绿僵菌黏附素MAD1体外表达及诱导花生响应的作用

【目的】绿僵菌具有昆虫致病性和植物共生性,其黏附素MAD1在绿僵菌侵染寄主昆虫过程中起重要作用。通过体外真核表达MAD1蛋白,明确MAD1在绿僵菌与植物共生中发挥的作用。【方法】以绿僵菌转录组的Unigenes作为参考序列,在GenBank中进行同源性检索比对,设计引物,以金龟子绿僵菌（Metarhizium anisopliae）IPPM010202菌株的cDNA作为模板,克隆得到mad1,通过DNAMAN对mad1序列进行蛋白翻译,利用ProtParam在线工具预测MAD1蛋白氨基酸组成及其理化特性,利用SMART在线程序分析其结构域。构建mad1重组真核表达载体并转化毕赤酵母,获得重组子,通过甲醇诱导表达得到MAD1蛋白;通过Ni-NTA亲和层析获得纯化蛋白。以纯化蛋白溶液（20 μg·mL^-1）浸没处理花生根0.5、6、12和24 h,通过qPCR检测花生根膜受体基因（CERK1、RPK）、免疫相关级联基因（MAPK、MMK1、CDPK）和转录因子基因（MYB86）、细胞壁整合基因（SCW1）及防御相关基因（PTi1、RML1A）的转录水平。【结果】克隆得到了绿僵菌黏附素基因mad1,全长2 136 bp,编码711个氨基酸,分子量约为74.8 kD;生物信息学分析表明,MAD1的N端有信号肽,C端有糖基磷脂酰肌醇（GPI）细胞壁锚定位点,且含有CFEM功能结构域,属于亲水性蛋白。成功构建了MAD1真核表达系统,诱导表达并纯化出具有生物活性的MAD1蛋白,对花生根进行不同时间段的处理,发现MAD1处理后0.5 h,根尖细胞感知并启动膜类识别受体基因CERK1表达;0.5—6 h,根膜受体基因CERK1、RPK转录水平均上调,膜整合基因SCW1转录由下调转为上调,免疫反应相关基因MAPK、MMK1、CDPK、MYB86的转录受到短暂抑制,防御基因PTi1、RML1A转录下调,植物根部免疫防御反应受到抑制;6—12 h,膜受体基因维持上调表达,而防御基因RML1A从下调逆转为强烈上调;12—24 h,膜受体类基因CERK1、RPK,防御基因PTi1、RML1A均上调,免疫相关级联基因MAPK、MMK1、CDPK,转录因子基因MYB86也出现微弱的上调,植物根部启动免疫防御反应。【结论】在绿僵菌与花生根的互作早期,绿僵菌MAD1蛋白 6 h时已激活花生根膜受体基因CERK1和RPK的识别响应,同时抑制花生免疫级联基因MAPK、CDPK、MMK1及防御相关基因如PTi1、RML1A的表达,有助于绿僵菌在花生根组织上定殖;随后诱导花生细胞壁整合基因SCW1上调表达,参与根上受损细胞壁的修复与重建,促进绿僵菌与花生共生关系的建立。诱导植物的免疫抑制和细胞壁重建整合可能是绿僵菌与植物建立共生关系的重要步骤。     

Synergy research: approaching a new generation of phytopharmaceuticals

The longstanding, successful use of herbal drug combinations in traditional medicine demands that we find a rationale for their comparative pharmacological and therapeutic superiority to isolated single constituents. The synergistic efficacy of these combinations can be evaluated and verified by Berenbaum's isobole method, followed by clinical studies performed in comparison with synthetic standard drugs. There are many examples of mono- and multi-extract combinations used presently, which exhibit synergistic efficiency based on multi-target mechanisms of action. Among the natural products, gallocatechins of green tea and curcuminoids of ginger are the presently favoured polyphenols for a possible future use in co-medication with antibiotics and standard anticancer drugs. The main targets were found to be COX 1 + 2, NF-κB, and membrane glycoproteins that belong to the ATP-binding cassette (ABC) transporter family.     

