金属离子胁迫对Sphingomonas sp. Y2降解壬基酚聚氧乙烯醚特性的影响

壬基酚聚氧乙烯醚（NEPOs）是全球应用量最大的非离子型表面活性剂之一,具有环境雌激素毒性。NPEOs的中间代谢产物种类多、难降解,且毒性远高于其母系化合物。为研究金属离子对功能微生物Sphingomonas sp. Y2降解NPEOs特性的影响,分析了金属离子的最低抑制浓度（MIC）、细菌形态、NPEOs降解效率及代谢产物组成等变化。结果显示,菌株Y2对多种金属离子具有耐受性,在重金属培养基中对Mn²⁺、Zn²⁺具有较高的耐受性,MIC分别为500、90 mg/L;在500 mg/L Mn²⁺胁迫下,菌株Y2对NPEOs降解率为100.00%（3 d）;在90 mg/L Zn²⁺胁迫下,菌株Y2对NPEOs的降解率为20.62%（5 d）;两种离子双重胁迫下NPEOs降解率为15.65%（5 d）;Mn²⁺胁迫下菌株Y2细胞表面结构和形态发生明显变化,且改变了NPEOs代谢产物中组分的含量组成,其中短链NPEOs与短链壬基酚聚氧乙烯酸（NPECs）的比例为0.68,与对照相比,抑制/减缓了短链NPEOs的羧化反应。结果表明,菌株Sphingomonas sp. Y2对多种金属离子具有耐受性,Mn²⁺胁迫对细胞表面超微结构及NPEOs中间代谢产物组分组成产生显著影响。该研究将为表面活性剂类污染物的生物降解及相应代谢产物在环境中的毒性评价提供理论依据。     

基于碳硫模块平衡策略的大肠杆菌高产L-半胱氨酸菌株构建

l-半胱氨酸是一种重要的含硫氨基酸,因其多样的生理功能,l-半胱氨酸在医药、化妆品和食品工业中有着广泛的应用。模块化代谢工程策略在细胞工厂的构建中具有极大的潜力。本研究利用碳硫模块协同表达策略进行大肠杆菌的l-半胱氨酸合成途径构建,构建了一株l-半胱氨酸合成基因工程菌。首先,通过增强l-半胱氨酸前体物质l-丝氨酸(serA^f、serB和serC^Cg)的生物合成以及转录调控因子CysB的表达,l-半胱氨酸的产量由0提高到(0.38±0.02)g/L。然后,通过促进l-半胱氨酸转运和无机硫源的吸收同化、减弱l-半胱氨酸和l-丝氨酸的降解以及异源表达cysE^f和cysB^St,l-半胱氨酸的产量提升至(3.82±0.01)g/L。最后,为了优化碳模块和硫模块的代谢通量,协同表达硫酸盐同化途径与硫代硫酸盐同化途径的基因cysM、nrdH、cysK以及cysIJ,得到l-半胱氨酸高产菌株。在500mL摇瓶和2L发酵罐中分别实现了(4.17±0.07)g/L和(11.94±0.1)g/L的l-半胱氨酸积累。研究结果表明,在细胞内通过对硫碳模块间代谢通量的协调控制,可以实现l-半胱氨酸的高效生物合成。研究结果为微生物发酵生产l-半胱氨酸的产业化奠定了基础。     

