转基因小麦Puroindoline蛋白SDS-PAGE 方法的研究与应用

小麦嘌呤吲哚蛋白(Puroindoline)是优质遗传特性硬度的生化标记,对小麦磨粉品质起决定性作用.对目前分析此类蛋白的两类Triton-X-114和Tricine-SDS-PAGE系统进行了优化和比较,建立了详细的适合半子粒分离Pin蛋白的电泳方法.采用此法检测转pinA基因的硬粒小麦P34的后代以及转基因硬粒小麦之间杂交子代的外源基因pinA的表达情况,取得了良好的效果.该方法为Puroindoline蛋白的进一步研究奠定了基础,为小麦品质生化和分子标记辅助育种提供了方法.     

苹果砧木M26试管苗不定根发育过程中蛋白质的变化

为进一步了解木本植物不定根发生、发育的机理,对苹果砧木M26试管苗不定根的发生、发育过程进行解剖观察,利用十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)对M26试管苗在接入生根培养基前后,不同时期表达的蛋白进行了研究。结果发现:在不定根原基形成和发育的阶段性过程中,茎基部蛋白质的种类和数量出现明显差异。生根培养后,在继代苗茎基部表达的5条蛋白条带减弱或消失,其分子量分别为26.8 kDa、37kDa、40.3 kDa、43 kDa和66 kDa;明显表达了4条新的蛋白条带,其分子量分别为26 kDa、27.7 kDa、32.5 kDa、和45 kDa,同时有4条蛋白条带(38.5 kDa、42 kDa、53.2 kDa和55.5 kDa)其表达丰度提高。多种蛋白条带的消失和出现,可作为M26试管苗不定根产生的生化标志。在不定根原基逐步形成发育的同时,有3条蛋白条带的表达丰度也逐步提高,可作为不定根形成过程表达的特异标记蛋白带。     

编码杜果丝氨酸/苏氨酸蛋白激酶类蛋白抗病基因同源序列的克隆与分析

根据丝氨酸／苏氨酸蛋白激酶类催化结构域Ⅰ和Ⅸ的氨基酸保守序列设计简并引物,PCR扩增‘紫花芒’杧果嫩绿色叶片基因组DNA。在NCBI中用Blast X比对发现,有6个阳性克隆是抗病基因同源序列,并命名为PK1～PK6,GenBank注册序列号为AY693369-AY693371和AY776277-AY776279。在PK3、PK4和PK6中发现了丝氨酸／苏氨酸蛋白激酶的9个催化结构域Ⅰ～Ⅸ,而其它3个克隆具有部分结构域。氨基酸序列同源性分析还发现,6个克隆编码的氨基酸序列都与受体样蛋白激酶有较高的氨基酸序列同源性。系统进化树分析还表明它们都是可能的抗病基因同源序列。总之,6个克隆不仅是抗病基因同源序列而且是可能的受体样蛋白激酶基因同源序列。     

Identifying and Characterizing a Novel Peritrophic Matrix Protein (MdPM-17) Associated With Antibacterial Response From the Housefly,Musca domestica (Diptera: Muscidae)

Peritrophic matrix/membrane (PM) critically prevents the midgut of insects from external invasion by microbes. The proteins in the peritrophic membrane are its major structural components. Additionally, they determine the formation and function of this membrane. However, the role of PM proteins in immune regulation is unclear. Herein, we isolated a novel PM protein (MdPM-17) from Musca domestica larvae. Further, the function of MdPM-17 in regulating host innate immunity was identified. Results showed that the cDNA of MdPM-17 full is 635 bp in length. Moreover, it consists of a 477-bp open reading frame encoding 158 amino acid residues. These amino acid residues are composed of two Chitin-binding type-2 domain (ChtBD2) and 19 amino acids as a signal peptide. Moreover, tissue distribution analysis indicates that MdPM-17 was enriched expressed in midgut, and moderate levels in the fat body, foregut, and malpighian tubule. Notably, MdPM-17 recombinant protein showed high chitin-binding capacity, thus belongs to the Class III PM protein group. MdPM-17 protein silencing via RNA interference resulted in the expression of antimicrobial peptide (defensin, cecropins, and diptericin) genes, and this occurred after oral inoculation with exogenous microbes Escherichia coli (Enterobacteriales:Enterobacteriaceae), Staphylococcus aureus (Bacillales:Staphylococcaceae), and Candida albicans (Endomycetales:Saccharomycetaceae)). Therefore, all the antimicrobial peptide (AMP) gene expression levels are high in MdPM-17-depleted larvae during microbial infection compared to controls. Consequently, these findings indicate that MdPM-17 protein is associated with the antibacterial response from the housefly.     

