Establishment of a Genetic Transformation System and Its Application in Thermoanaerobacter tengcongensis

The whole-genome sequence of Thermoanaerobacter tengcongensis,an anaerobic thermophilic bacterium isolated from the Tengchong hot spring in China,was completed in 2002.However,in vivo studies on the genes of this strain have been hindered in the absence of genetic manipulation system.In order to establish such a system,the plasmid pBOLOl containing the replication origin of the T.tengcongensis chromosome and a kanamycin resistance cassette,in which kanamycin resistance gene expression was controlled by the ttel482 promoter from T.tengcongensis,was constructed and introduced into T.tengcongensis via electroporation.Subsequently,the high transformation efficiency occurred when using freshly cultured T.tengcongensis cells without electroporation treatment,suggesting that T.tengcongensis is naturally competent under appropriate growth stage.A genetic transformation system for this strain was then established based on these important components,and this system was proved to be available for studying physiological characters of T.tengcongensis in vivo by means of hisG gene disruption and complementation.     

灭蚊真菌－贵阳腐霉对植物的安全性试验

The aim of this study is to investigate whether Pythium guiyangense, a mosquito-killing fungus isolated in Guiyang, Guizhou Province of China in 1994, is pathogenic to plants. Six common crops, Cucumis sativa, Lycopersicon esculentum, Capsicum annuum, Nicotiana tabacum, Brassica campestris and Oryza sativa were used as subjects for test. Zoospores of the fungus were used to infect the plants with soil inoculation method, caudex injection method and foliage spray method. Both positive control (using P. aphanidermatum) and negative control (using sterile water) were set up in all the experiments. The results showed that no infection was found on the tested plants in soil inoculation experiments. In caudex injection test, callus grew around the wounded tissue in most of the plants. Brownish rottenness could be found only in the injected wounds in a few plants, probably caused by saprophytic bacteria or other fungi, and the germ-carrying plants grew normally. No abnormal appearance was found on the six crops in foliage spray test. It was demonstrated that P. guiyangense could hardly infect plants in nature, and was a safe and promising agent for mosquito biological control.     

Establishment of an efficient transformation protocol and its application in marine-derived Bacillus strain

Marine-derived Bacillus strains have been proved to be a very promising source for natural product leads.However,transformation of environmental strains is much more difficult than that of domesticated strains.Here,we report the development of an efficient and robust electroporation-based transformation system for marine-derived Bacillus marinus B-9987,which is a macrolactin antibiotics producer and a very promising biological control agent against fungal plant diseases.The transformation efficiency was greatly enhanced 103-fold by using unmethylated plasmid to bypass modification-restriction barrier,and using glycine betaine to protect cells from electrical damages during electroporation.Addition of HEPES and 2 mmol L?1MgCl2 further improved the efficiency by additional 2-fold,with a maximum value of 7.1×104 cfu/μg pHT3101.To demonstrate the feasibility and efficiency of the protocol,a green fluorescent protein reporter system was constructed;furthermore,phosphopantetheinyl transferase gene sfp,which is essential to the biosynthesis of polyketides and nonribosomal peptides,was overexpressed in B-9987,leading to increased production of macrolactin A by about 1.6-fold.In addition,this protocol is also applicable to marine-derived Bacillus licheniforms EI-34-6,indicating it could be a reference for other undomesticated Bacillus strains.To our knowledge,this is the first report regarding the transformation of marine-derived Bacillus strain.     

Roles of cortactin, an actin polymerization mediator, in cell endocytosis

Cortactin, an actin-binding protein and a substrate of Src, is encoded by the EMS 1 oncogene. Cortactin is known to activate Arp2/3 complex-mediated actin polymerization and interact with dynamin, a large GTPase and proline rich domain-containing protein. Transferrin endocytosis was significantly reduced in cells by knock-down of cortactin expression as well as in vivo introduction of cortactin immunoreagents. Cortactin-dynamin interaction displayed morphologically dynamic co-distribution with a change in the endocytosis level in cells treated with an actin depolymerization reagent, cytochalasin D. In an in vitro beads assay, a branched actin network was recruited onto dynamin-coated beads in a cortactin Src homology domain 3 （SH3）-dependent manner. In addition, cortactin was found to function in the late stage of clathrin coated vesicle formation. Taken together, cortactin is required for optimal clathrin mediated endocytosis in a dynamin directed manner.     

