Raft培养可诱导人正常上皮细胞分化

人的正常上皮细胞在一般的体外培养情况下很难分化成类似于机体的上皮样结构。为了得到上皮结构进行相关研究,近年来,国外建立了Raft（船式）培养方法。本文在长期从事细胞培养的工作中,摸索出了适合国内一般实验室条件的Raft细胞培养操作方案,模拟体内人皮肤结构的分化层次,以纤维细胞与胶原作为混合基质（作为上皮细胞的滋养物）,同时加入一些生长因子、激素和必需的蛋白等成分。将纤维细胞、胶原和添加成分做为基层过夜培养,次日将培养好的上皮细胞种到上面,过夜培养后即为“skin equivalent”（皮肤等价物）,将“皮肤等价物”移到一种网状金属架上以使细胞在气－液状态下培养,在这种条件下,培养中的上皮细胞与体内上皮细胞自然生长的状态、环境非常类似,在体外导致上皮细胞分化成清晰可见的层次。此项技术在临床治疗烧伤以及其它皮肤损伤的疾病方面有潜在的应用价值,也为研究皮肤的正常生理功能以及癌症的发生、发展以及治疗提供了一种模型。     

模拟增温对黄河三角洲滨海湿地非生长季土壤呼吸的影响

冬季土壤呼吸能释放生长季所固存的碳, 因而在陆地碳循环中占有重要地位。随着全球气候变暖, 平均地表温度将升高0.3-4.8 ℃, 且冬季增温更加明显, 而温度的升高会促进更多CO₂的释放。另外, 滨海湿地地下水位浅, 淡咸水交互作用明显, 增温能引起土壤表层盐分升高, 从而影响土壤呼吸。该研究以黄河三角洲滨海湿地为研究对象, 采用红外辐射加热器模拟增温, 研究了该地区非生长季土壤呼吸的日动态及季节动态, 同时探讨了土壤呼吸对环境因子的响应机制。结果显示: 日动态中, 增温与对照的土壤呼吸速率变化趋势一致, 为单峰曲线; 在平均日变化中, 整个非生长季不同处理的土壤呼吸速率无显著差异, 而土壤温度和土壤盐分均为增温大于对照, 并且土壤呼吸峰值时间均比土壤温度提前。季节动态中, 整个研究期分为非盐分限制阶段(2014年11月-2015年2月中旬)和盐分限制阶段(2015年2月中旬-2015年4月)。在整个非生长季, 土壤呼吸速率无显著差异; 在非盐分限制阶段, 当10 cm土壤温度升高4.0 ℃时, 土壤呼吸速率显著提高22.9%, 而土壤呼吸温度敏感性系数(Q₁₀)与对照相比有所降低; 在盐分限制阶段, 尽管土壤温度升高3.3 ℃, 土壤呼吸速率却降低了20.7%, 这可能是由于增温引起了土壤盐分的升高, 同时由增温引起的土壤含水量的升高在一定程度上也限制了土壤呼吸, 而此阶段增温对Q₁₀无显著影响。因此, 在滨海湿地中, 增温除了直接影响土壤温度, 还可通过影响土壤水盐状况来影响土壤呼吸, 进而影响滨海湿地土壤碳库。     

The breeding of two polyploid rice lines with the characteristic of polyploid meiosis stability

Polyploidization is a basic feature of plant evolution. Nearly all of the main food, cotton and oil crops are polyploid. When ploidy levels increase, yields double; this phenomenon suggested a new strategy of rice breeding that utilizes wide crosses and polyploidization dual advantages to breed super rice.Because low seed set rates in polyploid rice usually makes it difficult to breed, the selection of Ph-liked gene lines was emphasized. After progenies of indica-japonica were identified and selected, two polyploid lines, PMeS-1 and PMeS-2 with Polyploid Meiosis Stability (PMeS) genes were bred. The procedure included seven steps: selecting parents, crossing or multiple crossing, back-crossing, doubling chromosomes, identifying the polyploid, and choosing plants with high seed set rates that can breed themselves into stable lines. The characteristics of PMeS were determined by observing meiotic behaviors and by cross-identification of seed sets. PMeS-1 and PMeS-2, (japonica rice), have several characteristics different from other polyploid rice lines, including a higher rate of seed set (more than 65%, increasing to more than 70% in their F1 offspring); and stable meiotic behaviors (pairing with bivalents and quarivalents nearly without over-quarivalent in prophase, nearly without lagging chromosomes in metaphase and without micronuclei in anaphase and telophase). The latter was obviously different from control polyploid line Dure-4X, which displayed abnormal meiotic behaviors including a higher rate of multivalents, univalents and trivalents in prophase, lagging chromosomes in metaphase and micronuclei in anaphase and telophase. There were also three differences of the breeding method between PMeS lines and normal diploid lines: chromosomes doubling, polyploidism identifying and higher seed set testing. The selection of PMeS lines is the first step in polyploid rice breeding; their use will advance the progress of polyploid rice breeding, which will in turn offer a new way to breed super rice.     

DGP1, a drought-induced guard cell-specific promoter and its function analysis in tobacco plants

The genetic regulation of stomatal movement mainly depends on an efficient control system of gene expression, and guard cell-specific promoter is becoming the best choice. Here we combined the dehydration responsive element (DRE) with guard cell specific element (GCSE) to construct a novel promoter, DGP1. Histochemical assays in transgenic tobacco carryingβ-glucuronidase (gus) gene fused to DGP1 demonstrated that GUS activity was found to be highly inducible by drought treatment and specifically restricted to guard cells. No GUS activity was detected in roots, stems or flowers after treatment. Further quantitative analysis showed that GUS activity in the epidermal strips was apparently induced by dehydration and dramatically increased with the elongation of treatment. The GUS activity after 8 h treatment was 179 times that of those without treatment. Although GUS activity in roots, stems or mesophyll increased after treatment, no great changes were observed. These results suggested that DGP1 could drive target gene expressed in guard cells when plant is subjected to drought stress. And this gets us prepared to control opening and closing of stomata through plant gene engineering.     

