野生大豆育成品种与其亲本间的遗传多样性比较

[目的]鉴定大豆育成品种与其亲本间的遗传多样性差异和遗传多态性信息含量,为大豆育种提供参考。[方法]采用SSR标记技术对大豆育成品种与其亲本间的遗传多样性进行分析。[结果]野生大豆亲本总等位变异数和特有等位变异数分别是栽培大豆亲本的1.31倍和3.63倍;平均多态性信息含量由大到小依次为野生大豆亲本(0.545 0)、育成大豆品种(0.478 7)、栽培大豆亲本(0.415 6)。[结论]野生大豆的遗传背景复杂,遗传多样性丰富,其外部形态和内在遗传基础都与栽培大豆有明显差异。     

Effect of CHOP expression knock-down on apoptosis of renal tubular epithelial cells

AIM:To study the effect of C/EBP homologous protein (CHOP) on the apoptosis of renal tubular epithelial HK2 cells. METHODS:The serum mRNA levels of CHOP in the patients with acute kidney injury and healthy controls were detected by qPCR. In vitro, renal tubular epithelial HK2 cells were divided into control group, negative group (transfected with negative control siRNA), si-CHOP group (transfected with CHOP siRNA), and induced by transforming growth factor-β1 (TGF-β1). The viability of the cells was measured by MTT assay, and the apoptotic rate was analyzed by flow cytometry. The protein levels of nuclear antigen Ki-67, proliferating cell nuclear antigen (PCNA), caspase-3 and cleaved caspase-3 were determined by Western blot. RESULTS:Compared with the healthy controls, the serum mRNA levels of CHOP in the patients with acute kidney injury were increased significantly (P<0.05). Transfection with CHOP siRNA significantly decreased the expression of CHOP in the renal tubular epithelial HK2 cells (P<0.05). Knock-down of CHOP expression by siRNA significantly increased the viability of renal tubular epithelial HK2 cells (P<0.05), decreased the apoptotic rate (P<0.05), increased the expression of Ki-67 and PCNA (P<0.05), and down-regulated the protein level of cleaved caspase-3 (P<0.05). CONCLUSION:The serum mRNA levels of CHOP were increased in the patients with acute kidney injury. Knock-down of CHOP expression inhibits the apoptosis of renal tubular epithelial cells by regulating the expression of proliferation-and apoptosis-related proteins.     

F-box domain is involved in regulating the proliferation of MCF-7 cells by FBXO39 protein

AIM: To investigate the effect of F-box domain on the regulation of MCF-7 cell proliferation by FBXO39 protein. METHODS: The effect of F-box domain on the localization of FBXO39 protein in the MCF-7 cells was investigated. MCF-7 cell cDNA library was used as the template resource. The full-length cDNA sequence of FBXO39 was amplified by PCR method and subcloned into eukaryotic expression vector pEGFP-C2. The pEGFP-FBXO39ΔF (F-box domain deletion mutation) plasmid was successfully constructed with the template resource of pEGFP-FBXO39 plasmid. The recombinant plasmids were transfected into the MCF-7 cells, and then the expression of FBXO39 and FBXO39ΔF were determined by Western blot. The cellular localization of FBXO39 and FBXO39ΔF were observed by confocal microscopy. The localization of endogenous FBXO39 in the MCF-7 cells was detected by immunofluorescence staining. In addition, MTT and EdU assays were used to measure the cell proliferation, flow cytometry was used to measure the cell cycle distribution, and immunohistochemical staining was used to observe the expression of FBXO39 in the breast cancer and para-carcinoma tissues. RESULTS: The eukaryotic expression vector pEGFP-FBXO39 and pEGFP-FBXO39ΔF were constructed successfully. F-box domain had no effect on the cell localization of FBXO39. FBXO39 promoted MCF-7 cell proliferation but FBXO39ΔF did not. FBXO39 was highly expressed in the breast cancer tissues. CONCLUSION: F-box domain had no effect on the cellular localization of FBXO39 protein. However, it plays an important role in the biological function of FBXO39. FBXO39 may be related to breast cancer tumorigenesis.     