Histological and Ultrastructural Observation Reveals Significant Cellular Differences between Agrobacterium Transformed Embryogenic and Non-embryogenic Calli of Cotton

Over the past few decades genetic engineering has been applied to improve cotton breeding. Agrobacterium medicated transformation is nowadays widely used as an efficient approach to introduce exogenous genes into cotton for genetically modified organisms. However, it still needs to be improved for better transformation efficiency and higher embryogenic callus induction ratios. To research further the difference of mechanisms for morphogenesis between embryogenic callus and non-embryogenic callus, we carried out a systematical study on the histological and cellular ultrastructure of Agrobacterium transformed calli. Results showed that the embryogenic callus developed nodule-like structures, which were formed by small, tightly packed, hemispherical cells. The surface of some embryogenic callus was covered with a fibrilar-like structure named extracellular matrix. The cells of embryogenic calli had similar morphological characteristics. Organelles of embryogenic callus cells were located near the nucleus, and chloroplasts degraded to proplastid-like structures with some starch grains. In contrast, the non-embryogenic calli were covered by oval or sphere cells or small clusters of cells. It was observed that cells had vacuolation of cytoplasm and plastids with a well organized endomembrane system. This study aims to understand the mechanisms of embryogenic callus morphogenesis and to improve the efficiency of cotton transformation in future.     

Apoptosis commitment--translating survival signals into decisions on mitochondria

Most defective and unwanted cells die by apoptosis, cells without damaging the surrounding tissue. Once a an exquisitely controlled genetic programme for removing such cell has committed to apoptosis, the process is remarkably efficient, and is completed within a few minutes of initiation. This point of no retum for an apoptotic cell is commonly held to be the point at which the outer mitochondrial membrane is permeabilised, a process regulated by the Bcl-2 family of proteins. How these proteins regulate this decision point is central to diseases such as cancer where apoptotic control is lost. In this review, we will discuss apoptotic signalling and how a cell makes the irreversible decision to die. We will focus on one set of survival signals, those derived by cell adhesion to the extracellular matrix （ECM）, and use these to highlight the complexities of apoptotic signalling. In particular, we will illustrate how multiple signalling pathways converge to determine critical cell fate decisions.     

Construction of brewing-wine Aspergillus oryzaepyrG- mutant bypyrG gene deletion and its application in homology transformation

pyrG- host cells are indispensable for pyrG- based transformation system. Isolations ofpyrG- host cells by random mutations are limited by time-consuming, unclear genetic background and potential interferences of homogenous recombination. The purpose of this study was to construct brewing-wine Aspergillus oryzae pyrG- mutant by sitedirected mutation of pyrG gene deletion which would be used as a host for further transformation, pMD-pyrGAB, a vector carrying pyrG deletion cassette, was used to con- struct pyrG- mutant of A. oryzae. Three stable pyrG deletion mutants of A. oryzae were isolated by resistant to 5-fluoroorotic acid and confirmed by polymerase chain reaction analysis, indicating thatpyrG was completely excised. The ApyrG mutants were applied as pyrG- host cells to disrupt xdh gene encoding xylitol dehydrogenase, which involves in xylitol production ofA. Oryzae. The xdh disrup- tion mutants were efficiently constructed by transforming a pMD-pyrC-xdh disruption plasmid carrying pyrG, and the produced xylitol concentration of the Axdh mutant was three times as much as that of the ApyrG recipient. Site-directed pyrG gene deletion is thus an effective way for the isolation of pyrG- host cells, and the established host-vector system could be applied in further functional genomics analysis and molecular breeding ofA. Oryzae.     

