不同品种木薯叶绿体比较蛋白质组学初步研究

以“华南8号”木薯(SC8)和“华南124号”木薯(SC124)的叶绿体作为研究材料,采用改进酚抽提法提取蛋白,通过单向SDS-PAGE电泳和双向SDS-PAGE电泳,比较不同木薯品种叶绿体的蛋白表达谱,并对表达的差异蛋白进行MALDI-TOF MS质谱鉴定,获得15个差异蛋白,其中有6个蛋白在SC124木薯叶绿体中表达较高,9个蛋白表达很低.对蛋白进行功能分析,发现差异蛋白主要参与蛋白翻译后修饰、周转、分子伴侣、碳水化合物运输等过程.通过RT-PCR验证了木薯核酮糖1.5-二磷酸羧化酶、ATP合酶β亚基的基因表达情况,结果表明,ATP合酶β亚基基因表达与蛋白质的表达比较一致,而核酮糖1,5-二磷酸羧化酶基因与蛋白质表达变化不一致.     

GAPB基因原核表达载体的构建

以拟南芥cDNA为模板,用PCR扩增出GAPB的基因全长,然后将GAPB基因片段连接到PET28a（＋）载体上,构建重组质粒并转化大肠杆菌DH5α,经菌落PCR和酶切鉴定筛选出阳性克隆,测序正确后,再将阳性克隆的质粒转化大肠杆菌BL21（DE3）。结果表明：成功构建了原核表达载体PET28a（＋）-GAPB,为后续的G...     

Functional analysis of the venom allergen-like protein gene from pine wood nematode Bursaphelenchus xylophilus using a baculovirus expression system

The pine wood nematode, Bursaphelenchus xylophilus, infects pine trees, leading to fatal pine wilt disease. Here, recombinant venom allergen-like protein (VAP) was obtained by expressing Bx-vap-1 in insect cells. Three-year-old Pinus massoniana were inoculated with recombinant VAP, simulating B. xylophilus esophageal gland secretions. Recombinant VAP up-regulated α-pinene synthase gene expression, the trees showed disease symptoms 15 d after inoculation and the xylem pith revealed brown tissue discoloration, indicating that recombinant VAP could damage P. massoniana cells. Recombinant VAP did not, however, lead to cavitation, indicating that the VAP secreted from B. xylophilus acts as a defense response elicitor.     

西农莎能奶山羊遗传检测的研究

通过对西农莎能奶山羊九个血液蛋白位点和四项外形特征的遗传抽样检测，结果表明：西农莎能奶山羊在七个血液蛋白位点和四项外形特征上具有多态性，并存在有原种瑞士莎能奶山羊不具有的Ｈｂ￣Ｂ和Ｔｆ￣Ｂ基因，以及其他莎能羊种中不具有的Ａｃｐ￣Ａ和Ｃａｔ￣Ｂ基因。     

犬瘟热病毒F蛋白单克隆抗体的制备及鉴定

[目的]获得犬瘟热病毒F蛋白特异性单克隆抗体。[方法]用纯化的大肠杆菌BL21表达的犬瘟热病毒重组F蛋白作为抗原,免疫BALB/c小鼠,采用传统的杂交瘤技术制备单克隆抗体。用ELISAI、FA、Western-blot、细胞中和试验鉴定各株单抗的生物学特性。[结果]获得7株稳定分泌犬瘟热F蛋白单克隆抗体的杂交瘤细胞株(2D6,3E10,3A2,5B7,5F6,6H8,9B6);单抗亚类鉴定结果分别为IgG2a,IgG1,IgG2b,IgG1,IgG1,IgG2a和IgG1;细胞中和试验初步证实2株单抗(5B7,9B6)具有中和犬瘟热病毒感染Vero细胞的作用,中和效价分别为1∶320和1∶640;经ELISA相加试验证实7株单抗的作用位点不同,其中2D6,5F6的作用位点非常相近。[结论]该研究成功制备了7株抗犬瘟热F蛋白特异性单抗,为进一步研究犬瘟热病毒F蛋白和临床诊断打下良好的基础。     

Seasonal critical concentration and relationships of leaf phosphorus and potassium status with biomass and yield traits of soybean

Analysis of uppermost fully expanded leaves is useful to detect a deficiency of mineral nutrients such as phosphorus (P) and potassium (K) in soybean. Although, the leaf P or K status aids in fertilizer management, information on nutrient seasonal relationships with growth and yield traits at maturity are limited. To investigate this, soybean was grown under varying P or K nutrition under ambient and elevated CO₂ concentrations. Results show significant relationships of the relative total biomass and yield‐related traits with the foliar P and K concentrations measured several times in the season across CO₂ levels. However, the relationships established earlier in the season showed that the growth period between 25 and 37 d after planting (DAP), representing the beginning of flowering and pod, respectively, is the best for leaf sampling to determine the foliar P or K status. The leaf P and K status as well as the critical leaf P (CLPC) and K (CLKC) concentrations for traits such as seed yield peaked around 30 DAP (R2 stage) and tended to decline thereafter with the plant age. The CLPC and CLKC of seed yield indicate that the leaf P and K concentration of at least 2.74 mg g^?1 and 19.06 mg g^?1, respectively, in the uppermost fully expanded leaves are needed between 25 and 37 DAP for near‐optimum soybean yield. Moreover, the greatest impact of P and K deficiency occurred for the traits that contribute the most to the soybean yield (e.g., relative total biomass, seed yield, pod and seed numbers), while traits such as seed number per pod, seed size, and shelling percentages were the least affected and showed smaller leaf critical concentration. The CLPC or CLKC for biomass and seed yield was greater under elevated CO₂ 24–25 DAP but varied thereafter. These results are useful to researchers and farmers to understand the dynamics of the relationship of pre‐harvest leaf P and K status with soybean productivity at maturity, and in the determination of suitable growth stage to collect leaf samples.     

