Molecular Cloning of MYMV Genome and Infectivity of Yellow Mosaic Virus in Green Gram Using Different Viral Transmission Tools

Mungbean yellow mosaic virus (MYMV) causes massive crop losses in green gram. MYMV is a member of begomovirus with bipartite genome comprising DNA-A and DNA-B components, which is transmitted by whiteflies. Cloning and preparation of infectious clone is very much essential for screening germplasm or transgenic material of pulse crops since viruliferous whiteflies may not be available throughout the year. In the current work, we have amplified rolling circle mediated viral genome of MYMV using Φ29 DNA polymerase. The amplified products was digested and cloned into the plant expression vector pCAMBIA2301.The cloned constructs was then transformed into Agrobacterium LBA4404 through freeze thaw method. Further, three viral transmission techniques including mechanical rubbing, Agroinfiltration and Agroinoculation, were employed for assessing the mosaic symptoms in green gram. The molecular confirmation through polymerase chain reaction (PCR) indicated that the yellow mosaic symptoms were formed due to infectivity of MYMV in the green gram.

Molecular Cloning, Bioinformatics Analysis and Overexpression of the Tyrosine Aminotransferase Gene in Rehmannia Glutinosa

Rehmannia glutinosa is an important medicinal plant producing many bioactive compounds such as catalpol, acteoside and so on. Tyrosine aminotransferase (TAT) is the first key enzyme that catalyzes the reversible interconversion of tyrosine and 4-hydroxyphenylpyruvate in the tyrosine-derived branch pathway of acteoside biosynthesis. To confirm its role for acteoside accumulation, we isolated a full-length cDNA from Rehmannia glutinosa Libosch. Sequence analysis indicated that it contained a 1266 bp open reading frame, encoding a TAT of 421 amino acid residues. Multiple sequence alignment revealed that the homology of RgTAT amino acid sequence to that of Sesamum indicum(XP_011100354.1) was the highest (89.94%). Evolutionary tree showed that Sesamum indicum TAT and RgTAT were grouped together. Quantitative real-time PCR analysis indicated that the expression of RgTAT in leaves was much higher than in roots and stems,and that the expression levels of RgTAT in the tuberous roots, stems and leaves of high-acteoside cultivar BJ-3 were higher than in that of low-acteoside cultivar Wen85-5. A plant expression vector was constructed containing the RgTAT and hygromycin resistance gene (Hyg). Transgenic Rehmannia glutinosa Libosch overexpressing RgTAT was obtained via an Agrobacterium tumefaciens-mediated transformation system, in which Hyg expression was confirmed by PCR. RgTAT expression in transgenic plantlets measured by real-time quantitative PCR was 7.72 ± 0.17 times greater than its expression in the untransformed plantlets. Moreover, HPLC analysis indicated that enhanced RgTAT expression corresponded to significantly increased acteoside for transgenic plantlets. Our results elucidate the role of RgTAT in the acteoside biosynthesis in Rehmannia glutinosa.

The maize hexokinase gene ZmHXK7 confers salt resistance in transgenic Arabidopsis plants

The hexokinase (HXK) gene family, whose members play vital roles in sugar induction signals and glycolysis in organisms, is widely found in plants. Although some hexokinase genes have been studied in maize, a systematic report of the gene family and its role in plant resistance is lacking. In this study, 10 hexokinase genes were systematically identified in maize based on the maize genome-wide database. Phylogenetic analysis divides the maize HXK protein family into four clusters. Prediction of cis-regulatory elements showed that a number of elements responding to abiotic stress exist in the promoter of hexokinase genes. The expression profile of these genes, originated from B73, showed that different members of hexokinase genes are highly expressed in roots and leaves of maize under salt or drought stress, which is similar to that of Mo17.The coding sequence of ZmHXK7 gene, isolated from maize B73, was constructed into plant expression vector pMDC45 and then transformed into athxk3 (Salk_022188C). By hyg resistance detection, PCR analysis, and western blot confirmation, the homozygous progenies of transgenic Arabidopsis lines were identified. Subcellular localization analysis showed that the ZmHXK7 gene was located in cytosol. Seedling growth and salt stress inhibition in complementary mutant plants of ZmHXK7 gene were significantly improved, and enhanced salt tolerance was displayed. Our study provides insights into the evolution and expression patterns of the hexokinase gene and show that maize ZmHXK7 proteins play an important role in resisting salt stress, which will be useful in plant breeding for abiotic stress resistance.