Measurement and Prediction of Insertion Force for the Mosquito Fascicle Penetrating into Human Skin

Mosquitoes are exceptional in their ability to pierce into human skin with a natural ultimate painless microneedle, namedfascicle. Here the structure of the Aedes albopictus mosquito fascicle is obtained using a Scanning Electron Microscope (SEM),and the whole process of the fascicle inserting into human skin is observed using a high-speed video imaging technique. Directmeasurements of the insertion force for mosquito fascicle to penetrate into human skin are reported. Results show that themosquito uses a very low force (average 18 μN) to penetrate into the skin. This force is at least three orders of magnitude smallerthan the reported lowest insertion force for an artificial microneedle with an ultra sharp tip to insert into the human skin. In orderto understand the piercing mechanism of mosquito fascicle tip into human multilayer skin tissue, a numerical simulation isconducted to analyze the insertion process using a nonlinear finite element method. A good agreement occurs between thenumerical results and the experimental measurements.     

Profile of Dr. Shaojun Liu

正Dr. Shao-Jun Liu is an expert in fish genetic breeding, who was born in 1962 in Changsha, Hunan province, China. He received his B.S. in biology in 1986 and his M.S. in zoology in 1989 at Hunan Normal University. He obtained his Ph.D.in zoology in 2000 at Sun Yat-sen University. From 1998 to1999, he was supported by the China Scholarship Council to research fish molecular biology in France. In 2019, he was elected as an academician of the Chinese Academy of Engineering, and currently, he is a Professor at Hunan Normal University and the Director of the State Key Laboratory of the Developmental Biology of Freshwater Fish.     

Genetic diversity in Chinese modern wheat varieties revealed by microsatellite markers

Genetic diversity of 1680 modern varieties in Chinese candidate core collections was analyzed at 78 SSR loci by fluorescence detection system. A total of 1336 alleles were detected, of which 1253 alleles could be annotated into 71 loci. For these 71 loci, the alleles ranged from 4 to 44 with an average of 17.6, and the PIC values changed from 0.19 to 0.89 with an average of 0.69. (1) In the three genomes of wheat, the average genetic richness was B>A>D, and the genetic diversity indexes were B>D>A. (2) Among the seven homoeologous groups, the average genetic richness was 2=7>3>4>6>5>1, and the genetic diversity indexes were 7>3>2>4>6>5>1. As a whole, group 7 possessed the highest genetic diversity, while groups 1 and 5 were the lowest. (3) In the 21 wheat chromosomes, 7A, 3B and 2D possessed much higher genetic diversity, while 2A, 1B, 4D, 5D and 1D were the lowest. (4) The highest average genetic diversity index existed in varieties bred in the 1950s, and then it declined continually. However, the change tendency of genetic diversity among decades was not greatly sharp. This was further illustrated by changes of the average genetic distance between varieties. In the 1950s it was the largest (0.731). Since the 1960s, it has decreased gradually (0.711, 0.706, 0.696, 0.695). The genetic base of modern varieties is becoming narrower and narrower. This should be given enough attention by breeders and policy makers.     

根癌农杆菌介导的Pr1基因在贵阳腐霉的组成性表达

【目的】构建组成性表达贵阳腐霉Pr1基因的载体pCambia-hph-Pr1,转化贵阳腐霉,以获得高毒力的基因工程转化菌株。【方法】以双元载体pCambia-Pnos-PNN-hph为基本骨架,将Pr1基因置于组成型启动子PgpdA和终止子TtrpC之间,获得含Pr1基因的表达元件。以贵阳腐霉菌丝体为受体材料,利用根癌农杆菌介导的遗传转化法转化贵阳腐霉,观察转化株的生物学特征和灭蚊毒力。【结果】转化株的菌丝生长速度和游动孢子产量与野生株无显著性差异。生物测定表明转化株对蚊幼虫平均致死率达到32.9%,比野生株提高了约一倍,并且使受试蚊幼虫生长速度明显减缓。【结论】利用根癌农杆菌介导的遗传转化获得了遗传稳定的高毒力菌株。     

