Identification of key gene networks controlling vernalization development characteristics of Isatis indigotica by full-length transcriptomes and gene expression profiles.

Isatis indigotica Fort., as a common Chinese medicinal raw material, will lose its medicinal value if it blooms early, so it is highly valuable to clarify the induction mechanism of the vernalization of I. indigotica at low temperature. In this study, the concentrations of soluble sugar, proline, glutathione and zeatin in two germplasms of I. indigotica with different degrees of low temperature tolerance (Y1 and Y2) were determined at 10 days, 20 days and 30 days of low-temperature treatment, and the full-length transcriptome of 24 samples was sequenced by Nanopore sequencing with Oxford Nanopore Technologies (ONT). After that, the data of transcripts involved in the vernalization of I. indigotica at low temperature were obtained, and these transcripts were identified using weighted gene co-expression network analysis (WGCNA). The results revealed the massive accumulation of soluble sugar and proline in Y1 and Y2 after low temperature induction. A total of 18,385 new transcripts, 6168 transcription factors and 470 lncRNAs were obtained. Differential expression analysis showed that gibberellin, flavonoids, fatty acids and some processes related to low temperature response were significantly enriched. Eight key transcripts were identified by WGCNA, among which ONT.14640.1, ONT.9119.1, ONT.13080.2 and ONT.16007.1 encodes a flavonoid transporter, 9-cis-epoxycarotenoid dioxygenase 3 (NCED3), growth factor gene and L-aspartate oxidase in plants, respectively. It indicated that secondary metabolites such as hormones and flavonoids play an important role in the vernalization of I. indigotica. qRT-PCR proved the reliability of transcriptome results. These results provide important insights on the low-temperature vernalization of I. indigotica, and provide a research basis for analyzing the vernalization mechanism of I. indigotica. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01110-2.

DCK is an Unfavorable Prognostic Biomarker and Correlated With Immune Infiltrates in Liver Cancer.

BACKGROUND: The biological function of deoxycytidine kinase in tumor is not yet clear, and there are a few studies relating to the correlation of deoxycytidine kinase gene with the occurrence and development of liver cancer. METHODS: The messenger RNA expression of deoxycytidine kinase was analyzed with the use of the UALCAN and GEPIA database. Moreover, we assessed the function of deoxycytidine kinase on clinical prognosis with Kaplan-Meier plotter database. The relationship between deoxycytidine kinase and cancer immune infiltrates was investigated via Tumor Immune Estimation Resource site. Furthermore, Tumor Immune Estimation Resource was also used to evaluate the correlations between the expression of deoxycytidine kinase and gene marker sets of immune infiltrates. RESULTS: The deoxycytidine kinase messenger RNA level significantly upregulated in patients with liver cancer compared to normal liver samples. Moreover, the increased expression of deoxycytidine kinase messenger RNA was closely associated with reduced overall survival and disease-free survival in all liver cancers. In addition, deoxycytidine kinase expression displayed a strong correlation with infiltrating levels of macrophages, neutrophils, and dendritic cells in liver cancer, and deoxycytidine kinase expression was positively correlated with diverse immune marker sets in liver cancer. CONCLUSIONS: All the above findings suggested that increased expression of deoxycytidine kinase was significantly related to unfavorable prognosis in patients with liver cancer. And deoxycytidine kinase is correlated with immune infiltrating levels, including those of B cells, macrophages, neutrophils, and dendritic cells in patients with liver cancer. These findings suggest that deoxycytidine kinase can be used as a prognostic biomarker for determining prognosis and immune infiltration in liver cancer. And deoxycytidine kinase is a potential target for liver cancer therapy, and these preliminary findings require further study to determine whether deoxycytidine kinase-targeting reagents might be developed for clinical application in liver cancer.

[Study on Genetic Stablity of Cryopreservation Regeneration Plant of Gentiana straminea with ISSR-PCR].