Expression of ZjPSY, A Phytoene Synthase Gene from Zoysia Japonica Leads to Leaf Yellowing and Plant Dwarfing in Arabidopsis Thaliana

Phytoene synthase (Psy) is a key limiting enzyme in the carotenoid biosynthesis pathway by regulating phytoene synthesis. In this study, ZjPSY was isolated and identified from an important lawn grass species, Zoysia japonica. ZjPSY cDNA was 1230 bp in length, corresponding to 409 amino acids. ZjPSY showed higher expression in young leaves and were down-regulated after GA3, ABA, SA, and MeJA treatments, exhibited a sensitivity to hormones. By analysis of cis-regulatory elements in ZjPSY promoter region, ZjPSY exhibited be regulated of light and multiple hormones. To investigate the functions of ZjPSY, the plant expression vector was constructed to obtain transgenic Arabidopsis thaliana. Overexpression of ZjPSY protein led to carotenoid accumulation and altered expression of genes involved in carotenoid contents. ZjPSY expressing Arabidopsis thaliana exhibited yellowing and dwarfing phenotypes and contained more carotenoids than the wild type. Yeast two-hybrid screening identified a novel interacting partner of ZjPSY, ZjJ2 (DNAJ homologue 2), which encodes heat-shock protein 40 (HSP40). Taken together, this study suggests that ZjPSY plays an important role in carotenoid synthesis, leaf color development and hormone response in transgenic plants. These results broadened the understanding of carotenoid synthesis pathways and laid a foundation for the exploration and utilization of PSY gene.

Effect of leaf position and days post-infiltration on transient expression of colorectal cancer vaccine candidate proteins GA733-Fc and GA733-FcK in Nicotiana benthamiana plant

Immunization with thetumor-associated antigen GA733 glycoprotein, which is highly expressed in colorectal cancer, is considered to be a promising strategy for cancer prevention and treatment. We cloned a fusion gene of GA733 and immunoglobulin Fc fragment (GA733-Fc), and that of GA733-Fc and an endoplasmic reticulum retention motif (GA733-FcK) into the Cowpea mosaic virus (CPMV)-based transient plant expression vector, pEAQ-HT. Agrobacterium tumefaciens (LBA4404) transformed with the vectors pEAQ-HT-GA733-Fc and pEAQ-HT-GA733-FcK was infiltrated into the leaves of Nicotiana benthamiana plants. To optimize harvesting of leaf to express therapeutic glycoproteins both spatially and temporally, protein expression levels at various leaf positions (top, middle, and base) and days post-infiltration (dpi) were investigated. The GA733-Fc and GA733-FcK genes were detected in leaves at 1–10 dpi using PCR. As assessed by western blot, GA733-Fc and GA733-FcK were expressed at the highest levels in the top leaf position at 5 dpi, and GA733-FcK was expressed more than GA733-Fc. The proteins were successfully purified from infiltrated N. benthamiana leaves using protein A affinity chromatography. ELISA verified that an anti-GA733 antibody recognized both purified proteins. Thus, a functional GA733-Fc colorectal cancer vaccine protein can be transiently expressed using a CPMV virus-based vector, with an optimized expression time and leaf position post-infiltration.

Activity and Function Studies of The Promoter Cis-Acting Elements of The Key Enzymes in Saponins Biosynthesis of DS From Panax Notoginseng

Background：Panax notoginseng is a traditional Chinese medicine for the treatment of blood diseases, in which saponins were the main active components. Dammarenediol synthase (DS) is a key enzyme in the saponin synthesis pathway of P. notoginseng.The promoter is an important region to regulate gene expression, and the study of the promoter sequence provides important evidence for revealing the mechanism of gene expression regulation. Methods: However, there was still little research on the promoter function of P. notoginseng. In this study, the 1382 bp promoter upstream of DS from P. notoginseng was cloned and sequenced. The promoter sequence was analyzed by online databases. The plant expression vector fused with the β-glucuronidase gene was constructed, transferred into Agrobacterium tumefaciens. Then tobacco was injected, and its response to exogenous hormones (gibberellin and abscisic acid) was studied by transient expression to verify its unique action elements. Results: The results showed that the tobacco leaves transferred with DS promoter had significantly increased GUS protease activity after spraying GA and ABA, indicating that both DS promoter can specifically and significantly respond to exogenous GA and ABA signal. Conclusions: These findings will help us to better understand the regulatory mechanisms of the upstream region of the DS gene and provide a basis for future research on the interaction of cis-acting elements of promoters with related transcription factors.

The Construction of Plant Expression Vector harbouring Carica Papaya L. WRKY Gene in Escherichia coli

Carica papaya is a well-liked and economically important fruit with outstanding nutritional and medicinal values. Its susceptibility to abiotic stress which affects the growth and harvest, causes significant yield loss to farmers. In recent years, significant progress has been made to understand the genes that play critical roles in abiotic stress response, especially some transcription factor (TF) encoding genes. Among all TFs, WRKY TF gene family is one of the best-studied TFs involved in various stress responses. To date, only limited information on functionally characterised WRKY TFs is available for C. papaya. The aim of this study was to produce a recombinant construct harbouring WRKY gene in pGEM®-T Easy cloning vector. The presence of a DNA band of the expected size of 465 bp on agarose gel electrophoresis indicated that WRKY gene was successfully amplified from all treated samples. DNA sequencing analysis revealed that the amplified sequence isolated from the treated samples were closely related to Carica papaya species with 97% similarity. Following transformation, 4 out of 5 colonies that were randomly selected showed the WRKY gene had been successfully inserted into pGEM®-T Easy vector and transformed into E. coli. In future, the WRKY gene from pGEMT-WRKY recombinant construct will be cloned into the plant expression vector pCAMBIA 1304 prior to transformation in the plant. The success of demonstrating the WRKY gene towards the response in abiotic stress will enable us to produce stress tolerant transgenic crops under unfavourable conditions via genetic engineering for sustained growth.