根癌农杆菌介导的香蕉遗传转化研究进展

香蕉的栽培品种绝大多数是三倍体, 常规育种难度大, 工作周期长, 难以采用传统的育种方法进行遗传改良, 因此转基因技术成为改良香蕉种质的有效方法。根癌农杆菌介导的香蕉的遗传转化从20世纪90年代起取得的了较大的进步, 但仍然存在着很多问题。文章分析了影响农杆菌介导香蕉转化的几个重要环节, 并对其研究现状、存在的问题及应用前景作简要概述。     

农杆菌介导GUS基因对多年生黑麦草转化的研究

通过检测愈伤组织中GUS基因的瞬间表达,研究农杆菌LBA4404/pCAMBIA1301介导多年生黑麦草的转化体系。通过对多年生黑麦草瞬间表达率的比较,确立了其遗传转化的最佳优化条件。研究发现,多年生黑麦草不同品种的转化率在25%~45%之间变化。多年生黑麦草遗传转化最佳优化条件是预培养10d的胚性愈伤组织、浓度为0.5~0.8OD的农杆菌菌液以及2d共培养时间。在共培养基中添加100μmol/L乙酰丁香酮能有效地提高植物瞬间表达率。两种侵染处理方法比较结果为滤纸滴加法比浸泡法更优。转化后对愈伤组织的干燥处理能抑制农杆菌过度繁殖,能改善愈伤状态,有利于提高转化率。     

农杆菌介导转基因植物T-DNA的整合方式

农杆菌介导的遗传转化已被广泛应用于植物转基因研究。作为外源基因的载体,农杆菌T-DNA片段在植物基因组中的整合方式,不仅影响外源基因的整合效率及稳定性,还会影响外源基因的表达特性。文章就农杆菌介导的T-DNA整合的两种主要模式、规律及相关研究手段进行综述,为农杆菌介导的转基因及T-DNA插入突变等相关研究提供借鉴。     

影响根癌农杆菌转化的因素及其在单子叶作物上的应用

在植物转基因方法中,根癌农杆菌介导的遗传转化应用最为广泛,进一步提高其转化频率并扩大其宿主范围到禾谷类作物是人们所关注的问题。有多种因素影响根癌农杆菌的转化频率,包括植物的受伤反应、细菌的吸附、致病基因的诱导、植物细胞ＤＮＡ合成及修复的活力、外植体的状态等。最近的研究结果证明在适宜的条件下,根癌农杆菌还是可以有效地转化禾谷类作物。本文试就这两方面的研究进展作一论述。     

Interaction of the virulence protein VirF of Agrobacterium tumefaciens with plant homologs of the yeast Skp1 protein

The infection of plants by Agrobacterium tumefaciens leads to the formation of crown gall tumors due to the transfer of a nucleoprotein complex into plant cells that is mediated by the virulence (vir) region-encoded transport system (reviewed in [1-5]). In addition, A. tumefaciens secretes the Vir proteins, VirE2 and VirF, directly into plant cells via the same VirB/VirD4 transport system [6], and both assist there in the transformation of normal cells into tumor cells. The function of the 22 kDa VirF protein is not clear. Deletion of the virF gene in A. tumefaciens leads to diminished virulence [7, 8] and can be complemented by the expression of the virF gene in the host plant. This finding indicates that VirF functions within the plant cell [8]. Here, we report that the VirF protein is the first prokaryotic protein with an F box by which it can interact with plant homologs of the yeast Skp1 protein. The presence of the F box turned out to be essential for the biological function of VirF. F box proteins and Skp1p are both subunits of a class of E3 ubiquitin ligases referred to as SCF complexes. Thus, VirF may be involved in the targeted proteolysis of specific host proteins in early stages of the transformation process.     