现代林业发展阶段的初步划分

以林业发展历程,两次现代化理论及林业的定位等为客观依据,从动态的、发展的视角,将我国现代林业的发展划分为5个阶段:起步阶段、发展阶段、初步实现阶段、基本实现阶段和全面实现阶段,并具体分析了各发展阶段的特点。     

VA菌根菌与宿主植物之间的相互作用

综述了VA菌根菌与宿主植物这一共生体系之间在生理、分子水平上的相互作用.     

基于共生理论的村域尺度下农村居民点空间重构策略与实现

农村居民点利用低效化是城乡转型进程中乡村地域系统演化的一种不良过程,对其进行空间重构成为合理利用土地资源、扎实推进新农村建设、重构乡村空间、促进城乡一体化发展的突破口。该研究引入共生理论,通过分析农村居民点空间重构共生系统,明确重构原则与步骤,构建农村居民点空间重构策略,对整村推进示范村重庆市合川区大柱村予以实证,构筑了生产功能型、服务功能型、生活功能型3种农村居民点组团并在村域尺度下予以空间表达,形成"一轴一带三团"的村域空间布局。结果表明,共生理论对于农村居民点空间重构具有较强的适用性;基于共生策略的农村居民点空间重构既能确保农村居民点的互联互通,又能尊重农户主体地位,实现村域"资源共享、环境共建",发挥村域资源优势,提高农村居民点利用效率,为丘陵山区整村推进与美丽乡村建设提供一种新思路。     

Research status of soybean symbiosis nitrogen fixation

Soybean (Glycine max) is one of the most important economic legume crops with largest planting area, and is also an important oil crop, as well as food and feed material. Soybean-rhizobia symbiosis plays important roles in plant cultivation and fertilizer application. However, there are many problems in agricultural application of soybean symbiotic nitrogen fixation. In this review, we summarized three restriction factors (host specificity, low nitrogen fixation efficiency and abiotic stress) and discussed research progresses of these factors. Clarification of host specific mechanism will help to select and apply rhizobia inoculants. Both maintaining high nitrogenase activity and delaying nodule senescence can improve the efficiency of symbiotic nitrogen fixation. Abiotic stress-tolerant rhizobia can improve the abiotic stress tolerance of soybean. Breeding stress tolerant genotypes of soybean and rhizobia, obtaining correlated genes are the common strategies to improve soybean symbiotic nitrogen fixation under extreme conditions. Regulatory mechanisms of these restriction factors are still poorly understood and needs further clarification.     

Assessing arbuscular mycorrhizal plant metal uptake and soil metal bioavailability among ‘dwarf’ sunflowers in a stratified compartmental growth environment

Using the micronutrient zinc (Zn) as a metal contaminant, a stratified compartmental pot greenhouse experiment involving ‘dwarf’ sunflowers and an arbuscular mycorrhizal (AM) fungus was designed to assess the role of AM symbiosis toward plant growth and metal uptake, and to differentiate its impact toward edaphic parameters across different soil strata. Consistent with previous hypotheses, AM plants contained up to 40% lower metal concentrations in their shoots than non-AM plants, particularly at the highest soil Zn levels (200 and 400 mg Zn kg^?1 dry soil); this, corresponding with an enhanced growth status among AM plants. Upon assessing the soil Zn concentrations and pH, AM treatments also tended to have higher soil Zn levels and more alkaline conditions compared to non-AM treatments. This was found especially in the topmost soil stratum where AM root colonization was deemed most active as evidenced by a higher frequency of extraradical hyphae, vesicles, and arbuscules. Together, these effects were putatively linked to the AM-induced mechanism of metal biosorption known to modulate soil nutrient bioavailability and even delay the onset of metal toxicity.     