OBJECTIVE: To establish ISSR-PCR system of cryopreservation regeneration plant of Gentiana straminea, and to select appropriate primers and analyze the genetic stability. METHODS: DNA was extracted by CTAB, the optimal ISSR-PCR system was established by orthogonal experiment,and genetic stability was analyzed. RESULTS: The optimal ISSR-PCR system (25 µL) was established: dNTPs 0.50 µL, Mg2+ 1.00 µL, 10 x PCR Buffer 2.00 µL, primer 0.60 µL, Taq DNA polymerase 1.25 µL, template DNA 1.30 µL, and ddH2O 18.35 µL. The amplification program was devised: 94 degrees C for 5 min, denaturing at 94 degrees C for 30 s, annealing of 1 min due to denaturing temperature of different primer,extension at 72 degrees C for 1.5 min, 35 cycles, last extension at 72 degrees C for 7 min, conservation at 4 degrees C . The DNA mutation rate of cryopreservation regeneration plant of Gentiana straminea was 1.05%. CONCLUSION: The cryopreservation regeneration plant of Gentiana straminea retains very good genetic stability, there is little variation between each plant, so the cryopreservation can be used as a feasible method for resource protection of Gentiana straminea.

[Study on rapid propagation of Scutellaria baicalensis variation].

OBJECTIVE: To establish and optimize the technology of taking root and promoting seedlings of white flower Scutellaria baicalensis test tube plantlet, and provide the theory and technology base for efficient factorization production system of white flower Scutellaria baicalensis. METHODS: Stem segments with axillary bud were cultured onto the different basic medium with different kinds and concentration of cytokinin and auxins to take root and produce strong seedling. RESULTS: The suitable culture medium for taking root of white flower Scutellaria baicalensis was 1/2 MS (all substance reduced half) + IBA 0.02 mg/L + sucrose 2%, the induction rate of root was 100%; The best medium for promoting seedling was 1/2 MS (all substance reduced half) + PP333 0.2 mg/L + IBA 0.02 mg/L + sucrose 2%, the seedling was green, the internode was normal, and its growth was vigorous and healthy. CONCLUSION: 1/2 MS (all substance reduced half) culture medium and relatively low concentration of sucrose is beneficial to inducing roots; Media adding appropriate concentration of IBA can significantly increase the root induction rate, the seedling has many stout roots; PP333 has dwarfing effect on seedlings and suitable concentration of PP333 can significantly improve the quality of the plantlets. A good technology of taking root and producing strong test tube plantlets is established.

[Comparison of content of total alkaloids and aconitine in Aconitum szechenyianum from different producing areas on basis of macroporous adsorption resin purification technology].

OBJECTIVE: To optimize the conditions of purifying the total alkaloids in Aconitum szechenyianum with macroporous adsorption resin, and compare the content of total alkaloids and aconitine in A. szechenyianum from different producing areas, in order to provide basis for further studies. METHOD: The orthogonal experiment method was adopted for optimizing the conditions of purifying the total alkaloids in A. szechenyianum with macroporous adsorption resin. The content of total alkaloids and aconitine were determined by using the titration method. The total alkaloids in A. szechenyianum from different producing areas were purified under optimum processing conditions. Aconitine was determined by HPLC. RESULT: Different processing conditions showed different influences on the purification of total alkaloids. The optimum conditions were resin type HPD-722, ethanol concentration of 80% , and ethanol elution volume of 80 mL x min(-1). The contents of aconitine in A. szechenyianum from different producing areas--Qinghai, Maxianshan, Ningxia and Yongdeng were 0.493 5, 0.883 5, 1.527 8, 1.664 4 mg x g(-1), respectively. CONCLUSION: The optimum processing conditions used in this essay could be used for purifying the total alkaloids and aconitine. A. szechenyianum from Yongdeng and Gansu contains the highest content of aconitine.

[Relationship between seedling grade of angelica and biomass accumulation and quality of product].

