Real-time Quantitative PCR Analysis of Vascular Endothelial Growth Factor Receptor-2(VEGFR-2) Expression at Zebrafish Different Developmental Stages

[Objective]To investigate the expression of zebrafish vascular endothelial growth factor-2（VEGFR-2） at different developmental stages.[Method]Total RNAs were extracted from 12,24,48,72 and 96 hpf stage zebrafish embryos and larvae.Real-time quantitative RT-PCR was performed to examine the expression of VEGFR-2.The data were analyzed by 2^-△△Ct method.[Result]The expression level of VEGFR-2 gene increased gradually from 12 to 72 hpf,and subsequently decreased at 96 hpf.The expression level was lowest at 12 hpf,highest at 72 hpf,and had significant differences when compared with that of other developmental stages.[Conclusion]The expression level of VEGFR-2 increases gradually before blood vessel maturation and decreases as blood vessels mature.

Analysis of Seed-specificity of Silencing fad_2 Gene Expression in Transgenic Rapeseed Line W-4(Brassica napus L.)

This study was to investigate the efficiency and specificity of RNAi silencing on the expression of endogenous fad2 gene in transgenic line W-4. [Method] The relative expression of fad2 gene in seeds at different developmental stages of 7th, 14th, 21st and 28th day after flowering （DAF） as wel as the root, stem, leaf at winter seedling stages of both the transgenic line W-4 and non-transgenic control Westar by real-time fluorescence quantitative PCR. [Results] The results showed the relative expression of fad2 gene was gradual y increasing with the days after flowering in the seeds of the control Westar, while it was found decreasing significantly since the 21st DAF in the seeds of the line W-4. The decline was up to 60% in comparison with the control Westar. However, no significant difference in the relative expression of fad2 gene in other organs like root, stem and leaf was observed between transgenic line W-4 and non-transgenic control Westar. Fatty acid composition analysis showed the oleic acid desaturation parameter（ODP） in seeds of the line W-4 was 0.07 in average, decreased by nearly 75% than control Westar which was 0.24 in average, while no significant difference in the seedling root, stem and leaf was measured between transgenic rapeseed and control. [Conclusion] The results above validated that RNA interference in transgenic rapeseed W-4 is at a seed-specific manner, not interfering with fad2 gene expression in organs such as the root, stem and leaf. The study also found that the period of fad2 gene expres-sion decline was wel coincided with the expression of napin gene, both appeared at the 21st DAF, indicating that the expression of dsRNA of fad2 gene is precisely control ed by the napin promoter.

Changes of the Transcriptional Levels of Molecules Associated with Endogenous Antigen Processing and Presentation in Porcine Skin-derived Dendritic Cells Infected with PCV2 in vivo

[Objective] This study aimed to investigate the changes of the transcriptional levels of molecules associated with endogenous antigen processing and presenta- tion in porcine skin-derived dendritic cells infected with PCV2 in vivo. [Method] Healthy 40-day-old Landrace piglets were infected with porcine circovirus type 2 （PCV2） and euthanized on the 34, 7rd, 14th, 21st and 35th d post inoculation （DPI）. The porcine skin-derived dendritic cells （DCs） were collected to analyze the transcrip- tional levels of molecules （LMP7, UBP, MHC-I, calreticulin） associated with endogenous antigen processing and presentation by using real-time fluorescent quantitative PCR （real-time FQ-PCR）. [Result] The results showed that the level of LMP7 mR- NAs was reduced significantly on the 3DPI （P〈0.05）; the level of UBP mRNAs was consistently up-regulated, which increased significantly on the 21DPI and 35DPI （P〈 0.05）; the level of MHC-I mRNAs was significantly down-regulated on the 7DPI （P〈 0.05）; the level of calreticulin mRNAs was up-regulated slightly without significant dif- ference. [Conclusion] PCV2 can inhibit the endogenous antigen processing and presentation ability of porcine skin-derived DCs at early stages of infection.

A new approach to promote astaxanthin accumulation via Na_2WO_4 in Haematococcus pluvialis

The optimum concentration of Na_2 WO_4 was explored in relation to the cell density and astaxanthin content in Haematococcus pluvialis. Then, the cellular morphology, nitrate reductase(NR) activity, soluble sugar and protein contents, and chlorophyll ?uorescence were measured, and the transcriptional expression of carotenogenic genes was determined by quantitative real-time PCR. The results showed that 3.0 mmol/L of Na_2 WO_4 was the optimum concentration to induce astaxanthin accumulation, with a maximum content of 49.41±0.13 pg/cell reached on the tenth day. The NR activity decreased signi?cantly and continually after Na_2 WO_4 treatment. The soluble sugar content increased gradually during the experimental period and was eventually signi?cantly higher than that in the control. The soluble protein content increased rapidly,reached a maximum in day 0.5 and day 1 and then decreased. The ef fective photochemical effciency of PSII( F v'/F m') and light saturation( E k) ?rst decreased and then tended to stabilize, and NADP +-glyceraldehyde-3-phosphate dehydrogenase(GAPDH) gene expression was correlated with photosynthesis. The transcriptional expression of ipi, psy and bkt was signi?cantly increased compared with that in the control after application of Na_2 WO_4, and the relative expression of ipi reached the highest level on the ?fth day, with a 98.03±1.92-fold increase. Our results describe a new approach to promote the ef fective accumulation of astaxanthin in H. pluvialis by NR inhibitor Na_2 WO_4.

