海洋微生物纤维素酶的研究进展及其应用

海洋极端酶因在极端环境中具有更高的酶活性及更好的稳定性,因而具有重要的理论价值和工业应用前景.随着海洋微生物极端酶的研究开发,海洋微生物纤维素酶的研究也逐渐受到学者们的关注,并取得了较大进展.综述迄今为止分离出的产纤维素酶的海洋微生物种群及其酶学特性,海洋微生物纤维素酶基因克隆与表达的国内外研究现状,分析海洋微生物纤维素酶潜在的应用价值及未来发展前景.     

CRISPR Cas9- and Cas12a-mediated gusA editing in transgenic blueberry

To develop an effective genome editing tool for blueberry breeding, CRISPR-Cas9 and CRISPR-Cas12a were evaluated for their editing efficiencies of a marker gene, beta-glucuronidase (gusA), which was previously introduced into two blueberry cultivars each a single-copy transgene. Four expression vectors were built, with CRISPR-Cas9 and CRISPR-Cas12a each driven by a 35S promoter or AtUbi promoter. Each vector contained two editing sites in the gusA. These four vectors were respectively transformed into the leaf explants of transgenic gusA blueberry and the resulting transgenic calli were induced under hygromycin selection. GUS staining showed that some small proportions of the hygromycin-resistant calli had non-GUS stained sectors, suggesting some possible occurrences of gusA editing. We sequenced GUS amplicons spanning the two editing sites in three blueberry tissues and found about 5.5% amplicons having editing features from the calli transformed with the 35S-Cas9 vector. Further, we conducted a second round of shoot regeneration from leaf explants derived from the initial Cas9- and Cas12a-containing calli (T₀) and analyzed amplicons of the target editing region. Of the newly induced shoots, 15.5% for the 35S-Cas9 and 5.3% for the AtUbi-Cas9 showed non-GUS staining, whereas all of the shoots containing the Cas12a vectors showed blue staining. Sanger sequencing confirmed the editing-induced mutations in two representative non-GUS staining lines. Clearly, the second round of regeneration had enriched editing events and enhanced the production of edited shoots. The results and protocol described will be helpful to facilitating high-precision breeding of blueberries using CRISPR Cas technologies.

     

温度对大菜粉蝶生长发育的影响

为明确温度对大菜粉蝶生长发育的影响,在16、20、22、25、28和31℃共6个恒温条件下以白菜为寄主进行饲养,测定了温度对大菜粉蝶各虫态的发育历期、发育速率、存活率及成虫寿命等的影响,并采用直接最优法计算其各虫态的发育起点温度和有效积温。结果表明：在16～28℃温度范围内,大菜粉蝶的卵、幼虫、蛹及全世代平均发育速率具有随温度升高而加快的趋势,存活率则随着温度的升高而降低;卵、幼虫、蛹和成虫的发育起点温度分别为11.76℃、6.87℃、5.30℃和14.91℃,有效积温分别为38.14、213.90、169.68和43.01 d·℃。在28℃时大菜粉蝶卵的孵化率、各龄幼虫存活率、化蛹率、蛹重及羽化率均显著低于其他温度,31℃条件下不能完成个体发育。由此可见,高温环境不利于大菜粉蝶的存活和生长发育。研究结果可为大菜粉蝶田间发生期预测及综合防治提供科学依据。     