Preliminary investigation of sequence-independent DNA binding proteins in rat skeletal muscle sarcoplasmic reticulum and their function

To observe the binding of plasmid DNA to non-nuclear DNA binding proteins in sarcoplasmic reticulum (SR) and the effects of this binding on SR function, sarcoplasmic reticulum proteins in rat skeletal muscle were isolated by differential centrifuge and sucrose density-gradient centrifuge. The results showed that there are two sequence-independent DNA binding proteins in SR proteins, the molecular weights of which are 83 and 58 ku, respectively. Ca²⁺ uptake and release of SR were remarkably promoted by the binding of plasmid DNA to DNA binding proteins in SR, the mechanism is probably through increasing of Ca²⁺-ATPase activity in SR and changing of character of Ca²⁺ release channel ryanodine receptors induced by the binding. These results suggest that there exist DNA binding proteins in SR and its binding to DNA may affect Ca²⁺ transport of SR.     

Cloning, sequencing and function ofsanA, a gene involved in nikkomycin biosynthesis ofStreptomyces ansochromogenes

Several genetically stable mutants blocked in nikkomycin biosynthesis were obtained after the slightly germinated spores ofStreptomyces ansochromogenes, a nikkomycin producer, were treated with ultra violet radiation. One of the mutants is the same in morpholotical differentiation as the wild type strain and is designated as NBB19. A DNA library was constructed using plasmid pIJ702 as cloning vector, NBB19 as cloning recipient. A 6 kb DNA fragment which can genetically complement NBB19 was cloned when screening the library for antifungal activity. Sequence analysis showed that the 3 kbBgl II -Sal I fragment contains one complete ORF (ORF1) and one partial ORF (ORF2). ORF1 is designated assanA. sanA is 1 365 bp, encoding a protein consisting of 454 amino acid residues. Database searching indicated thatsanA is homologous to the hypothetical methyltransferase inPyrococcus horikoshii with 25% identities and 41% positives. Disruptant ofsanA lost the ability to synthesize nikkomycin. It indicated thatsanA is a novel gene which is essential for nikkomycin biosynthesis.     

REPEAT SEQUENCE PRIMER‐PCR STUDY ON DNA POLYMORPHISM OF GEOGRAPHIC POPULATIONS OF COTTON APHID,APHIS GOSSYPII,IN CHINA*

Abstract The repeat sequence primer‐PCR technique was used to determine DNA polymorphism of the aphid Aphis gossypii (Glover) collected from 8 different localites in China. Three primers were selected and used for similarity index (SI) and cluster analysis based on the data of Nei's genetic distance (D). We found that the populations in the north and northwest of China was linked before they were joined by those in southern China. A proposed explanation emphasizes the populations in the north and northwest of China were located near — 4 °C isotherm or lower in January with less than 200 frost‐free days per year, whereas populations in southern China were located near 0 °C isotherm or higher with more than 300 frost ‐ free days per year. So it may be the over‐winter host plants instead of the geographic distance or gene flow, exert pronounced influence on the geographic population differentiation of A. gossypii. Species on different over‐winter host plants may directly lead to the genetic differentiation of their summer offsprings.     

牛脊髓神经丝的体外组装与结构

利用差速离心法从牛脊髓中分离神经丝 ,在电镜下观察了其形态 ;应用扫描隧道显微镜 (STM)研究了它的结构 ,发现神经丝具有长短 2种侧臂 ,二者相间排列 ,相邻长侧臂或相邻短侧臂的间距都是 2 0～ 2 2nm ;由此推测神经丝内部存在 3 /4分子交错 ;还研究了神经丝蛋白的体外组装 ,以胶体金标记的方法证明 ,中等分子量与高分子量的神经丝蛋白 ,都能同低分子量的神经丝蛋白共同装配成 10nm的纤维 ;同时发现 ,中等分子量与高分子量的神经丝蛋白能够组装成一种较细的纤维 ,不同于中间纤维 .     

水稻黄矮弹状病毒的leader区和trailer区的序列分析及其体内转录物的鉴定

克隆及测定了水稻黄矮病毒基因组RNA的 3′端leader区和 5′端trailer区的序列 .结果表明 ,RYSV的 3′leader长 2 0 3个核苷酸 ,5′trailer长 191个核苷酸 .两者末端的 9个核苷酸完全互补 ,可形成弹状病毒基因组RNA的典型的锅柄结构 .与其他弹状病毒的leader和trailer相比较 ,RYSV的leader和trailer较长 ,除leader的 3′末端和trailer的 5′末端序列具有一定的保守性外 ,其余的核苷酸序列无明显的同源性 .用 3′RACE方法在感染RYSV的水稻中检测出leader的转录物 ,并证明该leaderRNA具有polyA尾巴 .这是继苦苣菜黄脉病毒的leaderRNA后第 2例弹状病毒leaderRNA带polyA尾巴的报道 .用类似的方法没有检测到带polyA尾巴的trailer的正链转录物 .     

优化AFLP阳性带回收克隆技术

通过将AFLP聚丙烯酰胺变性胶切小、适当曝光过量、以聚丙烯酰胺凝胶电泳代替琼脂糖电泳检测克隆片段大小的方法 ,增加了回收克隆AFLP阳性带的准确性。该方法对于从变性和非变性大page胶上回收克隆目的片段具有普遍意义。