The Expression and Clinical Significance of ABCG2, Oct4 and Nanog in Laryngeal Carcinoma

Objective: To investigate the protein expression and clinicopathological characteristics of ABCG2, Oct4, Nanog in laryngeal cancer tissues, and to seek new molecular markers for the diagnosis of laryngeal cancer. Methods: The laryngeal cancer tissues and paracancerous tissues of 87 patients with laryngeal carcinoma diagnosed in the department of otorhinolaryngology and head and neck surgery in our hospital from April 2016 to April 2018 were selected as the subjects. QRT-PCR, Real-time PcR, Western blot and immunohistochemical staining were used to detect the expression of ABCG2, Oct4 and Nanog in (Tumor Tissue) and (Adjacent Tissue) in tumor tissue and paracancerous tissue. Results: The results of RT-PCR showed that the positive rates of ABCG2, Oct4 and Nanog in laryngeal carcinoma tissues were 49.30%, 45.07% and 52.11%, respectively, while those in paracancerous tissues were 22.54%, 21.13% and 15.49%, respectively (P < 0.01). The expression of ABCG2, Oct4 and Nanog in laryngeal carcinoma was correlated with tumor differentiation, depth of invasion, age and sex (P < 0.05), but not with tumor size and TNM stage. Conclusion: The expressions of ABCG2, Oct4 and Nanog in cancer tissues are related to tumor differentiation status, and they can be used as new molecular markers for the diagnosis of laryngeal cancer.

Cryptochromes mRNA Expression Under Mating and Black light Treatment on Helicoverpa armigera

[ Objective ] The paper was to confirm the effect of mating and black light treatment on cryptochromes mRNA expression of Hclicoverpa armigera. [ Method ] Quantitative real-time PCR （ SYBR Green） technique was applied to detect the expression of cryptochromes gene （ cryl and cry2 ） of H. armigera under different conditions. Total RNA was extracted from the head of H. armigera, and carried out reverse transcription to synthesize cDNA after digested by DNase I. Specific primers were used to carry out quantitative real-time PCR on cryl, cry2 and EF-Ict gene, respectively. [ Result] The expression of cryl mRNA of H. ar- migera significantly decreased after exposure to black light for 2 h, the mRNA expression of cry2 was smaller than control, they had no significant difference with each other. Mating had significant effect on mRNA expression of cryl and cry2 of H. armigera, and the mRNA expression of cryl and cry2 of male and female adults showed decreasing trend with the prolongation of time after mating. [ Conclusion] The result had important significance for further studying on function of cry gene and the control of cotton bollwonn.

Differential gene expression profiling of gastric intraepithelial neoplasia and early-stage adenocarcinoma

AIM: To investigate the differentiated whole genome expression profiling of gastric high- and low-grade intraepithelial neoplasia and early-stage adenocarcinoma.

Comparative proteomics analysis of maize(Zea mays) leaves infected by small brown planthopper(Laodelphax striatellus)

Maize rough dwarf disease(MRDD) is a viral disease caused by brown planthopper infestation, and leads to great yield loss, especially in China. Comparative proteomics was performed using maize inbred line Zheng 58 and LN 287. MRDD pathogen was detected as rice black-streaked dwarf virus(RBSDV) by quantitative real time PCR(q RT-PCR) in Shandong Province, China. The modified trichloroacetic acid(TCA)/acetone method was used for soluble protein extraction from leaves. Two-dimensional electrophoresis(2-DE) analysis was performed on 24-cm long, p H 4-7 linear immobilized p H gradient(IPG) strips, and gels were stained with silver and coomassie brilliant blue. We identified 944 proteins expressed in RBSDV infected maize leaves by proteomics approaches. Among these, 44 protein spots that revealed a 1.5-fold difference in intensity were identified by mass spectrometry between mock-inoculated and RBSDV infected samples. Among these, 17 and 26 spots were up-regulated, and 27 and 18 spots were down-regulated in the virus infected samples of Zheng 58 and LN 287, respectively. Differential protein spots were analyzed by mass spectrometry identification, which could be divided into six categories. Furthermore, the expression of stress-related proteins was detected and confirmed by q RT-PCR. This study lays the foundation for further investigations, enabling the enhancement of MRDD resistance in maize.

Proteomic analysis of differentially expressed proteins between Xiangyou 15 variety and the mutant M15

A high oleic acid rapeseed material MI5 （derived from Xiangyou 15 variety） has been received more attention for its significant effect for human health. And it has almost the same physiological characteristic with Xiangyou 15 variety. To find out the difference between high oleic acid rapeseed material and Xiangyou 15 seedling, a comparative proteomic approach based on 2-DE and mass spectrometry was adopted. A total of 277 protein spots showed a significant change in intensity by more than 2.0-fold from M15 compared with Xiangyou 15 variety. Among them, 48 spots that changed at least 3.0-fold were excised from gels and successfully identified by MALDI-TOF/TOF MS. The identified proteins involved in metabolism of carbohydrate and energy （75%）, stress and defense （8.3%）, photosynthesis （6.3%）, protein metabolism （2.1%） and other functions （8.3%）. Then real-time quantitative PCR （qPCR） analysis was used to verify the expression levels of differentially expressed proteins, but the results did well agree with the proteomic results. In this work, most of the proteins involved in metabolism of carbohydrate and energy have higher expression in M15, which may reveal M15 has higher metabolism ability. These results provided much information to understand the differences between high oleic acid rapeseed material and Xiangyou 15 variety, which will be useful to screen high oleic rapeseed materials in seedling period.