Gene markers for grain polyphenol oxidase activity in common wheat

Polyphenol oxidase (PPO) in grain is regarded as a major factor resulting in time-dependent darkening of wheat end products, particularly for Asian noodles and steamed bread. Breeding wheat cultivars with low PPO activity using efficient and reliable markers is one of the best ways to reduce the undesirable darkening. In the present study, we developed a gene-specific marker (PPO05) for low PPO activity from the sequence AY515506. This marker detected double PCR fragments (<750 and >750 bp) in the cultivars with low PPO activity and a single PCR fragment (<750 bp) in the cultivars with high PPO activity. Screening of this marker on 235 Chinese wheat micro-core collections showed that the double fragments were present in 113 genotypes and the single fragments in the remaining 122 genotypes. Statistic analysis revealed that the cultivars with the double fragments had significantly lower mean PPO activity than those with single fragments. Through sequence analysis and blast search in NCBI, we found that the cultivars with the double fragments contained the PPO-2Ab allele, while the cultivars with the single fragments contained the PPO-2Aa allele. The PPO-2Ab and PPO-2Da alleles were associated with the low grain PPO activity and the PPO-2Aa and PPO-2Db alleles associated with the high PPO activity. The genotypes carrying both PPO-2Ab and PPO-2Da showed the lowest PPO activity, while the genotypes carrying both PPO-2Aa and PPO-2Db showed the highest PPO activity. Comparison of PPO05 and STS01 with the STS markers PPO18 and PPO29 showed that the larger and small fragments of PPO05 were equivalent to the 876- and 685-bp fragments of PPO18, respectively, and that STS01 was the complementary marker of PPO29. Thus, the STS markers PPO05 and STS01 along with PPO18 and PPO29 are the efficient and reliable markers for the evaluation of PPO activity and can be used in wheat breeding programs to improve the quality of noodles and other end products.     

一株珊瑚礁-海草床复合生态系统固氮菌的分离与鉴定

三亚珊瑚礁自然保护区部分珊瑚礁退化后逐渐演替为以泰来藻(Thalassia hemprichii)为优势种的海草床群落,采用选择性无氮培养基从泰来藻植株的根际,分离得到一株固氮菌,编号为G33-1,经形态学、生理生化鉴定和16SrDNA及固氮基因nifH的序列分析,初步鉴定为成团泛菌(Pantoea agglomerans)。该菌为革兰氏阴性菌,以周生鞭毛运动,呈直杆状,菌落圆形,半透明乳白色,比较湿润,有光泽,直径约1mm,低凸,光滑,边缘比较整齐。最适培养条件为:氯化钠浓度25‰,生长温度为37°C,起始pH值为8。与成团泛菌标准菌株(ATCC27155TM)相比较,在碳源利用、精氨酸双水解、苯丙氨酸脱胺酶、鸟氨酸脱胺酶、以及生长温度和盐度等方面都具有较高的相似性,以16SrDNA为基础构建的系统进化树分析结果,表明其与成团泛菌属Pantoeaag-glomeransWAB1870进化距离最近,相似性大于99%。此外,利用乙炔还原法对固氮活性进行测定,其具有较高的固氮活性,达299.16nmolC2H2/(mL.h)。     

运用重叠延伸PCR技术构建在甘丙肽全长cDNA嵌入第二内含子的重构分子

构建嵌入第二内含子的甘丙肽(Galanin,GAL)全长基因组cDNA的重构分子。通过RT-PCR扩增出cDNA编区的序列,分别从基因组中扩增出cDNA的5′和3′端部分非编码序列;使用重叠延伸PCR(overlap extention PCR,OE-PCR)方法将三个片段重叠获得全长cDNA序列;再将全长cDNA从第三外显子第15个碱基处分成两部分,分开的cDNA前半部分和后半部分以及第二内含子进行重叠延伸获得重构分子,含有第二内含子的甘丙肽(GAL)全长基因组cDNA;将重构分子连入pMDI9-Tsimple载体。电泳分析观察到清晰的重构分子片段;测序显示重构分子由所设计的序列组成,第二内含子插入的位置准确,且无移码。使用重叠延伸PCR能够成功在cDNA中插入内含子获得一段重构基因。     

