation of resistance to gray mold disease in strawberry fruit.

Molecular Characterization and Expression Patterns of Shabby-Related Kinase (MmSK) Gene of Mulberry (Morus multicaulis)

Shaggy-related protein kinase (SK) plays important roles in the plant growth development, signal transduction, abiotic stress and biotic stress and substance metabolism regulation. In the present paper, a cDNA sequence encoding MmSK (GenBank accession No: KY348867) was cloned from the leaves of mulberry based on mulberry expressed sequence tags (EST) and homologous cloning technology using RT-PCR, which was 1705 bp in length with a full open reading frame (ORF) of 1236 bp encoding a protein of 411 amino acids. The estimated molecular weight and isoelectric point (pI) of the putative protein were 46.55 kDa and 8.61, respectively. Conservation domain structure analysis indicated that MmSK protein had typical structure of the protein kinase domain and belonged to GSK3/shaggy protein kinase family. Multiple sequence alignment and phylogenetic analysis showed that the homology between the amino acid sequences encoded by the MmSK gene and various species was more than 89%. Quantitative real-time PCR (qRT-PCR) analysis revealed that MmSK was expressed in all the tested tissues including leaf, bud, fruit, stem, phloem and xylem of the mulberry with the highest expression in the phloem. The expression level of the mRNA has changed significantly under salt, drought, cold and ABA stress treatments compared to the normal growth environment. Overall, these results showed a better understanding of the molecular basis for the signal transduction mechanism during the stress responses in mulberry trees.

Bioengineering a Cadmium Sensing Green Fluorescent Protein based Whole-Cell Biosensor from Pseudomonas aeruginosa

Heavy metal toxicity remediation is a major issue due to the increase in industrial waste production, with cadmium being one of the major pollutants. An approach involving homologous cloning was made to bioengineer a microbial cadmium biosensor from a strain of Pseudomonas aeruginosa. The promoter pCadR from P. aeruginosa was cloned into a vector pEGFP-N2. Gibson assembly cloning method was chosen considering the several advantages it holds over the traditional cloning techniques. Escherichia coli DH5 alpha strain was used as the host cell, which on sensing cadmium, produced fluorescence using the reporter gene called green fluorescent protein (GFP). The clone, pEGFP-N2CadR, was subjected to increasing concentrations of cadmium chloride to determine the sensitivity. It was observed that pEGFP-N2CadR responded well to micromolar concentrations of cadmium chloride, and hence it may be used to detect low concentrations of cadmium. To determine the specificity of the sensor, it was also tested with lead nitrate and copper sulfate solutions. It was determined that the biosensor had non-specific interactions with other metals. The interaction of the promoter with the metals was weak as compared to previous studies, and this was attributed to slight variations and mutations observed in the promoter sequence following subsequent rounds of sequencing. As the fluorescent intensities of the sensor in response to the metals were not highly distinguishable, this biosensor was not classified as an effective cadmium sensor. Further studies for determining the promoter interaction and affinity towards various metals at varying concentrations are required to validate the results obtained.

Bioengineering, a Cadmium Sensing Green Fluorescent Protein, Based Whole-Cell Biosensor From Pseudomonas aeruginosa

Heavy metal toxicity is a significant issue due to an increase in industrial waste production, with cadmium being one of the major pollutants. An approach involving homologous cloning was made to bioengineer a microbial cadmium biosensor from a strain of Pseudomonas aeruginosa. The promoter pCadR from P. aeruginosa was cloned into a vector pEGFP-N2 using Gibson assembly. Escherichia coli DH5 alpha strain was used as the host cell, which on sensing cadmium, produced fluorescence using the reporter gene called green fluorescent protein (GFP). The clone, pEGFP-N2CadR, was subjected to increasing concentrations of cadmium chloride to determine the sensitivity. It was observed that pEGFP-N2CadR responded to micromolar concentrations of cadmium chloride; however, it was determined that the biosensor tested it with lead nitrate and copper sulfate solutions had non-specific interactions with other metals. The interaction of the promoter with the metals was weak compared to previous studies, which was attributed to several reasons mentioned in the paper. As the sensor's fluorescent intensities were dull and indistinguishable, it was not classified as a useful cadmium biosensor. Further studies for determining the promoter interaction and affinity towards various metals at varying concentrations are required to validate the results obtained.

Overexpression of Medicago sativa LEA4-4 can improve the salt, drought, and oxidation resistance of transgenicArabidopsis.

Late embryogenesis abundant (LEA) proteins are widely involved in many adverse conditions among plants. In this study, we isolated a LEA4 gene from alfalfa (Medicago sativa L.) termed MsLEA4-4 via a homology cloning strategy. MsLEA4-4 encodes 166 amino acids, and the structural analysis showed that the gene contained five repeating TAQAAKEKTQQ amino acid motifs. There were a large number of α-helix in MsLEA4-4, and belongs to hydrophilic amino acid. Subcellular localization analysis showed that MsLEA4-4 was localized in the nucleus. The MsLEA4-4 promoter consisted of G-box and A-box elements, abscisic acid-responsive elements (ABREs), photo regulation and photoperiodic-controlling cis-acting elements, and endosperm expression motifs. The MsLEA4-4 overexpressing in Arabidopsis conferred late-germination phenotypes. Resistance of the overexpressed plants to abiotic stress significantly outperformed the wild-type (WT) plants. Under salt stress and abscisic acid treatment, with more lateral roots and higher chlorophyll content, the overexpressed plants has a higher survival rate measured against WT. Compared to those in the WT plants, the levels of soluble sugar and the activity of various antioxidant enzymes were elevated in the overexpressed plants, whereas the levels of proline and malondialdehyde were significantly reduced. The expression levels of several genes such as ABF3, ABI5, NCED5, and NCED9 increased markedly in the overexpressed plants compared to the WT under osmotic stress.