Soluble Klotho protein suppresses THP-1-derived foam cell formation

AIMTo investigate the role of soluble Klotho protein in THP-1-derived foam cell formation. METHODSTHP-1 monocytes were induced into macrophages by treatment with 160 nmol/L phorbol myristate acetate for 48 h, and then were divided into 6 groups: negative control group (THP-1-derived macrophages), positive control group [THP-1-derived foam cells induced by oxidized low-density lipoprotein (ox-LDL) for 48 h], and 25, 50, 100 and 200 μg/L soluble Klotho protein groups (THP-1-derived macrophages pretreated with soluble Klotho protein at the indicat?ed concentraions for 2 h and then induced by ox-LDL for 48 h). Lipid droplets in cytoplasm were observed by oil red O staining. The cholesterol outflow rate was detected by scintillation counting technique. The content of intracellular total cholesterol, free cholesterol and cholesterol ester was detected by enzyme fluorescence analysis. The expression of acyl-coenzyme A:cholesterol acyltransferase 1 （ACAT1） and ATP-binding cassette transport?er A1 (ABCA1) at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. RESULTSOil red O staining and lipid mass quantification showed that THP-1-derived foam cell formation was dose-dependently suppressed by soluble Klotho protein. The cholesterol efflux rate of THP-1-derived foam cells was increased by soluble Klotho protein in a dose-dependent manner (P<0.05). In addition, soluble Klotho protein decreased the expression of ACAT1 and increased the expression of ABCA1 in a dose-dependent manner (P<0.05). CONCLUSION The soluble Klotho protein inhibits THP-1-derived foam cell formation in a dose-dependent manner by down-regulating the expression of ACAT1 and up-regulating the expression of ABCA1.     

Crude garlic extract significantly inhibits replication of grapevine viruses

Efforts to control viral diseases of grapevine include the production of certified material and development of virus-resistant transgenic grapevines. However, effective antiviral agents, once the viruses have infected the plants, are still lacking. This study shows that a crude garlic extract has significant antiviral activity against grapevine viruses. Replication of grapevine leafroll-associated virus 2 (GLRaV-2) was obviously inhibited in grapevine cv. Cabernet Sauvignon calli treated with diluted (1:100) garlic extract. The relative RNA levels of GLRaV-2 and grapevine fleck virus (GFkV) in cv. Summer Black grapevine in in vitro-grown plantlets 10 days after treatment with diluted (1:100) garlic extract were about 22% and 20%, respectively, of that in controls. The viral RNA accumulation of GLRaV-2, GFkV, grapevine virus A (GVA), grapevine fanleaf virus (GFLV) and grapevine rupestris stem pitting-associated virus (GRSPaV) in field-grown grapevine cv. Centennial Seedless plants sprayed with diluted (1:100) garlic extract were about 31–40%, 26–38%, 18–31%, 17–42% and 15–18%, respectively, of that in controls. Moreover, the garlic extract treatment led to a significant decrease in viral RNA accumulation of GLRaV-3, GLRaV-2, GVA, GFkV, GFLV, GRSPaV and grapevine Pinot Gris virus in pot-grown grapevine cv. Shine Muscat plants, and viral disease symptoms in these plants were obviously attenuated. In addition, this extract significantly induced expression of pathogenesis-related protein genes and stimulated activity of antioxidant enzymes in grapevines. Taken together, these results indicate that the crude garlic extract acts as a significant inhibitor against a broad range of grapevine viruses.     