大豆GmNRT1.2a和GmNRT1.2b基因的克隆及功能探究

拟南芥中硝酸盐的吸收、转运和分配是通过硝酸盐转运蛋白(nitrate transporter, NRT)实现的。尽管之前的生物信息学分析推测大豆GmNRT1.2s可能参与共生固氮过程,但尚未开展相应的功能研究。本研究通过对其表达模式分析収现,GmNRT1.2a和GmNRT1.2b分别在根和叶中高表达,且受硝酸盐诱导,在接种根瘤菌与结瘤因子(nodfactors,NFs)后表达量明显升高。功能研究结果显示,过表达GmNRT1.2a或GmNRT1.2b后大豆根瘤数目显著增加。本研究为深入探究GmNRT1.2a和GmNRT1.2b调控大豆共生固氮过程的分子机制提供了一定的数据支持。     

SPI凝胶颗粒制备及其Pickering高内相乳液特性研究

制备了大豆分离蛋白（Soy protein isolate，SPI）凝胶颗粒，并使用该颗粒制备了稳定的Pickering高内相乳液。通过粒径和ζ 电位测定、冷冻扫描电镜和光学显微镜观察以及外观分析，以流变特性为指标，对SPI凝胶颗粒和Pickering高内相乳液特性进行研究。结果表明，SPI凝胶颗粒的粒径和ζ 电位随pH值的变化而变化，当SPI凝胶颗粒pH值为4.0～5.0时，临近SPI等电点，ζ 电位的绝对值最小，此时凝胶颗粒相互聚集，不能成功制备稳定的高内相乳液。在pH值为 9.0时，大豆分离蛋白凝胶颗粒紧密结合在一起，呈凝胶网络状结构。在碱性条件下，蛋白质质量分数为1.00%、内相体积分数为78%～82%时，可以制备稳定的Pickering高内相乳液。通过增加内相体积分数，使大豆分离蛋白凝胶颗粒稳定的Pickering乳液体系分布更加均匀，不易发生聚集，形成更加致密稳定的多孔结构，且具有更强的弹性凝胶乳液特性。     

大鼠成熟的肺表面活性蛋白B的原核表达及纯化

为表达和纯化SP-B蛋白,先对SP-B基因进行稀有密码子优化,PCR反应获得SP-B片段,构建重组质粒pGEX4T-1/SP-B,转化到E.coli BL21(DE3)中诱导表达;用SDS-PAGE与Western blot进行检测;用GSTPrep FF 16/10对融合蛋白进行纯化。经双酶切鉴定证实质粒中插入基因长250bp,测序结果与大鼠SP-B cDNA序列相符;质粒转化后用IPTG进行诱导,在分子质量约34ku处出现1个新条带,与pGEX4T-1/SP-B蛋白预期大小一致,且该融合蛋白能可溶性表达并被纯化。结果表明成功构建了pGEX4T-1/SP-B重组质粒,表达、纯化得到GST/SP-B蛋白,为研究肺表面活性物质替代药物奠定了基础。     

高压电场干燥和热风干燥对马铃薯蛋白质二级结构的影响

以自然风干为对照组,采用SDS-PAGE凝胶电泳和圆二色光谱法研究了高压电场干燥(HVEF)和热风干燥对马铃薯蛋白质分子量的影响以及两种干燥方法对马铃薯蛋白质二级结构的影响。SDS-PAGE凝胶电泳结果显示:两种干燥方法对马铃薯蛋白质分子量基本不产生影响。圆二色光谱检测结果表明:经高压电场干燥后,马铃薯蛋白质的α-螺旋所占比例下降1.30%,β-折叠所占比例上升1.60%,β-转角所占比例上升0.30%,无规卷曲所占比例上升1.20%;经热风干燥后,马铃薯蛋白质的α-螺旋所占比例上升了12.50%,β-折叠所占比例下降了10.70%,β-转角所占比例上升了4.30%,无规卷曲所占比例下降4.40%。通过比较:HVEF对马铃薯蛋白质二级结构中α-螺旋、β-折叠、无规卷曲的作用趋势与热风干燥正好相反;如,热风干燥使α-螺旋的比例显著上升,而HVEF则使α-螺旋的比例下降,并且HVEF对马铃薯蛋白质二级结构的影响比热风干燥对马铃薯蛋白质二级结构的影响较小。     