Characterization of transcription activation domain of EcaICE1 and its interaction with EcaSIZ1 in Eucalyptus camaldulensis

SUMOylation in plants is associated with biotic and abiotic stress responses, flowering, and other aspects of development. ICE1 protein SUMOylation by the SUMO E3 ligase SIZ1 plays important roles in plant cold tolerance. Here, we reported the subcellular localization of EcaICE1 and its interaction with EcaSIZ1 in Eucalyptus camaldulensis. The genes EcaICE1 and EcaSIZ1 were isolated by homologous cloning. The subcellular localization analysis showed that EcaICE1 was located in nucleus. Bimolecular fluorescence complementation (BiFC) analysis revealed that EcaICE1 could interact with EcaSIZ1 in the nucleus of Nicotiana benthamiana leaves. Moreover, yeast two-hybrid assay confirmed that the amino acid region from position 84 to 126 in EcaICE1 was critical for the strong transactivation activity of EcaICE1 and that the C-terminal region from position 361 to 557 in EcaICE1 was the key region for its interaction with EcaSIZ1 using different truncated lengths of non-transactivation activity of EcaICE1 as the bait protein. Collectively, our results showed that EcaICE1 may have a SUMOylation pathway similar to Arabidopsis thaliana.

Molecular cloning and functional characterization of CmFT (FLOWERING LOCUS T) from Cucumis melo L.

CmFT homologous gene in muskmelon was obtained by homologous cloning, introducing CmFT gene by Agrobacterium mediated transformation. The results of subcellular localization showed that CmFT protein was expressed in cytoplasm and nucleus. qRTPCR results showed that the expression levels of AtLFY, AtFT, AtCO, AtFLC, AtSOC1 and AtAP1 were upregulated in the 35S::MeFT Arabidopsis line. The CmFT gene was introduced into wild-type Arabidopsis by Agrobacterium-mediated transformation, and the growth status of T2 transgenic Arabidopsis thaliana and wild-type A. thaliana was observed. The results showed that wild-type Arabidopsis began to bolt on the 25th day after sowing, we can initially confirm that the FT gene of melon can promote the early flowering of melon in the growth and development of melon.

Proline metabolism and molecular cloning of AmP5CS in the mangrove Avicennia marina under heat stress.

Proline is one of the most important compatible osmolyte in cells, which accumulates in response to various stresses, including salt, water deficit, heavy metal, pathogen infection and extreme temperature. In this study, a growth chamber was employed to simulate heat environment for Avicennia marina seedlings. We detected some physiological indices in the leaves of A. marina at 40 °C, including the activity of delta-1-pyrroline-5-carboxylate synthase (P5CS), the content of free proline and soluble protein, transpiration rate and membrane permeability, and discussed the relationship between these five indices and heat resistant ability. And then a P5CS gene was cloned from A. marina using homologous cloning and rapid amplification of cDNA ends methods. It was designated as AmP5CS, encoding a protein that contained a feedback inhibition site of proline, proA, proB, conserved Leu zipper, GSA-DH domain and other functional domains of P5CS protein in high plants. Expression analysis of AmP5CS gene indicated it was involved in heat stress response. It is the first time that P5CS from A. marina has been cloned and the findings laid the foundation of figuring out heat resistant mechanisms and relieving heat damage, which is significant during global warming.

Secretory expression of the recombinant FGF-2 protein in Pichia pastoris carrying multiple copies of target gene

Introduction: Fibroblast growth factor-2 (FGF-2) is a multifunctional protein that plays an important role in the regulation of proliferation, differentiation and migration of a variety of cells. The recombinant human FGF-2 (rhFGF-2) is currently used in stem cell culture, medicine and cosmetic products. In this study, we aim to produce secreted rhFGF-2 protein from a Pichia pastoris strain containing multiple copies of the fgf-2 gene to eliminate the disadvantages of intracellular expression systems.&#x0D; Methods: The recombinant Pichia pastoris carrying the fgf-2 gene was cloned by using homologous cloning method. The recombinant strains were screened by PCR reactions using specific primers for the target gene and the AOX1 promoter sequence. Moreover, the copy number of the fgf-2 gene inserted into the P. pastoris genome was identified by semi-quantitative PCR method. The secreted rhFGF-2 protein was collected in the induced BMMY medium at a final methanol concentration of 0.5%, and purified by one-step heparin affinity chromatography. The quantity and biological activity of the purified protein were determined by competitive ELISA method and MTT assay on NIH-3T3 cell line, respectively.&#x0D; Results: Various recombinant P. pastoris clones carrying different copy numbers of the fgf-2 gene were obtained and categorized into 3 groups: the low copy strains (4-5 copies), medium copy strains (8-11 copies), and high copy strains (more than 20 copies). Among those strains, the 4-copy one produced the rhFGF-2 protein at the highest expression level. After purification, the purity of rhFGF-2 protein reached 98.8%, and the recovery yield was 179.2 µg of protein from 200 mL of flask culture (equivalent to 850 µg/L). The purified rhFGF-2 protein showed similar biological activity on NIH-3T3 cell line with the commercial FGF-2 protein.&#x0D; Conclusion: The recombinant FGF-2 protein was successfully secretory expressed from Pichia pastoris, and successfully purified by only one-step chromatography.