桔小实蝇14-3-3基因的克隆和表达谱分析

【目的】克隆桔小实蝇14-3-3蛋白的基因（Bactrocera dorsalis 14-3-3,Bdor 14-3-3）,研究Bdor 14-3-3 mRNA在不同组织和不同发育时期的表达情况,探索其是否参与了桔小实蝇的发育过程。【方法】采用反转录聚合酶链式反应(RT-PCR)和快速扩增cDNA末端(RACE)技术克隆Bdor 14-3-3;采用实时荧光定量PCR（real-time PCR）方法,研究Bdor 14-3-3 mRNA在桔小实蝇不同组织及不同发育时期的相对表达量。【结果】克隆获得了Bdor 14-3-3基因,其编码区长度为747 bp,编码248个氨基酸残基。氨基酸序列一致性分析表明,在昆虫纲中,桔小实蝇与刺舌蝇14-3-3蛋白的序列一致性最高,为98.8%,与豌豆蚜的序列一致性最低,为85.4%。实时荧光定量PCR分析表明,Bdor 14-3-3 mRNA在桔小实蝇不同组织和发育时期都有表达;在雌虫头（去除触角）和翅中的相对表达量均较高,分别是触角的1.39和1.44倍;在雄虫胸和后足中的相对表达量均较高,分别是前足的1.28 和1.23倍。在蛹的早期发育过程中Bdor 14-3-3 mRNA表达量逐渐升高,到7 d蛹期相对表达量达到最高峰,是10 d蛹的4.91倍。【结论】克隆获得了Bdor 14-3-3基因,其表达的14-3-3蛋白可能参与了桔小实蝇的变态发育过程,尤其在蛹的发育过程中Bdor 14-3-3可能发挥着重要作用。     

西农莎能奶山羊乳蛋白遗传学特性的研究

利用聚丙烯酰胺凝胶电泳和统计模型方法对２２５只西农莎能奶山羊乳中主要蛋白质的特性进行了系统的遗传分析。结果表明，西农莎能奶山羊酪蛋白基因位点间具有显著的连锁关系；β－乳球蛋白（β－ＬＧ）和Ｋ－酪蛋白（Ｋ－ＣＮ）基因位点分别对３０５ｄ产奶量、乳蛋白率和于物质率有显著影响，其中β－ＬＧＢＢ型和Ｋ－ＣＮＡＡ型个体表现出较好的泌乳性能。     

Effects of CENP-W down-regulation on human glioma U87 cells

AIM: To study the effect of centromere protein W (CENP-W) down-regulation on human glioma U87 cells.METHODS: Small interfering RNA (siRNA) was used to inhibit the expression of CENP-W in the U87 cells. The interference effect of siRNA was evaluated by RT-qPCR and Western blot. The proliferation of the cells was analyzed by MTT assay, BrdU staining and colony formation experiment. Transwell chamber assay was used to detect the invasion ability of the cells. The cell migration ability was measured by a scratch test. The changes of the cell cycle distribution and apoptosis were analyzed by flow cytometry.RESULTS: The results of MTT assay, colony formation experiment and BrdU staining showed that the cell proliferation and colony formation abilities in experimental group were significantly lower than those in control group and negative control group. The results of Transwell and scratch experiments showed that the migration and invasion abilities in experimental group were weaker than those in blank control group and negative control group. The results of flow cytometry analysis showed that the cell cycle distribution in experimental group was arrested in G₀/G₁ phase. The percentage of apoptotic cells in experimental group was higher than that in control group (P<0.05).CONCLUSION: Down-regulation of CENP-W expression inhibits the proliferation, migration and invasion of human glioma cells and promotes the apoptosis of the cells, suggesting that CENP-W may be a potential target of gene therapy for human glioma.     

四个家鸡群体血液蛋白质多态性分析

本实验采用醋酸纤维薄膜电泳和聚丙烯酰胺凝胶电泳方法测定了新兴黄鸡，矮脚鸡，竹丝鸡，粤黄鸡麻羽系血液蛋白质多态性，观察了以上群体多态性血液蛋白质基因频率的区别，根据基因频率计算出４个群体的平均杂合度，用主分量分析方法对它们之间的区别与联系进行了研究。主要分析表明竹丝鸡与新兴黄鸡的关系相对新兴鸡与矮脚鸡，竹丝鸡与矮脚鸡之间的关系更为疏远：矮脚鸡则与粤黄鸡麻羽系关系密切。