拟南芥乙烯应答基因HLS1的原核表达及多克隆抗体制备

【目的】制备乙烯应答基因HLS1多克隆抗体,在过表达植物中检测HLS1的积累,为深入研究HLS1响应乙烯信号通路的分子机制奠定基础。【方法】构建pET28a-HLS1c原核表达载体,转化到大肠杆菌BL21(DE3)中,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导获得重组蛋白HLS1c-His;镍柱亲和纯化重组蛋白后免疫家兔获得抗HLS1血清,利用抗原亲和纯化抗血清获得高纯度的HLS1多克隆抗体;用HLS1抗体和GFP抗体,检测原生质体瞬时转化体系中GFP-HLS1c融合蛋白的表达;用农杆菌蘸花法获得过表达GFP-HLS1g的转基因植物,用HLS1抗体和GFP抗体检测GFP-HLS1g融合蛋白的表达。【结果】成功构建了原核表达载体pET28a-HLS1c,并在大肠杆菌系统中诱导获得重组蛋白HLS1c-His,且多以包涵体形式存在。纯化的HLS1c-His多克隆抗体,能清晰检测到约80 ng原核表达的HLS1抗原。纯化的HLS1抗体不仅能检测到原生质体中瞬时表达的GFP-HLS1c融合蛋白,也能检测到过表达转基因植物株系中的GFP-HLS1g融合蛋白,并且与标签蛋白GFP对应抗体所检测到的特异性条带大小一致。【结论】成功制备了拟南芥HLS1蛋白多克隆抗体,为HLS1蛋白在植物发育中的功能研究奠定了基础。     

Correlation between Mnk2 protein expression and prognosis of patients with esophageal squamous cell carcinoma

AIM: To investigate the protein expression of mitogen-activated protein kinase-interacting kinase-2 (Mnk2) and its prognostic effect in the patients with resected esophageal squamous cell carcinoma (ESCC). METHODS: A total of 86 informative patients with surgically resected ESCC and 54 normal esophageal tissues were enrolled. Western blot and immunohistochemistry (IHC) were utilized to assess the protein expression of Mnk2, and its correlation with prognosis was statistically analyzed by the methods of Kaplan-Meier curve and Cox proportional hazard mode. RESULTS: The protein expression of Mnk2 was elevated in most of tumor tissues compared with the adjacent tissues. Clinicopathologic analysis showed that Mnk2 expression was significantly correlated with the TNM stage (P<0.05). Both disease-free survival (DFS) and overall survival (OS) of Mnk2 over-expression patients were shorter than those in Mnk2 negative expression group. Multivariate analysis confirmed that Mnk2 expression, as an independent and significant factor for both DFS and OS, predicted a poor prognosis of the patients with resected ESCC (P<0.05). CONCLUSION: The expression of Mnk2 was significantly related to the TNM stages, and might be a novel predictor for prognosis in ESCC.     

酸模叶蓼对大豆生长的影响及其经济阈值

酸模叶蓼是大豆田的恶性杂草,严重危害大豆生长发育及产量,为明确酸模叶蓼在大豆田中的经济危害允许水平和经济阈值,在大田条件下采用添加系列试验和拟合函数关系模型的方法,研究了酸模叶蓼与大豆的竞争关系。结果表明,在酸模叶蓼的竞争干扰下,大豆单株荚数,产量均随酸模叶蓼密度的增加而逐渐降低,而株高没有显著变化。线性函数模型y=-0.443x+39.453(R2=0.962,F=75.887,P=0.003)能较好地拟合酸模叶蓼对大豆单株荚数的影响;二次曲线函数模型y=0.702x~2-54.395x+3027.810(R~2=0.972,F=34.843,P=0.028)能较好地拟合大豆产量与酸模叶蓼密度之间的关系,对数函数y=16.131lnx-23.458(R2=0.911,F=20.532,P=0.045)能较好的拟合大豆产量损失与酸模叶蓼密度之间的关系。大豆田酸模叶蓼人工拔除的经济阈值为10.22株/m2,使用75%噻吩磺隆水分散粒剂、80%阔草清水分散粒剂、95%精异丙甲草胺乳油防除的经济阈值分别为4.75、5.03、5.25株/m~2。酸模叶蓼对大豆产量有明显影响,通过对经济阈值分析,在大豆田化学除草剂防治酸模叶蓼具有明显的经济优势。