一株C3形态多重耐药大肠杆菌噬菌体生物学特性和基因组分析

【目的】多重耐药菌的出现对公共卫生安全构成严重威胁,本研究分离多重耐药大肠杆菌噬菌体,研究其生物学特性和基因组特征,为耐药菌的噬菌体疗法提供理论依据。【方法】使用双层平板法从污水样本中分离纯化大肠杆菌噬菌体;磷钨酸染色后通过透射电镜观察形态;测定其宿主范围,测定温度和pH稳定性、一步生长曲线和体外抑菌效果等生物学特性;体内抑菌试验评估噬菌体对多重耐药大肠杆菌N1203-1Af感染的大蜡螟幼虫的保护作用;基于全基因组测序对其基因组特点进行分析。【结果】本研究分离共得到5株大肠杆菌噬菌体,分别命名为pEC-S163-2.1、pEC-S163-2.2、pEC-M1167-5Ar.1、pEC-m1291-2Dr.1和pEC-N1203-2Af.1;电镜结果显示噬菌体pEC-N1203-2Af.1属于短尾噬菌体中罕见的C3形态型,头部较长,长是宽的2–3倍;pEC-N1203-2Af.1可裂解受试15株大肠杆菌中的3株;感染10 min后进入指数增长期,–20-50℃、pH值为4.0–10.0的环境下均能够保持稳定活性;大蜡螟幼虫感染大肠杆菌N1203-2Af后噬菌体pEC-N1203-2Af....     

链霉菌形态分化与次级代谢产物合成的研究进展

链霉菌是一类具有复杂的形态分化周期和强大的次级代谢能力的高GC含量的放线菌,能够利用其初级代谢产生的前体化合物和能量,合成多种结构复杂、功能多样的具有生物活性的次级代谢产物,在农业、食品、畜牧业、工业以及医药研究等领域都具有重要的价值。在链霉菌的形态分化后期常常伴随着次级代谢产物的生物合成,并且两者都受到复杂的网络调控;同时链霉菌的形态对次级代谢产物的产量和种类造成很大影响。对链霉菌生长周期的全面理解将加深对链霉菌形态分化与次级代谢产物合成关系的认识。本文将对链霉菌的形态分化过程、形态分化和抗生素合成两者共同的调控因子以及链霉菌形态与抗生素产量之间的关系进行综述,这将有助于理解抗生素的合成过程,也将会在缩短发酵周期、构建高产工程菌株、新型杀菌剂的研发以及新型抗生素的合成等方面给予我们启发。     

ARPC4 promotes bladder cancer cell invasion and is associated with lymph node metastasis

The significance of actin-related protein 2/3 complex subunit 4 (ARPC4) expression in bladder cancer, and its potential role in the invasion and migration of bladder cancer cells, has yet to be determined. This study was to identify the correlation between ARPC4 and lymph node metastasis, and to determine the role of ARPC4 in the invasive migration of T24 bladder cancer cells. One hundred and ninety-eight bladder cancer tissues and 40 normal bladder and lymph node tissues were examined. Tissue microarrays were constructed and subjected to immunohistochemical stating for ARPC4. Multiple logistic analysis was used to determine risk factors associated with bladder cancer metastasis. ARPC4 expression in T24 bladder cancer cells was suppressed using small interfering RNA and changes in protein levels were determined by Western blot analysis. The proliferation of bladder cancer cells after knocking down of ARPC4 was determined by cell counting kit-8. The effects of ARPC4 knockdown on T24 cell invasion and migration was determined using transwell and wound healing assays. Immunofluorescence analysis was performed to examine changes in pseudopodia formation and actin cytoskeleton structure. The expression of ARPC4 was elevated in bladder cancer tissues than normal tissues (84.3% vs 27.5%, P < 0.001). The multivariate logistic analysis demonstrated that the level of ARPC4, as a risk factor, was correlated with lymphatic metastasis (P < 0.05). ARPC4 knockdown attenuated proliferation, migration, invasion, and pseudopodia formation in T24 cells. ARPC4 expression, as a risk factor, is associated with lymphatic metastasis and is upregulated in bladder cancer tissues in comparison with normal tissues. Inhibition of ARPC4 expression significantly attenuates the proliferation, migration, and invasion of bladder cancer cell, possibly due to defects in pseudopodia formation.     

Responses of litter decomposition and nutrient release rate to water and nitrogen addition differed among three plant species dominated in a semi-arid grassland

Background and aims

Precipitation and nitrogen (N) deposition are predicted to increase in northern China. The present paper aimed to better understand how different dominant species in semi-arid grasslands in this region vary in their litter decomposition and nutrient release responses to increases in precipitation and N deposition.

