Effects of Heterologously Overexpressing PIP5K-Family Genes in Arabidopsis on Inflorescence Development

Castor is one of the top 10 oil crops in the world and has extremely valuable uses.Castor inflorescences directly affect yield,so the study of inflorescence development is very important in increasing castor yield.Our previous studies have shown that the PIP5K gene family(PIP5Ks)is associated with inflorescence development.In this study,to determine the function of each PIP5K gene in castor,a female Lm-type castor line,aLmAB2,was used to determine the relative expression levels of the PIP5Ks in castor inflorescences.Six PIP5K genes were heterologously overexpressed in Arabidopsis thaliana,the relative expression of each gene and the effect on plants was determined in A.thaliana,and the relationships among the PIP5Ks in castor were inferred.The expression levels of the PIP5Ks in the female Lm-type castor line aLmAB2 were analyzed.The relative expression levels of the PIP5K9 and PIP5K11 genes were high(p<0.05)in isofemale inflorescences,and those of PIP5K1,PIP5K2,PIP5K6,and PIP5K8 were high(p<0.05)in female inflorescences but low(p<0.05)in bisexual inflorescences.The PIP5Ks were heterologously overexpressed in A.thaliana,and T3-generation plants with stable genetic resistance,i.e.,AT-PIP5K^(+)plants(AT-PIP5K1^(+),AT-PIP5K2^(+),AT-PIP5K6^(+),AT-PIP5K8^(+),AT-PIP5K9^(+),and ATPIP5K11^(+) plants),were obtained.Biological tests of the AT-PIP5K+plants showed that the growth of the main stem was significantly delayed in AT-PIP5K+plants compared with Columbia wild-type(WT)A.thaliana plants;the PIP5K1 and PIP5K2 genes promoted lateral stem growth and flower and silique development;and the PIP5K6,PIP5K8,PIP5K9 and PIP5K11 genes inhibited lateral stem growth and flower and silique development.The correlations among PIP5Ks in castor suggest that there may be a synergistic relationship among PIP5K1,PIP5K2,and PIP5K6 in castor inflorescences,and PIP5K8,PIP5K9,and PIP5K11 are complementary to the other three genes.

Overexpression of a Foreign Bt Gene in Cotton Affects the Low-Molecular-Weight Components in Root Exudates

Most research in the past using genetically modified crops (GM crops) has focused on the ecological safety of foreign gene (i.e., the gene flow), gene products (for example, Bt (Bacillus thuringiensis) protein), and the safety of transgenic food for humans. In this study, changes in both the species and amounts of low-molecular-weight components in cotton (Gossypium hirsutum L.) root exudates after foreign Bt gene overexpression were investigated under different nutritional conditions. Transgenic cotton containing Bt (Bt-cotton), supplemented with all the mineral nutrients, secreted more organic acids than the wild-type cotton (WT). When nitrogen was removed from the full-nutrient solution, the amount of organic acids secretion of Bt-cotton was lesser than that of WT. The roots of the transgenic cotton secreted lesser amounts of amino acids and soluble sugars than the WT roots in the full-nutrient solution. Deficiencies of P and K caused a large increase in the total amino acid and soluble sugar secretions of both Bt-cotton and WT, with larger increases observed in Bt-cotton. Because transferring the foreign Bt gene into cotton can result in alterations in the components of the root exudates, with the effect varying depending on the nutritional status, the cultivation of genetically modified crops, such as Bt-cotton, in soil environments should be more carefully assessed, and the possible effects as a result of the alterations in the root exudate components should be considered.