干酪乳杆菌arat基因的敲除及其对苯乳酸合成代谢的影响

苯乳酸（PLA）作为一种新型的广谱抑菌物质,在食品行业具有巨大的发展潜力,作为乳酸菌的天然代谢产物之一,对其代谢机理的研究具有非常重要的意义。通过CRISPR/Cas9基因编辑系统对一株干酪乳杆菌（Lactobacillus casei）1.8727的芳香族氨基转移酶基因arat进行敲除,得到一株arat缺失菌株（L. casei）1.8727Δarat。通过HPLC方法分析其代谢产物,发现发酵72 h时,PLA产量比出发菌株提高约66.7%,而苯丙氨酸（Phe）比出发菌株提高约57.8%。首次在L. casei 1.8727中成功应用基因编辑手段敲除了arat基因,研究表明该菌株具有自身合成代谢Phe的能力,arat作为PLA代谢途径的关键酶基因,其缺失并未使PLA产量下调,而是促进了PLA与Phe的合成代谢,证明干酪乳杆菌中可能存在其他的代偿途径,arat基因缺失所造成的代谢流的改变最终造成PLA与Phe产量的提高,同时PLA的合成代谢可能涉及多个基因的参与,是一个复杂的代谢网络。研究结果对于深入研究PLA的合成代谢机制具有重要意义,为后续PLA合成代谢途径提供了新的思路及方法。     

一株防治香蕉枯萎病的短密木霉筛选及代谢物木霉素作用评价

由尖孢镰刀菌古巴专化型热带四号小种(Fusarium oxysporum f. sp. cubense tropical race4, FocTR4)引起的香蕉枯萎病(banana Fusarium wilt, BFW)是全世界范围内难以防治的真菌病害,给香蕉产业造成巨大的经济损失。本研究旨在筛选高效拮抗FocTR4的木霉生防菌株,并对其发酵代谢产物进行分离、提纯和鉴定,为香蕉枯萎病的高效生物防治提供重要生防菌株和活性化合物资源。从作物根际土壤中分离出木霉菌株,通过平板对峙培养、发酵液对病原菌孢子萌发及菌丝生长抑制,测试筛选出高效抑制FocTR4的生防木霉菌株;通过构建系统发育树明确生防菌株的分类地位;通过柱色谱法分离纯化菌株发酵液中活性成分,通过核磁共振波谱法(nuclear magnetic resonance spectroscopy, NMR)解析活性成分的结构;通过香蕉苗感病盆栽实验检测生防木霉菌株对香蕉枯萎病的防治效果。结果表明,本研究筛选到了1株拮抗FocTR4的菌株JSHA-CD-1003,平板对峙抑制率为60.6%;发酵液在24 h内能完全抑制FocTR4孢子萌发,7 d内对FocTR4菌丝生长的抑制率为52.6%;基于内转录间隔区(internal transcribed spacer, ITS)和tef1-α基因串联序列构建系统发育树,该菌株鉴定为短密木霉(Trichoderma brevicompactum),通过柱色谱法分离提纯和NMR鉴定单一活性化合物为木霉素(trichodermin),最小抑菌浓度(minimum inhibitory concentration, MIC)为25 μg/mL;盆栽生防实验表明,菌株JSHA-CD-1003发酵液对香蕉枯萎病的叶片黄化防治率为47.4%,球茎褐化防治率为52.0%。因此,JSHA-CD-1003通过产生木霉素有效抑制FocTR4孢子萌发和菌丝生长,对FocTR4引起的香蕉枯萎病具有良好的生物防治效果,是一株具有生防潜力的菌株。     

L-丝氨酸生产菌的选育及不同碳源对发酵的影响

以一株黄假单胞属(Pseudomonas flava)菌株A3为出发菌株,经过紫外(UV)诱变和硫酸二乙酯(DES)逐级诱变处理和选育,选育出一株能够以甲醇为唯一碳源的兼性甲基营养型菌JW-01(Mth^R、Gly^R)。在含甘氨酸30g/L、甲醇1%的发酵培养基中发酵3d后L-丝氨酸产量为6．2g/L,较出发菌株提高了67．6%。该菌具有较好的传代稳定性。     