Identification and characterization of plant genes involved in Agrobacterium-mediated plant transformation by virus-induced gene silencing

Genetic transformation of plant cells by Agrobacterium tumefaciens represents a unique case of trans-kingdom sex requiring the involvement of both bacterial virulence proteins and plant-encoded proteins. We have developed in planta and leaf-disk assays in Nicotiana benthamiana for identifying plant genes involved in Agrobacterium-mediated plant transformation using virus-induced gene silencing (VIGS) as a genomics tool. VIGS was used to validate the role of several genes that are either known or speculated to be involved in Agrobacterium-mediated plant transformation. We showed the involvement of a nodulin-like protein and an alpha-expansin protein (alpha-Exp) during Agrobacterium infection. Our data suggest that alpha-Exp is involved during early events of Agrobacterium-mediated transformation but not required for attaching A. tumefaciens. By employing the combination of the VIGS-mediated forward genetics approach and an in planta tumorigenesis assay, we identified 21 ACG (altered crown gall) genes that, when silenced, produced altered crown gall phenotypes upon infection with a tumorigenic strain of A. tumefaciens. One of the plant genes identified from the screening, Histone H3 (H3), was further characterized for its biological role in Agrobacterium-mediated plant transformation. We provide evidence for the role of H3 in transfer DNA integration. The data presented here suggest that the VIGS-based approach to identify and characterize plant genes involved in genetic transformation of plant cells by A. tumefaciens is simple, rapid, and robust and complements other currently used approaches.     

灭蚊真菌贵阳腐霉孢子囊和游动孢子形成的观察

贵阳腐霉是很有应用前景的灭蚊真菌.其无性繁殖的游动孢子是其侵犯蚊虫体壁的生理阶段.观察了孢子囊形成的时间及影响因素,并观察了游动孢子形成的动态过程.研究结果为其在灭蚊的实际应用提供了有价值的参考资料.     

灭蚊真菌贵阳腐霉Pythium guiyangense对大鼠的长期安全性测试

为了解灭蚊真菌贵阳腐霉Pythium guiyangense对大鼠是否有长期毒性作用。将大鼠120只随机分4组,经饮水口服贵阳腐霉菌丝体。菌丝剂量分别为200mg/kg、100mg/kg、50mg/kg,对照组饮自来水。分别于试验期的90d和180d各组取鼠总数的1/3处死进行检查,剩余1/3大鼠待停用菌丝体悬液2周后处死。观察大鼠一般情况、血常规、肝肾功能等各项生化和组织学指标。结果表明各组大鼠均发育正常,精神食欲好,体重增加;血液学、生化指标变化无明显的剂量-效应关系;内脏系数无异常;对重要脏器的解剖学及组织学检查未见明显异常。本研究结果证明灭蚊真菌贵阳腐霉对大鼠是安全的,从而为其在防治蚊虫的推广应用中的安全性提供了重要的毒理学依据。     

NaCl溶液对贵阳腐霉菌丝生长、孢子囊产生及灭蚊能力的影响

研究不同浓度NaCl溶液对贵阳腐霉菌丝生长、产孢能力及灭蚊能力的影响,探讨贵阳腐霉对NaCl溶液的耐受性,为其在含盐水体中控制蚊虫种群的应用提供理论依据。结果表明：低浓度NaCl溶液可以促进贵阳腐霉菌丝生长及孢子囊形成,高浓度NaCl溶液则抑制菌丝生长及孢子囊形成。于0.01%浓度NaCl溶液中培养24h菌丝生长速度最快,产孢子囊数量最多,与对照相比差异显著。随着NaCl浓度升高,菌丝及孢子囊形成均受影响。菌丝生长速度降低,孢子囊形成数量减少。在0.75% NaCl溶液中贵阳腐霉的菌丝生长严重受抑制,且产孢子囊能力为零。灭蚊实验表明,0.01% NaCl溶液中贵阳腐霉的灭蚊能力在72–96h时均比蒸馏水对照组强,但二者未有显著差异,120h蒸馏水对照组灭蚊能力均明显高于各浓度NaCl溶液处理组;蚊虫总感染率蒸馏水对照组均显著高于NaCl溶液处理组。