Self-assembled terplexes for targeted gene delivery with improved transfection

To improve transfection efficiency and to incorporate target ligands to the gene delivery systems, heparin and heparin-biotin were introduced to complexes of polyamidoamine dendrimer and DNA (PAMAM/DNA) via electrostatic interactions to form self-assembled PAMAM/DNA/heparin and PAMAM/DNA/heparin-biotin terplexes, respectively. The self-assembled terplexes were characterized by agarose gel electrophoresis and particle size analysis. The MTT assay indicated that, after incorporation of heparin and heparin-biotin, the terplexes exhibited decreased cytotoxicity. In addition, as compared with PAMAM/DNA and PAMAM/DNA/heparin complexes, the PAMAM/DNA/heparin-biotin complexes exhibited much higher cellular uptake into HeLa cells due to the specific interactions between biotin and biotin receptors on HeLa cells, which led to the enhanced transfection activity. The PAMAM/DNA/heparin-biotin complexes would be a promising targeting gene delivery system.     

Recombinant PBD‐1 (porcine beta‐defensin 1) expressed in the milk by transplanting transgenic mES‐like‐derived cells into mouse mammary gland

ES (embryonic stem)‐derived cells have been investigated in many animal models of severe injury and degenerative disease. However, few studies have examined the ability of ES‐derived cells to improve functional outcome following partially damaged breast and also the modification of mammary tissue to produce costly proteins. This study investigates the feasibility of implanting mES‐dK (mouse ES‐derived keratinocytes‐like) cells stably transfected with a mammary gland special expression vector for the PBD‐1 (porcine beta‐defensin 1) in developing mammary glands. Our aim was to assess the ability of cell grafting to improve functional outcome following partial damage of the breast, also on the breast modification mammary tissue in mice for the production of PBD‐1 protein secreted in the milk. Our results showed that the ratios of the surviving cells labelled with the myoepithelial or luminal cell markers, EMA (epithelial membrane antigen) and CALLA, were 41.7±15.2% and 28.4±9.6%, respectively, which revealed that transplanted mES‐dK cells survived, integrated in vivo and differentiated into myoepithelial or luminal cells. In addition, Western blot analysis showed that 37.5% (3 out of 8) female transplanted mice had PBD‐1 expression in their milk and reached 0.4998, 0.5229 and 0.5195 μg/ml, respectively.     

Detection of Helicobacteraceae in intestinal biopsies of children with Crohn's disease

Background: Given that members of Helicobacteraceae family colonize the intestinal mucus layer, it has been hypothesized that they may play a role in Crohn’s disease. This study investigated the presence of Helicobacteraceae DNA in biopsies collected from children with Crohn’s disease and controls. Materials and Methods: The presence of Helicobacteraceae DNA was investigated in intestinal biopsies collected from 179 children undergoing colonoscopy (Crohn’s disease n = 77, controls n = 102) using a Helicobacteraceae‐specific PCR. Results: Members of the Helicobacteraceae were detected in 32/77 children with Crohn’s disease (41.5%) and 23/102 controls (22.5%). Statistical analysis showed the prevalence of Helicobacteraceae detected in patients to be significantly higher than that in controls (p = .0062). Analysis of non‐pylori Helicobacteraceae showed that their prevalence was also significantly higher in patients than in controls (p = .04). Helicobacter pylori was detected in 14.0% of the biopsies across all groups. Given that all children tested were negative for gastric H. pylori, this was a surprising finding. Phylogenetic analysis of H. pylori sequences detected in the biopsies showed that the H. pylori strains identified in the patients did not group with gastric H. pylori included in the analysis, but rather with other H. pylori strains detected in the intestine, gall bladder, and liver. Conclusions:  The higher prevalence of Helicobacteraceae DNA in Crohn’s disease patients would suggest that members of this family may be involved in this disease. In addition, phylogenetic analysis of H. pylori strains showed that extragastric sequences clustered together, indicating that different H. pylori strains may adapt to colonize extragastric niches.     

丝裂原活化蛋白激酶信号通路相关研究

丝裂原活化蛋白激酶信号通路是生物体内重要的信号转导系统之一,参与介导细胞生长、发育、分裂、分化等多种生理反应过程。在哺乳动物细胞中存在5个MAPK亚族,分别是ERK1／2、JNK、p38、ERK3／4和ERK5。MAPK通常定位于细胞质中,受激活后移行进入细胞核,并产生相应的生理作用。