一株内生真菌Talaromyces sp.次级代谢产物及其活性

为了研究红树杨叶肖槿来源内生真菌Talaromyces sp. YX-001的次级代谢产物及其乙酰胆碱酯酶(acetylcholinesterase, AChE)抑制活性,该文运用硅胶柱层析和HPLC等手段从菌株YX-001的PDB培养基发酵提取物中分离得到单体化合物,而后结合1/2D NMR和MS等现代波谱技术并与文献数据对比鉴定其结构,最后通过比色法测定各单体化合物的AChE抑制活性。结果表明:(1)共分离得到9个单体化合物,分别为asterrelenin(1),aszonalenin(2),cladosporisteroid C(3),sitosterol(4),ergosterol(5),cyclo-Ile-Pro-diketopiperazine(6),cyclo(-Pro-Val)(7),4-methoxy-2-methylisoquinolin-1-one(8)和allantoin(9)。(2)其中化合物1和2显示出一定的AChE抑制活性,IC₅₀值分别为81.5、105.8 μmol·L^-1。该文首次对杨叶肖槿来源内生真菌次级代谢产物及其AChE抑制活性进行了研究,为后续红树植物杨叶肖槿来源内生真菌资源的开发与再利用奠定了基础。     

多细胞耦合转化N-乙酰氨基葡萄糖和乳糖生产唾液酸乳糖

唾液酸乳糖是母乳寡糖(human milk oligosaccharides, HMOs)中含量最丰富的唾液酸化低聚糖之一，对婴幼儿的健康发育有重要作用，但是其目前缺乏高效、廉价的生产工艺。针对该问题，本研究建立了多菌株两步法耦合生物合成唾液酸乳糖方法。第一步构建2株工程菌，大肠杆菌JM109(DE3)/pET28a-BT0453和JM109(DE3)/pET28a-nanA，用于耦合合成中间产物N-乙酰神经氨酸，在2株工程菌株细胞生物量比为1:1，反应时间为32 h的条件下，得到的N-乙酰神经氨酸最高产量为20.4 g/L。第二步向上述发酵液中添加大肠杆菌JM109(DE3)/pET28a-neuA、JM109(DE3)/ pET28a-nst和面包酵母，通过三菌株耦合发酵合成3ʹ-唾液酸乳糖(3ʹ-sialyllactose, 3ʹ-SL)。在底物N-乙酰氨基葡萄糖、乳糖浓度均为200 mmol/L，面包酵母细胞生物量为150 g/L，辅因子Mg²⁺浓度为20 mmol/L的优化条件下，发酵24 h，发酵液中3ʹ-唾液酸乳糖的最高产量达到55.04 g/L，底物N-乙酰氨基葡萄糖的转化率为43.47%。研究结果为低成本生产3ʹ-唾液酸乳糖提供了一条新的技术路线。     

ycjX和ycjF基因在布鲁氏菌热应激中的作用

【背景】细菌应对环境压力的能力对其存活和增殖及引起感染具有重要意义。充分揭示布鲁氏菌应激机制,可为防控布鲁氏菌病提供理论依据。研究发现,ycjX基因在细菌热应激时高表达且可能受σ³²调节,ycjF基因在细菌败血症期间表达显著上升,二者在革兰阴性菌中可能构成操纵子。【目的】研究ycjX和ycjF基因在布鲁氏菌热应激中的作用。【方法】对布鲁氏菌(Brucella)及其ycjX和ycjF双基因缺失株(△ycjXF)和回补株(C△ycjXF)进行热应激试验,计算3株菌的存活率。利用RT-PCR和β-半乳糖苷酶活性检测试验鉴定ycjX和ycjF的操纵子模式和启动子区域活性。利用ChIP试验分析σ³²与ycjX和ycjF的靶向调节关系。表达并纯化pGEX-4T-1-σ³²重组蛋白,凝胶电泳迁移试验分析σ³²与ycjX和ycjF之间的结合关系。【结果】热刺激后△ycjXF存活显著低于Brucella suis S2和C△ycjXF。明确布鲁氏菌ycjX和ycjF为同一转录本,确定其启动子区域具有活性。ChIP试验表明,σ³²可靶向富集在ycjXF启动子区,凝胶电泳迁移试验确定σ³²可与ycjXF体外直接结合。【结论】在σ³²的调节下,ycjX和ycjF在布鲁氏菌热应激中发挥正向作用。     