Pyramiding of Cry Toxins and Methanol Producing Genes to Increase Insect Resistance in Cotton

The idea of enhanced methanol production from cell wall by pectin methyl esterase enzymes (PME) combined with expression of cry genes from Bacillus thuringiensis as a strategy to improve pests control in cotton is presented. We constructed a cassette containing two cry genes (cry1Fa and Cry32Aa) and two pme genes, one from Arabidopsis thaliana (AtPME), and other from Aspergillus niger (AnPME) in pCAMBIA1301 plant expression vector using CAMV-35S promoter. This construction was transformed in Eagle-2 cotton variety using shoot apex-cut Agrobacterium-mediated transformation. The expression of cry genes and pme genes was confirmed by qPCR. Methanol production was measured in control and in the cry and pme transformed plants showing methanol production only in transformed plants, then the non-transgenic cotton plants. Finally, insect bioassays performed with transgenic plants expressing cry and pme genes, showed 100 % mortality for Helicoverpa armigera (cotton bollworm) larvae, 70% mortality for pectinophore gossypiella (pink bollworm) larvae and 95% mortality of Earias fabia, (spotted bollworm) larvae, that was higher than the transgenic plants expressing only cry genes that showed 84%, 49% and 79% mortality, respectively. These results demonstrate that Bt. cry-genes coupled with pme genes is an effective strategy to improve the control of different insect pests.

Expression of a NGATHA1 Gene from Medicago truncatula Delays Flowering Time and Enhances Stress Tolerance

Shoot branching is one of the most variable determinants of crop yield, and the signaling pathways of plant branches have become a hot research topic. As an important transcription factor in the B3 family, NGATHA1 (NGA1), plays an important role in regulating plant lateral organ development and hormone synthesis and transport, but few studies of the role of this gene in the regulation of plant growth and stress tolerance have been reported. In this study, the NGA1 gene was isolated from Medicago truncatula (Mt) and its function was characterized. The cis-acting elements upstream of the 5′ end of MtNGA1 and the expression pattern of MtNGA1 were analyzed, and the results indicated that the gene may act as a regulator of stress resistance. A plant expression vector was constructed and transgenic Arabidopsis plants were obtained. Transgenic Arabidopsis showed delayed flowering time and reduced branching phenotypes. Genes involved in the regulation of branching and flowering were differentially expressed in transgenic plants compared with wild-type plants. Furthermore, transgenic plants demonstrated strong tolerances to salt- and mannitol-induced stresses, which may be due to the upregulated expression of NCED3 (NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3) by the MtNGA1 gene. These results provide useful information for the exploration and genetic modification use of MtNGA1 in the future.

Expression of Recombinant Human Glucocerebrosidase Protein in Sunflowers

Molecular farming has become one of the most significant implementations of modern biotechnology to generate modified plant crops to produce medicinal proteins. Agrobacterium is one plant genetic engineering tool that integrates genes of interest inside a host plant. In recent years, the need to produce recombinant proteins as therapeutics has growing rapidly, and human glucocerebrosidase is one of the proteins that is need to treat disease. In this study, specific primers were designed to amplify Hu-GBA1 gene from constructed pGEM-GBA plasmid which was cloned into the plant expression vector pCAMBIA1304. The generated recombinant pCAMBIA1304-GBA plasmid was used to transform A. tumefaciens LBA4404 and applied for transformation of sunflower cotyledon explants. Colony PCR technique was used to confirm the presence of Hu-GBA1 gene in transformed A. tumefaciens. Agrobacterium containing pCAMBIA1304-GBA was suspended in Infection Medium (IM) supplement with 200 mM acetosyringone. A bacterial suspension was used to transform sunflower cotyledons. After infection, cotyledons were co-cultivated in Co-cultivation medium (CCM), supplied with 200 mM acetosyringone without antibiotics. The cotyledons were then transferred to selection media containing 7.5 mg/L Hygromycin and 250 mg/L Cefotaxime and grown for additional 14 days at 25℃ in photoperiod of 16h L/8h D. The transformed sunflower cotyledons were successfully generated complete plant with used 6-Benzylaminopurine and Naphthalene acetic acid as growth hormones. The presence of the Hu-GBA1 gene in the genomic DNA of transgenic sunflower plant was proven by PCR as a band of 1561bp size. The GBA mRNA expression in modified sunflowers was detected by qRT-PCR compared with control GBA mRNA. Enzyme Linked Immunoassay was done on crude recombinant protein that extracted from transformed sunflower using Human Glucosylceramide ELISA Kit, the Elisa test results confirmed the production of recombinant glucocerebrosidase and the concentration of crude recombinant enzyme extracted from transformed sunflower with GBA1 gene was 0.45 ng/µl