Methods

Above-ground litter of three dominant species (two grasses, Agropyron cristatum and Stipa krylovii, and one forb, Artemisia frigida) was collected from areas without experimental treatments in a semi-arid grassland in Inner Mongolia. Litter decomposition was studied over three years to determine the effects of water and N addition on litter decomposition rate and nutrient dynamics.

Results

Litter mass loss and nutrient release were faster for the forb species than for the two grasses during decomposition. Both water and N addition increased litter mass loss of the grass A. cristatum, while the treatments showed no impacts on that of the forb A. frigida. Supplemental N had time-dependent, positive effects on litter mass loss of the grass S. krylovii. During the three-year decomposition study, the release of N from litter was inhibited by N addition for the three species, and it was promoted by water addition for the two grasses. Across all treatments, N and potassium (K) were released from the litter of all three species, whereas calcium (Ca) was accumulated. Phosphorus (P) and magnesium (Mg) were released from the forb litter but accumulated in the grass litter after three years of decomposition.

Conclusions

Our findings revealed that the litter decomposition response to water and N supplementation differed among dominant plant species in a semi-arid grassland, indicating that changes in dominant plant species induced by projected increases in precipitation and N deposition are likely to affect litter decomposition, nutrient cycling, and further biogeochemical cycles in this grassland. The asynchronous nutrient release of different species’ litter found in the present study highlights the complexity of nutrient replenishment from litter decomposition in the temperate steppe under scenarios of enhancing precipitation and N deposition.

     

In vitro assembly of minicellulosomes with two scaffoldins on the yeast cell surface for cellulose saccharification and bioethanol production

The present paper reports in vitro strategies for assembly of minicellulosomes with two miniscaffoldins on the Saccharomyces cerevisiae cell surface. It was carried out through incubation of the yeast cells displaying scaffoldins with Escherichia coli lysates containing recombinant cellulases, or using a four-population yeast consortium. The results showed that the display level of miniscaffoldin II was distinctly increased by moving the cellulases production into E. coli or other yeast cells, indicating that the metabolic burden of the yeast host was decreased. The yeast consortium did not show any cellulolytic activity, while the E. coli lysates-treated yeast, whose anchoring miniscaffoldin length was optimized, was able to produce ～1138 mg/L ethanol from microcrystalline cellulose within 4 days. We also confirmed that the yeast-associated minicellulosome moreover showed both higher thermal stability and lower protease accessibility than free minicellulosome. This research promotes the application of S. cerevisiae as a consolidated bioprocessing (CBP) microorganism in cellulosic bioethanol production.     

Maternal Deprivation Enhances Behavioral Vulnerability to Stress Associated with miR-504 Expression in Nucleus Accumbens of Rats

Objective

In this study, the effect of maternal deprivation (MD) and chronic unpredictable stress (CUS) in inducing depressive behaviors and associated molecular mechanism were investigated in rats.

Methods

Maternal deprivation was established by separating pups from their mothers for 6 hours daily from postnatal day 1 to day 14. Chronic unpredictable stress was established by water deprivation, elevated open platform, food deprivation, restraint stress and electric foot shock. The depressive behaviors were determined by use of sucrose preference test and forced swim test.

Results

Rats in MD/CUS group exhibited lower sucrose preference rate, longer immobility time, and lighter body weights than rats in other groups (MD/control, non-MD/CUS and non-MD/control group). Meanwhile, higher miR-504 expression and lower dopamine receptor D1 (DRD1) and D2 (DRD2) expression were observed in the nucleus accumbens of rats in the MD/CUS group than in the other three groups. MiR-504 expression correlated negatively with DRD1 gene expression and sucrose preference rate in the sucrose preference test, but correlated positively with immobility time in forced swim test. Both DRD2 mRNA and protein expression correlated negatively with immobility time in forced swim test.

Conclusion

These results suggest that MD enhances behavioral vulnerability to stress during adulthood, which is associated with the upregulation of miR-504 and downregulation of DRD2 expression in the nucleus accumbens.     

膜翅目昆虫干标本的基因组DNA提取

提出了膜翅目昆虫干标本基因组DNA的提取方法,通过对实验结果和所测得的基因片段（28S D2 rDNA)的分析,表明该方法可行。