谷物蛋白对白酒发酵过程中微生物群落及其代谢多样性的调控

[背景] 在白酒发酵过程中,原料中的谷物蛋白可为微生物的生长提供氮源等营养物质,进而形成多种代谢产物。谷物蛋白可分为清蛋白、球蛋白、醇溶蛋白和谷蛋白。然而,谷物蛋白对微生物多样性及其代谢产物多样性的调控尚不明确。[目的] 揭示白酒发酵过程中与微生物多样性及其代谢产物多样性显著相关的关键谷物蛋白种类及其调控作用。[方法] 通过Osborne法测定不同品种高粱中谷物蛋白的组成;采用多组学联用技术解析4种高粱在发酵过程中的微生物菌群多样性及代谢产物多样性;通过模拟发酵揭示原料中影响微生物群落及其代谢多样性的关键蛋白。[结果] 4种高粱中的谷物蛋白组成存在显著差异（ANOSIM：R=0.85,P=0.001）;4种高粱在发酵第5天时,S4高粱的细菌多样性显著（P<0.05）高于其他3种高粱,S3高粱中微生物的代谢产物多样性显著（P<0.05）高于其他3种高粱;清蛋白和球蛋白含量与发酵第5天的优势细菌多样性（R²=0.34,P<0.05;R²=0.58,P<0.05）和代谢产物多样性呈显著正相关（R²=0.58,P<0.05;R²=0.36,P<0.05）,被定义为关键蛋白;模拟发酵实验验证了优势细菌多样性和代谢产物多样性可随着2种关键蛋白即清蛋白和球蛋白含量的升高而升高。当清蛋白含量在3.0 g/L时,优势细菌多样性及代谢产物多样性可分别达到0.72和0.65;当球蛋白含量在3.0 g/L时,优势细菌多样性及代谢产物多样性可分别达到0.66和0.81。[结论] 研究揭示了酿造原料中的清蛋白和球蛋白对发酵过程中细菌多样性及代谢产物多样性的调控作用,为提高白酒发酵的可控性及质量提供了依据。     

红树林沉积物中多环芳烃降解菌Aquabacter sediminisP-9T的分离、鉴定及降解机制的研究

多环芳烃(polycyclic aromatic hydrocarbons, PAHs)是一类具有致畸、致癌和致突变特性的高风险有毒污染物,近年来,关于红树林生态系统受到PAHs污染的报道引起广泛关注,微生物降解被认为是处理PAHs污染的有效、经济和多功能替代方法。目前,研究者发现了大量细菌利用PAHs污染作为碳源和能源,然而其普遍存在降解效率偏低、降解谱系窄、对高盐环境适应性差等问题,红树林来源的PAHs降解菌的降解机理尚待充分挖掘。【目的】在红树林沉积物中定向筛选针对PAHs的高效广谱降解菌,并深入探讨其降解效能与作用机制,以期为红树林生态系统中微生物污染修复技术的创新研发提供坚实的科学基础。【方法】从深圳福田红树林沉积物筛选出一株降解PAHs的潜在新种菌株P-9^T,通过形态学观察、生理生化特性检测和16S rRNA基因序列分析,对该菌株进行鉴定;基于菌株基因组测序与分析预测该菌株的PAHs代谢潜能;在不同温度(25-40 ℃)、不同pH (5.0-9.0)和不同底物条件下,对菌株P-9^T的降解能力进行测定;利用高效液相色谱-质谱联用(HPLC-ESI-MS/MS)技术检测菌株降解PAHs的中间代谢产物,初步揭示P-9^T降解PAHs的代谢机制。【结果】菌株P-9^T为黄色杆菌科(Xanthobacteraceae)水杆菌属(Aquabacter)的潜在新种,暂命名为Aquabacter sediminis P-9^T,也是Aquabacter属中首次发现的PAHs降解菌种;在菌株A. sediminis P-9^T的基因组中则发现了一条完整的苯甲酸盐降解通路,以及参与萘和其他PAHs降解的脱氢酶、水杨酸羟化酶和细胞色素P450等关键基因。菌株P-9^T可以在25-40 ℃和pH 5.0-8.0的条件下降解菲,并且可分别以萘、菲、芘为唯一碳源生长繁殖。在菲初始浓度为50 mg/L下培养5 d降解率达100%。利用HPLC-ESI-MS/MS技术检测发现,该菌株对萘、菲、芘降解的中间代谢产物包括1-羟基-2-萘甲酸、1,2-萘二酚和儿茶酚和1-羟基-2-萘醛。推断菌株P-9^T是采用水杨酸途径降解PAHs。【结论】A. sediminis P-9^T是Aquabacter属的新种,而且是该属中首次发现的PAHs降解菌,最佳降解条件为37 ℃和pH 7.0,可通过水杨酸途径高效降解萘、菲及芘。     

