过表达腺苷生物合成正相关基因GlPNP提高灵芝腺苷的含量

【背景】灵芝被纳入我国“药食同源”试点名单，腺苷作为其主要活性物质之一，在免疫调节、抗炎、抗癌等方面发挥着重要作用。【目的】调控腺苷生物合成关键酶基因的表达来提高灵芝腺苷产量。【方法】将不同培养时间阶段腺苷合成酶基因(包括5-氨基咪唑-4-甲酰胺核糖核苷酸甲酰转移酶GlATIC、嘌呤核苷磷酸化酶GlPNP、腺苷激酶GlADK)的表达量与腺苷含量相关联，筛选出与灵芝腺苷含量呈正相关的关键酶基因。克隆关键酶基因并在灵芝中过表达，探究关键酶基因过表达对灵芝腺苷积累的影响。【结果】GlPNP的表达与灵芝腺苷含量呈正相关。GlPNP的cDNA全长为969 bp，预测GlPNP蛋白的相对分子量为34.6 kDa，呈三聚体的四元结构。研究结果表明，过表达菌株中GlPNP的表达量在第4天比野生型菌株(WT)上调了2.9-3.9倍，与含空载体的菌株(CK)相比，腺苷含量分别提高了78%和63%。【结论】过表达嘌呤核苷磷酸化酶是提高灵芝腺苷产量的一种有效手段。     

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.     

Photosynthetic generation of heterologous terpenoids in cyanobacteria

The work aims to convert the secondary slow metabolism of the terpenoid biosynthetic pathway into a primary activity in cyanobacteria and to generate heterologous products using these photosynthetic microorganisms as cell factories. Case study is the production of the 10-carbon monoterpene β-phellandrene (PHL) in Synechocystis sp. PCC 6803 (Synechocystis). Barriers to this objective include the slow catalytic activity of the terpenoid metabolism enzymes that limit rates and yield of product synthesis and accumulation. “Fusion constructs as protein overexpression vectors” were applied in the overexpression of the geranyl diphosphate synthase (GPPS) and β-phellandrene synthase (PHLS) genes, causing accumulation of GPPS up to 4% and PHLS up to 10% of the total cellular protein. Such GPPS and PHLS protein overexpression compensated for their slow catalytic activity and enabled transformant Synechocystis to constitutively generate 24 mg of PHL per g biomass (2.4% PHL:biomass, w-w), a substantial improvement over earlier yields. The work showed that a systematic overexpression, at the protein level, of the terpenoid biosynthetic pathway genes is a promising approach to achieving high yields of prenyl product biosynthesis, on the way to exploiting the cellular terpenoid metabolism for commodity product generation.     

A putative 6‐transmembrane nitrate transporter OsNRT1.1b plays a key role in rice under low nitrogen

OsNRT1.1a is a low-affinity nitrate(NO_3~-) transporter gene. In this study, another mRNA splicing product, OsNRT1.1b,putatively encoding a protein with six transmembrane domains, was identified based on the rice genomic database and bioinformatics analysis. OsNRT1.1a/OsNRT1.1b expression in Xenopus oocytes showed OsNRT1.1a-expressing oocytes accumulated ~(15)N levels to about half as compared to OsNRT1.1bexpressing oocytes. The electrophysiological recording of OsNRT1.1b-expressing oocytes treated with 0.25 mM NO_3~- confirmed ~(15)N accumulation data. More functional assays were performed to examine the function of OsNRT1.1b in rice. The expression of both OsNRT1.1a and OsNRT1.1b was abundant in roots and downregulated by nitrogen(N) deficiency. The shoot biomass of transgenic rice plants with OsNRT1.1a or OsNRT1.1b overexpression increased under various N supplies under hydroponic conditions compared to wild-type(WT). The OsNRT1.1a overexpression lines showed increased plant N accumulation compared to the WT in 1.25 mM NH_4NO_3 and 2.5 mM NO_3~- or NH_4~+ treatments, but not in 0.125 mM NH_4NO_3.However, OsNRT1.1b overexpression lines increased total N accumulation in all N treatments, including 0.125 m M NH_4NO_3,suggesting that under low N condition, OsNRT1.1b would accumulate more N in plants and improve rice growth, but also that OsNRT1.1a had no such function in rice plants.     

Ligation of newly replicated DNA controls the timing of DNA mismatch repair

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Analyses of Sexual Reproductive Success in Transgenic and/or Mutant Plants

The pistil, the female reproductive organ of plants, is a key player in the success of sexual plant reproduction. Ultimately, the production of fruits and seeds depends on the proper pistil development and function. Therefore, the identification and characterization of pistil expressed genes is essential for a better understanding and manipulation of the plant reproduction process. For studying the function of pistil expressed genes, transgenic and/or mutant plants for the genes of interest are used. The present article provides a review of methods already exploited to analyze sexual reproductive success. We intend to supply useful information and to guide future experiments in the study of genes affecting pistil development and function.     

Over‐expressed MST1 impaired spatial memory via disturbing neural oscillation patterns in mice

The activated mammalian Ste20‐like serine/threonine kinases 1 (MST1) was found in the central nervous system diseases, such as cerebral ischemia, stroke and ALS, which were related with cognitions. The aim of this study was to examine the effect of elevated MST1 on memory functions in C57BL/6J mice. We also explored the underlying mechanism about the pattern alteration of neural oscillations, closely associated with cognitive dysfunctions, at different physiological rhythms, which were related to a wide range of basic and higher‐level cognitive activities. A mouse model of the adeno‐associated virus (AAV)‐mediated overexpression of MST1 was established. The behavioral experiments showed that spatial memory was significantly damaged in MST1 mice. The distribution of either theta or gamma power was clearly disturbed in MST1 animals. Moreover, the synchronization in both theta and gamma rhythms, and theta‐gamma cross‐frequency coupling were significantly weakened in MST1 mice. In addition, the expressions of GABAA receptor, GAD67 and parvalbumin (PV) were obviously increased in MST1 mice. Meanwhile, blocking MST1 activity could inhibit the activation of FOXO3a and YAP. The above data suggest that MST1‐overexpression may induce memory impairments via disturbing the patterns of neural activities, which is possibly associated with the abnormal GABAergic expression level.     

Expression of the INU2 gene for an endoinulinase of Aspergillus ficuum in Saccharomyces cerevisiae

The INU2 gene encoding an endoinulinase of Aspergillus ficuum was expressed by the Kluyveromyces marxianus INU1 promoter in a SUC2-deleted Saccharomyces cerevisiae to produce the endoinulinase preparation free of an exoinulinase and an extracellular invertase in the culture medium. A recombinant yeast strain produced the sufficient amount of the enzyme to make a halo around its colony, when inulin was included in the medium.