Profiling influences of gene overexpression on heterologous resveratrol production in Saccharomyces cerevisiae

Metabolic engineering of heterologous resver- atrol production in Saccharomyces cerevisiae faces challenges as the precursor L-tyrosine is stringently regulated by a complex biosynthetic system. We over- expressed the main gene targets in the upstream pathways to investigate their influences on the downstream resver- atrol production. Single-gene overexpression and DNA assembly-directed multigene overexpression affect the production of resveratrol as well as its precursor p-coumaric acid. Finally, the collaboration of selected gene targets leads to an optimal resveratrol production of 66.144-3.74 mg.L-1, 2.27 times higher than the initial production in YPD medium （4% glucose）. The newly discovered gene targets TRP1 expressing phosphoribosy- lanthranilate isomerase, AR03 expressing 3-deoxy-D- arabino-heptulosonate-7-phosphate synthase, and 4CL expressing 4-coumaryl-CoA ligase show notable positive impacts on resveratrol production in S. cerevisiae.

Cloning and Overexpressing Apo-phycoerythrocyanin α-subunit Gene of Mastigocladus laminosus in E.coli

By PCR method, apo phycoerythrocyanin α subunit gene (pecA) of Mastigocladus laminosus (M. laminosus) was amplified from its genomic DNA, and then cloned in pBluescript. The pecA gene was subcloned into the expression vector pGEMD, and then transformed into E.coli BL21 (DE3). After induction, a new protein of molecular weight 19×10 3 existing in inclusion body was overexpressed. The expressed product was confirmed to be apo phycoerythrocyanin α subunit by Dot ELISA.

In vivo protective effect of late embryogenesis abundant protein(ApSK3 dehydrin)on Agapanthus praecox to promote post-cryopreservation survival

Dehydrins(DHNs),as members of the late embryogenesis abundant protein family,play critical roles in the protection of seeds from dehydration and plant adaptation to multiple abiotic stresses.Vitrification is a basic method in plant cryopreservation and is characterized by forming a glassy state to prevent lethal ice crystals produced during cryogenic storage.In this study,ApSK3 type DHN was genetically transformed into embryogenic calluses(EC)of Agapanthus praecox by overexpression(OE)and RNA interference(RNAi)techniques to evaluate the in vivo protective effect of DHNs during cryopreservation.The cell viability showed a completely opposite trend in OE and RNAi cell lines,the cell relative death ratio was decreased by 20.0%in ApSK3-OE EC and significantly increased by 66.15%in ApSK3-RNAi cells after cryopreservation.Overexpression of ApSK3 increased the content of non-enzymatic antioxidants(AsA and GSH)and up-regulated the expression of CAT,SOD,POD,and GPX genes,while ApSK3-RNAi cells decreased antioxidant enzyme activities and FeSOD,POD,and APX genes expression during cryopreservation.These findings suggest that ApSK3 affects ROS metabolism through chelating metal ions(Cu^(2+)and Fe^(3+)),alleviates H_(2)O_(2)and OH·excessive generation,activates the antioxidant system,and improves cellular REDOX balance and membrane lipid peroxidation damage of plant cells during cryopreservation.DHNs can effectively improve cell stress tolerance and have great potential for in vivo or in vitro applications in plant cryopreservation.

A toolbox for genetic manipulation in intestinal Clostridium symbiosum

Gut microbes are closely related with human health,but remain much to learn.Clostridium symbiosum is a conditionally pathogenic human gut bacterium and regarded as a potential biomarker for early diagnosis of intestinal tumors.However,the absence of an efficient toolbox that allows diverse genetic manipulations of this bacterium limits its in-depth studies.Here,we obtained the complete genome sequence of C.symbiosum ATCC 14940,a representative strain of C.symbiosum.On this basis,we further developed a series of genetic manipulation methods for this bacterium.Firstly,following the identification of a functional replicon pBP1 in C.symbiosum ATCC 14940,a highly efficient conjugative DNA transfer method was established,enabling the rapid introduction of exogenous plasmids into cells.Next,we constructed a dual-plasmid CRISPR/Cas12a system for genome editing in this bacterium,reaching over 60% repression for most of the chosen genes as well as efficient deletion(>90%)of three target genes.Finally,this toolbox was used for the identification of crucial functional genes,involving growth,synthesis of important metabolites,and virulence of C.symbiosum ATCC 14940.Our work has effectively established and optimized genome editing methods in intestinal C.symbiosum,thereby providing strong support for further basic and application research in this bacterium.