Effects of MULTIFOLIATE-PINNA, AFILA, TENDRIL-LESS and UNIFOLIATA genes on leafblade architecture in Pisum sativum

In order to dissect the genetic regulation of leafblade morphogenesis, 16 genotypes of pea, constructed by combining the wild-type and mutant alleles of MFP, AF, TL and UNI genes, were quantitatively phenotyped. The morphological features of the three domains of leafblades of four genotypes, unknown earlier, were described. All the genotypes were found to differ in leafblade morphology. It was evident that MFP and TL functions acted as repressor of pinna ramification, in the distal domain. These functions, with and without interaction with UNI, also repressed the ramification of proximal pinnae in the absence of AF function. The expression of MFP and TL required UNI function. AF function was found to control leafblade architecture multifariously. The earlier identified role of AF as a repressor of UNI in the proximal domain was confirmed. Negative control of AF on the UNI-dependent pinna ramification in the distal domain was revealed. It was found that AF establishes a boundary between proximal and distal domains and activates formation of leaflet pinnae in the proximal domain.     

Presence of Rhizobium Etli bv . Phaseoli and Rhizobium Gallicum bv . Gallicum in Egyptian Soils

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.     

Characterisation of cell wall polysaccharides, arabinogalactans-proteins (AGPs) and phenolics of Cola nitida, Cola acuminata and Garcinia kola seeds

Many Cola plant species are endemic to West and Central Africa. Cola acuminata and Cola nitida are used as masticatory when fresh, while the dried nuts are used for beverages and pharmaceutical purposes in Europe and North America. Garcinia kola seeds, that serve as a substitute for the true kola nuts, are used in African traditional medicine for the treatment of various diseases, including colic, headache and liver cirrhosis. Seeds extracts of G. kola are also known for their anti-inflammatory, antimicrobial and antiviral properties. To gain information on the chemical properties of the kolas, we have isolated and analyzed cell wall polysaccharides, arabinogalactan-proteins and phenolic substances from the seeds of the three kola species. The sugar composition of cell wall material of C. acuminata, C. nitida and G. kola revealed that Gal (up to 30%), Ara, GalA and Glc as the predominant monosaccharides, representing approximately 90% by mol of the total hydrolysable sugar present in this material. In Ammonium oxalate cell wall fraction, GalA was found to be the major sugar present in all kola species. In the alkali-soluble fraction, there were significant differences in the level of Glc and Gal. The level of Glc was high in C. acuminata and C. nitida while the level of Gal and Xyl were high in C. nitida and G. cola. Isolation and quantification of arabinogalactan-proteins demonstrate that G. kola seeds contained four to eight times more of these proteoglycans than the seeds of the other two species. Finally, analysis of soluble phenolic substances shows that caffeine and catechin were largely represented in C. acumina and C. nitida seeds, with caffeine accounting for 50% of all soluble phenolics. These findings indicate that the three Kola seeds are highly enriched in pectins and proteoglycans and that C. acuminata and C. nitida can be used as a possible source of caffeine and catechin.     

