OBJECTIVETo develop a hydroponic Nicotiana cultivation system for rapid and high-yield transient expression of recombinant proteins under laboratory conditions.

METHODSTo establish the hydroponic cultivation system, several parameters were examined to define the optimal conditions for the expression of recombinant proteins in plants. We used the green fluorescent protein (GFP) and the geminiviral plant transient expression vector as the model protein/expression vector. We examined the impact of Nicotiana species, the density and time of Agrobacterium infiltration, and the post-infiltration growth period on the accumulation of GFP. The expression levels of GFP in Nicotiana leaves were then examined by Western blotting and ELISA.

RESULTSOur data indicated that a hydroponic Nicotiana cultivation system with a light intensity of 9000 LX/layer, a light cycle of 16 h day/8 h night, a temperature regime of 28 degrees celsius; day/21 degrees celsius; night, and a relative humidity of 80% could support the optimal plant growth and protein expression. After agroinfiltration with pBYGFPDsRed.R/LBA4404, high levels of GFP expression were observed in both N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants cultured with this hydroponic cultivation system. An optimal GFP expression was achieved in both Nicotiana species leaves 4 days after infiltration by Agrobacterium with an OD(600) of 0.8. At a given time point, the average biomass of N. tobaccum (cv. Yuyan No.5) was significantly higher than that of N. benthamiana. The leaves from 6-week-old N. benthamiana plants and 5-week-old N. tobaccum (cv. Yuyan No.5) plants could be the optimal material for agroinfiltration.

CONCLUSIONWe have established a hydroponic cultivation system that allows robust growth of N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants and the optimal GFP expression in the artificial climate box.

Gene Expression Regulation, Plant ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Hydroponics ; methods ; Plants, Genetically Modified ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Tobacco ; genetics ; growth & development

OBJECTIVETo study the effect of RNA interference (RNAi) on small heat shock protein (sHSP) Sjp40 of Schistosoma japonicum and its synergistic effect on the expression of SjHSP60, SjHSP70, and SjHSP90 mRNA, and observe the mRNA expression levels of Sjp40, SjHSP60, SjHSP70, and SjHSP90 in different stages of S.japonicum.

METHODSDouble-stranded RNA (dsRNA) of Sjp40 (dsSjp40) and a control dsRNA of green fluorescent protein (dsGFP) were generated by in vitro transcription and transfected into adult worm by immersing the worm in dsRNA solution. The total RNA and proteins were isolated simultaneously from the adult worms using TRIzol reagent 7 days after transfection. The expression levels of Sjp40, SjHSP60, SjHSP70, and SjHSP90 mRNA and the expression level of Sjp40 protein were determined by quantitative real-time PCR (qPCR) and Western blotting, respectively. The mRNA expression of HSPs of S. japonicum in different stages was evaluated by qPCR.

RESULTSCompared with those in the control worms transfected with dsGFP, Sjp40 mRNA level was decreased by 80% in the worms transfected with dsSjp40, and the level of Sjp40 protein showed also a significant decrease. The mRNA expression levels of SjHSP60, SjHSP70, and SjHSP90 did not show an obvious synergism after Sjp40 RNAi. The expression profiles of Sjp40, SjHSP60, SjHSP70, and SjHSP90 showed significant differences in different stages of S. japonicum, and the expression level of Sjp40 mRNA in the egg stage was much higher than that of other HSP genes.

CONCLUSIONdsSjp40-RNAi can induce effective suppression of Sjp40 gene expression at both mRNA and protein levels, but no obvious synergism occurs in the mRNA expressions of SjHSP60, SjHSP70, and SjHSP90.

Animals ; Gene Expression Profiling ; Heat-Shock Proteins, Small ; genetics ; Helminth Proteins ; genetics ; RNA Interference ; RNA, Double-Stranded ; RNA, Messenger ; genetics ; Schistosoma japonicum ; genetics

Objective:To explore relationship between serum 25-hydroxy vitamin D ( 25OHD ) level and ketosis-prone in patients with newly-diagnosed type 2 diabetes mellitus(T2DM).Methods:One hundred and thirty-four patients with newly diagnosed T2DM (103 males, 31 females) admitted in The Third Affiliated Hospital, Southern Medical University from January 2017 to January 2019 were enrolled into this study, including 36 patients with ketosis-prone (KPDM group) and 98 patients without ketosis(NKPDM group). Clinical characteristics, including height, weight, and history of hypertensive disease were collected. Serum 25OHD levels, lipid profile, islet function and glycosylated hemoglobin (HbA 1C)levels, ICA, IAA, GAD-Ab, etc., were measured. Results:Among the 134 patients, the patients with vitamin D deficiency, insufficiency, and sufficiency were 71 cases(52.99%), 52 cases(38.81%), and 11 cases(8.20%), respectively. KPDM group had significantly lower serum 25OHD level than NKPDM group[(44.12±9.77) nmol/L vs (55.35±15.31) nmol/L, P<0.01]. The rate of vitamin D deficiency was significantly higher in KPDM group compared to that in NKPDM group [(77.78% vs 43.88%), P<0.01]. The prevalence of KPDM varied significantly in different vitamin D status groups( P<0.01). Logistic regression analysis suggested that low serum 25OHD, younger age, high HbA 1C, and triglyceride were risk factors to ketosis-prone in newly diagnosed T2DM( P<0.01). Conclusion:Vitamin D deficiency is a common problem in newly diagnosed T2DM, especially in KPDM. Low serum 25OHD level seems to be an independent risk factor for ketosis-prone in patients with newly diagnosed T2DM.