Inhibitor tolerance and bioethanol fermentability of levoglucosan-utilizing Escherichia coli were enhanced by overexpression of stress-responsive gene ycfR:The proteomics-guided metabolic engineering

Pretreatment of lignocellulosic biomass is crucial for the release of biofermentable sugars for biofuels production,which could greatly alleviate the burgeoning environment and energy crisis caused by the massive usage of traditional fossil fuels.Pyrolysis is a cost-saving pretreatment process that can readily decompose biomass into levoglucosan,a promising anhydrosugar;however,many undesired toxic compounds inhibitory to downstream microbial fermentation are also generated during the pyrolysis,immensely impeding the bioconversion of levoglucosan-containing pyrolysate.Here,we took the first insight into the proteomic responses of a levoglucosan-utilizing and ethanol-producing Escherichia coli to three representative biomass-derived inhibitors,identifying large amounts of differentially expressed proteins(DEPs)that could guide the downstream metabolic engineering for the development of inhibitor-resistant strains.Fifteen up-and eight down-regulated DEPs were further identified as the biomarker stress-responsive proteins candidate for cellular tolerance to multiple inhibitors.Among these biomarker proteins,YcfR exhibiting the highest expression fold-change level was chosen as the target of overexpression to validate proteomics results and develop robust strains with enhanced inhibitor tolerance and fermentation performance.Finally,based on four plasmid-borne genes encoding the levoglucosan kinase,pyruvate decarboxylase,alcohol dehydrogenase,and protein YcfR,a new recombinant strain E.coli LGE-ycfR was successfully created,showing much higher acetic acid-,furfural-,and phenol-tolerance levels compared to the control without overexpression of ycfR.The specific growth rate,final cell density,ethanol concentration,ethanol productivity,and levoglucosan consumption rate of the recombinant were also remarkably improved.From the proteomics-guided metabolic engineering and phenotypic observations,we for the first time corroborated that YcfR is a stress-induced protein responsive to multiple biomass-derived inhibitors,and also developed an inhibitors-resistant strain that could produce bioethanol from levoglucosan in the presence of inhibitors of relatively high concentration.The newly developed E.coli LGE-ycfR strain that could eliminate the commonly-used costly detoxicification processes,is of great potential for the in situ cost-effective bioethanol production from the biomass-derived pyrolytic substrates.

Overexpression of the nuclear protein gene AtDUF4 increases organ size in Arabidopsis thaliana and Brassica napus

Organ size is an important trait of many crops that is influenced by internal and environmental signals and controlled by a combina- tion of factors during organogenesis （I（rizek, 2009）. The final size of plant organs is determined by two successive but overlapping pro- cesses： cell division, which increases cell number, and cell expan- sion, which determines final cell size （Anastasiou and Lenhard, 2007）. Some genes have been identified to control organ size by regulating cell division and for cell expansion in plants.

HLA-G inhibits xenogenetic cytotoxicity mediated by human NK cells and T lymphocytes against PECs

In order to investigate whether the non-classi- cal HLA-G classⅠmolecule protects the porcine endothelial cells (PECs) from the lysis mediated by human immune cells in pig to human discordant xenotransplantation, we have cloned HLA-G cDNA from a human placenta by RT-PCR. Mammalian expression vector, pEFG-neo, was constructed by insertion of HLA-G cDNA in pEF-neo. We obtained effi-ciently expressed PECs by stable transfection. Cytotoxicity assay showed that overexpression of HLA-G on PECs was sufficient to inhibit human NK-92 cell lysis. The level of lysis was equal to or less than that of the lysis of human umbilical vein endothelial cells mediated by human NK-92 cells. It also indicated that HLA-G inhibited the lysis of PECs mediated by xeno-antigen specific T lymphocytes. The reduction of lysis ranged between 59.1% and 88.9%. These findings sug-gest that the transgenic approach to overexpress HLA-G is believed to be a new immunotherapy in overcoming the im-mune rejections in xenotransplantion, including delayed xenograft rejection and cell-mediated rejection.