荒漠草原建群种及其枯落物的C、N、P生态化学计量特征

以希拉穆仁草原建群种羊草(Leymuschinensis)、短花针茅(Stipa breviflora Griseb)、芨芨草(Achnatherum splendens nevski)及其枯落物为研究对象,通过测定其C、N、P质量分数,并计算生态化学计量比,分析建群种养分限制格局及养分再吸收规律。结果表明:①植物叶片的C、N、P质量分数均值分别为398.74、24.41、1.55 g/kg,枯落物的为362.53、14.79、1.17 g/kg,3种植物叶片的C、N、P质量分数与其枯落物的差异显著(P<0.05)。②植物在枯落之前会将养分转移,防止养分的流失,N、P的养分再吸收效率范围分别在13.51%~77.49%、2.65%~49.51%,N的回流率大于P,进而使其具备了较强的适应干旱环境的能力。③3种植物叶片的N/P、C/N、C/P变化范围分别在11.81~21.70、15.76~16.59、195.47~273.37,枯落物的变化范围分别在7.72~17.05、17.71~64.46、215.43~487.46,3种植物叶片与枯落物的N/P、C/P差异显著(P<0.05)。④植物叶片N/P均值为16.95,枯落物N/P均值为12.31,说明植物的生长主要受P的限制。研究结果可为荒漠草原提供草地管理指导。     

不同海拔生态区移栽唐古特大黄株系性状表现

为探究唐古特大黄移栽到不同海拔区域后的性状表现,确定合理的种植海拔,同时对唐古特大黄进行品种选优的初期筛选。选用唐古特大黄10个株系温室育苗移栽到青海5个不同海拔生态区进行适应性研究,利用灰色关联分析法确定10个株系植株的综合表现排名。结果表明,海拔对移栽第1年的株高、叶片数、最大叶叶长、最大叶叶宽、最大叶叶柄长、产生叶裂程度、最大叶叶裂长、最大叶叶裂宽、根茎长、根茎数、根茎粗、根茎鲜质量、根茎鲜质量产量有极显著影响（P＜0.01）,表现为随海拔升高各项指标极显著降低;10个株系中较优株系为L2、L9、L1、L5和L10,其次为L3、L6和L8,而L4和L7表现最差。综上所述,海拔影响栽培唐古特大黄的性状表现,在2 016～3 763 m内,海拔越高,植株性状表现越差;较优株系L1、L2、L5、L9、L10可作为品种选育进一步研究材料。     

蛋白酶解对鲣鱼肉乳化特性的改善

天然鱼蛋白乳化性不佳,难以满足实际生产需求,但鱼蛋白的酶解改性可改善其功能特性,并拓展应用范围。以鲣鱼白色肉为原料,以水解度和乳化性为主要指标,比较了不同蛋白酶(胰蛋白酶、风味蛋白酶、木瓜蛋白酶)和水解时间对酶解液制备和鱼蛋白乳化性的影响,优化了酶解工艺参数。将鱼蛋白进行冷冻干燥后,分析鱼蛋白酶解前后溶解性、乳化性和起泡性等功能特性的差异。结果表明,当胰蛋白酶添加量2 000 U·g^-1,酶解10 min时,鱼蛋白水解度(DH)达到8.2%,此时鱼蛋白乳化特性最佳。与酶解前样品相比,酶解后鱼蛋白的乳化活性和乳化稳定性显著提高,其溶解度、起泡性和起泡稳定性均有一定程度的改善。酶解制备的鲣鱼蛋白在广泛pH范围内具有更好的功能性质,在食品工业具有更高的应用价值。