OBJECTIVE: To research the relationship between the seedling grade of angelica and the biomass accumulation, output and quality of product, so as to provide base for establishing seedling standard. METHOD: Thirty seedlings of Angelica were collected from the main production area in Gansu province, such as Minxian, Zahngxian, Dangchang and Weiyuan county, all the samples were measured with weigh of single seedling and the seedling were divided into three grade by the clustering analysis results, the grade were made to treatment to do field test and laboratory experiment. RESULT: The weigh of dry root, above ground and the whole plant in growth period of treatment 2 (the weigh of single seedling between 0.74-1.38 g) were all superior to other treatments and the ck. The treatment 2 had low bolting percentage, the weigh of single root was higher and the yield was the highest. The characters of product from the treatment 2 was well and the content of ferulic acid was higher than the standard of Chinese pharmacopoeia (2010 year part 1). CONCLUSION: The plant from the grade 2 seedling with larger growth increment, higher output and better quality, which can be the best seedling in production.

[Cryopreservation and plantlet regeneration of dormant buds of Gentiana straminea by vitrification].

OBJECTIVE: To investigate the detailed techniques for cryopreservation of Gentiana straminea dormant buds by vitrification. METHODS: Dormant buds as an experimental material,the influence of the different size of dormant buds,preculture and PVS etc. on cryopreservation of Gentiana straminea were studied. RESULTS: The optimal procedures were as follows: 10-11 mm long dormant buds which were cultured on MS medium supplemented with different sucrose concentration (0. 3,0. 5 or 0.7 mol/L) for 1 day respectively. The buds were immersed in loading solution for 20 min at 20 degrees C, and then treated in PVS2 solution for 40 min at 0 degrees C and finally plunged into liquid nitrogen quickly. After 24 hours, the buds were rapidly thawed in a water bath at 40 degrees C for 2 - 3 min and washed twice with MS medium supplemented with 1/2 MS liquid medium containing 1. 2 mol/L sucrose. Finally the buds were transferred to regeneration medium (MS + 0.5 mg/L 6-BA + 0.1 mg/L NAA + 3% sucrose + 0.7% agar), the survival rate was up to 83.3%. CONCLUSION: A high-efficiency cryopreservation protocol of Gentiana straminea is set up.

Proteomic analyses of Sirt1-mediated cisplatin resistance in OSCC cell line.

Oral squamous cell carcinoma (OSCC) accounts for about 90% of malignant oral lesions, and is recognized as the third most common cancer in developing nations and the sixth most common cancer worldwide. While chemotherapy remains the primary treatment for both resectable and advanced OSCC, most OSCC are naturally resistant to anticancer drugs, rendering new therapeutic avenues in dire need. Sirt1, a class III histone deacetylase, was linked to cisplatin resistance in several cancer types; however, the underlying mechanism is still unclear. Here, we demonstrated that overexpression of Sirt1 survived OSCC cell line Tca8113 under cisplatin treatment. Notably, BML-210, a chemical inhibitor of class III histone deacetylase, significantly abolished Sirt1-mediated cisplatin resistance in Tca8113 cells. Further, inactivation of endogenic Sirt1 by nicotinamide markedly increased chemo-sensitivity in cisplatin resistant sub-cell line Tca8113/CDDP. Proteomic strategy was applied to profile the differentially expressed proteins between pcDNA3.1-Sirt1- and mock vector-treated Tca8113 cells. Among 54 spots identified, 31 proteins were up-regulated and 23 proteins were down-regulated upon Sirt1 expression. Expression of four proteins with most significant alteration, including Annexin A4, Stathmin, SOD2 and thioredoxin, were validated by both RT-PCR and Western blot. Finally, we showed that Sirt1 could prevent cisplatin-induced ROS accumulation in Tca8113 cells. Our findings are considered as a significant step toward a better understanding of Sirt1-mediated cisplatin resistance.