Transport of cadmium from soil to grain in cereal crops: A review

Due to rapid urbanization and industrialization, many soils for crop production are contaminated by cadmium(Cd), a heavy metal highly toxic to many organisms. Cereal crops such as rice, wheat, maize, and barley are the primary dietary source of Cd for humans, and reducing Cd transfer from soil to their grains is therefore an important issue for food safety. During the last decade, great progress has been made in elucidating the molecular mechanisms of Cd transport, particularly in rice. Inter-and intraspecific variations in Cd accumulation have been observed in cereal crops. Transporters for Cd have been identified in rice and other cereal crops using genotypic differences in Cd accumulation and mutant approaches. These transporters belong to different transporter families and are involved in the uptake, vacuolar sequestration, root-to-shoot translocation, and distribution of Cd. Attempts have been made to reduce Cd accumulation in grains by manipulating these transporters through overexpression or knockout of the transporter genes, as well as through marker-assisted selection breeding based on genotypic differences in Cd accumulation in the grains. In this review, we describe recent progress on molecular mechanisms of Cd accumulation in cereal crops and compare different molecular strategies for minimizing Cd accumulation in grains.

Metabolic engineering of Yarrowia lipolytica for scutellarin production

Scutellarin related drugs have superior therapeutic effects on cerebrovascular and cardiovascular diseases.Here,an optimal biosynthetic pathway for scutellarin was constructed in Yarrowia lipolytica platform due to its excellent metabolic potential.By integrating multi-copies of core genes from different species,the production of scutellarin was increased from 15.11 mg/L to 94.79 mg/L and the ratio of scutellarin to the main by-product was improved about 110-fold in flask condition.Finally,the production of scutellarin was improved 23-fold and reached to 346 mg/L in fed-batch bioreactor,which was the highest reported titer for de novo production of scutellarin in microbes.Our results represent a solid basis for further production of natural products on unconventional yeasts and have a potential of industrial implementation.

Tailoring the Immune Microenvironment of Dendritic Cells by Targeting C-type Lectin Receptor

Background:The C-type lectin receptor(CLR)expressed by DCs participates in the recognition and capture of various glycosylated self-antigens and pathogens.Understanding the diversity of the CLR expressed by DCs,as well as their role in maintaining the balance between Th1-type and Th2-type cytokines would promote the understanding of the pathogenesis of many diseases including preeclampsia(PE).Methods:DCs were isolated from the placentae of healthy women who underwent normal pregnancies and infected with a CLR lentiviral(LV)vector for gene overexpression or small interfering RNA(siRNA)knockdown.DCs were cocultured with T-cells and EVCTs,and five groups were established as follows:Group 1-DCs from healthy women who underwent normal pregnancies,Group 2-DCs from women with preeclampsia(PE),Group 3-DCs infected with empty LV vectors,Group 4-DCs infected with a CLR LV vector for gene overexpression,and Group 5-DCs infected with a CLR LV vector for siRNA knockdown.The levels of Th1-and Th2-type cytokines were measured in all groups.Results:The levels of Th1-type cytokines were significantly higher in women with PE than in those with normal pregnancies(P<0.05).Among these five groups,the Th1/Th2 ratio of Group 5 was highest(P<0.05).There was no difference in the Th1/Th2 ratio between Groups 1 and 3.Conclusions:There was a Th1/Th2 imbalance in women with PE displaying Th1-type immunity.CLR-overexpressing DCs showed a diminished capacity to polarize naïve T-cells into Th1 effector cells.The impaired Th1 response in DCs was rescued by CLR siRNA knockdown.In conclusion,DCs may affect the production of cytokines and the migration of T-cells through CLR-mediated signaling pathways during pregnancy.