Arbuscular mycorrhizal dependency of different plant species and cultivars

The degree of plant growth change associated with arbuscular mycorrhizal (AM) colonization is expressed as mycorrhizal dependency (MD). In this review, previous reports on the differences in MD among plant species or cultivars were surveyed, and the factors affecting the differences are discussed. Mean values of MD were 44% for field crops (37 species), 56% for forage crops (46 species), 70% for wild grasses and forbs (140 species), 79% for trees (26 species), and 56% for all plants (250 species), indicating that the cultivated plant species showed a lower MD than the wild plant species. MD was negatively correlated with root morphological characteristics such as root length, root dry weight, root hair length, density of root hairs, the ability of roots to acquire phosphate from soil, and the phosphorus utilization efficiency of the host plant. Inoculation of arbuscular mycorrhizal fungi (AMF) for low input systems should be carried out in considering differences in MD.     

High and low nodulation in relation to molecular diversity of chickpea mesorhizobia in Indian soils

Chickpea (Cicer arietinum L.) nodulation variants of two cultivars ICC 4948 and ICC 5003 were used as trap plants to isolate 385 native rhizobia from CCS Haryana Agricultural University, Hisar farm soil. After authentication and considering growth characteristics, selected 110 rhizobia revealed immense molecular diversity using the profiles of DNA fragments generated by Polymerase chain reaction (PCR) with enterobacterial repetitive intergeneric consensus (ERIC) sequences. Low nodulating variants of cvs ICC 4948 and ICC 5003 were able to trap more numbers of rhizobial genotypes, namely seven as compared four to five by high nodulating variants of these cultivars. Overall eight rhizobial genotypes were trapped by the chickpea cultivars. Rhizobial isolates from same nodule or same plants were present in the same or different clusters and few isolates showed 100% similarity also. Based on nodules from a plant, nodulation variant or cultivar, rhizobia could not be differentiated and no exclusive cluster was formed by either rhizobial isolates from low or high nodulating variants of both the cultivars. Two most efficient rhizobial isolates LN 707b and LN 7007 were characterized by amplification and sequencing of 16S rRNA gene. Rhizobial isolate LN 707b showed more than 98% similarity with Mesorhizobium sp SH 2851 and Mesorhizobium mediterraneum. Another isolate LN 7007 showed more than 99% similarity with the sequence of 16S r RNA gene of Mesorhizobium sp STM 398, and M. mediterraneum. So the chickpea rhizobia from Northern Indian subcontinent are proposed to be kept under M. mediterraneum strain LN707b and LN 7007.     

金龟子绿僵菌黏附素基因mad1的敲除及功能分析

金龟子绿僵菌能够寄生昆虫,也能与植物共生。黏附素MAD1是绿僵菌与宿主互作初期的黏附因子。已知它在昆虫的侵染和致病中起重要作用,但与植物的互作机制报道甚少。为了研究MAD1在绿僵菌与植物共生中的作用,我们通过同源重组构建了金龟子绿僵菌mad1敲除株,并检测了敲除株的生长、产孢、孢子萌发及毒力等生物学特性,进一步利用qRT-PCR分析了MAD1在调节花生免疫响应中的作用。结果显示,与野生型菌株相比,敲除株产孢量降低了43.67%,分生孢子萌发中时为27.69 h,显著长于野生型菌株的13.43 h。敲除株对家蚕的半致死时间LT50为8.9 d,较野生型菌株的7.62 d显著延长。敲除株处理花生6 h后,花生免疫类基因CNGC1、PCD4,抗病基因SWEET10及转录因子WRKY41、MYB86的转录水平出现显著上调,而钙调素CML5、CML19的转录表达受到明显抑制。本研究证明了mad1是金龟子绿僵菌产孢、孢子萌发及毒力的正相关基因,并且MAD1在金龟子绿僵菌与植物相互作用初期,抑制植物抗性级联反应,减弱过敏反应,降低对微生物的抵御能力,同时增强共生信号的传导,这些作用有助于金龟子绿僵菌在花生根组织上定殖。