Hydroxylation of steroids by Fusarium oxysporum, Exophiala jeanselmei and Ceratocystis paradoxa

The potential of Fusarium oxysporum var. cubense UAMH 9013 to perform steroid biotransformations was reinvestigated using single phase and pulse feed conditions. The following natural steroids served as substrates: dehydroepiandrosterone (1), pregnenolone (2), testosterone (3), progesterone (4), cortisone (5), prednisone (6), estrone (7) and sarsasapogenin (8). The results showed the possible presence of C-7 and C-15 hydroxylase enzymes. This hypothesis was explored using three synthetic androstanes: androstane-3,17-dione (9), androsta-4,6-diene-3,17-dione (10) and 3α,5α-cycloandrost-6-en-17-one (11). These fermentations of non-natural steroids showed that C-7 hydroxylation was as a result of that position being allylic. The evidence also pointed towards the presence of a C-15 hydroxylase enzyme.The eleven steroids were also fed to Exophialajeanselmei var. lecanii-corni UAMH 8783. The results showed that the fungus appears to have very active 5α and 14α-hydroxylase enzymes, and is also capable of carrying out allylic oxidations.Ceratocystis paradoxa UAMH 8784 was grown in the presence of the above-mentioned steroids. The results showed that monooxygenases which effect allylic hydroxylation and Baeyer–Villiger rearrangement were active. However, redox reactions predominated.     

Evaluation of Beauveria bassiana and B. brongniartii strains and four wild-type fungal species against adults of Bactrocera oleae and Ceratitis capitata

The virulence of two isolates of the hyphomycete fungi, Beauveria bassianaand B. brongniartii, and additional fungal species isolated from diseased Bactrocera oleae pupae and Sesamia nonagrioideslarvae were assessed against adults of the olive fruit fly B. oleae and the Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae). Contact and oral bioassays revealed that moderate to high mortality rates for the olive fruit fly occurred when the adults were exposed to conidia of Mucor hiemalis, Penicillium aurantiogriseum, P. chrysogenum and B. bassianaisolates. A strain of M. hiemalis isolated from S. nonagrioides larvae was the most toxic resulting in 85.2% mortality to the olive fruit fly adults. B. brongniartiiand B. bassiana were the most pathogenic to the C. capitataadults causing 97.4 and 85.6% mortality. Metabolites collected from the M. hiemalis and P. chrysogenum isolates were toxic to adults of both species.     

甘菊ClHSP70与ClHSP90基因的克隆及表达分析

该研究以甘菊(Chrysanthemum lavandulifolium)为实验材料,通过RT-PCR方法从甘菊转录组数据中分离出热激蛋白合成相关基因,命名为ClHSP70和ClHSP90。序列分析表明,ClHSP70基因ORF全长为2 559bp,编码852个氨基酸,蛋白功能区预测表明含有典型的HSP70蛋白NBD和SBD保守结构域;ClHSP90基因ORF全长为2 094bp,编码697个氨基酸,含有HATPase结构域和HSP90保守结构域。生物信息学分析表明,甘菊ClHSP70与大豆(Glycine max)和烟草(Nicotiana tomentosiformis)HSP70蛋白有较高的一致性,ClHSP90基因编码的氨基酸序列与紫茎泽兰(Ageratina adenophora)HSP90高度相似;实时荧光定量表达分析表明,在42℃处理不同时间,甘菊叶片中ClHSP70和ClHSP90基因表达均在0.5h时显著增加,1h达到最大值,2h后缓慢下降;不同组织表达分析表明,甘菊在42℃处理1h后,ClHSP70在成熟叶中的表达量显著高于嫩叶和根等其他组织;ClHSP90在成熟茎中的表达量最高。研究说明,ClHSP70和ClHSP90基因具有热激蛋白特征,参与了甘菊热胁迫应答过程,该研究结果为以后深入研究其基因功能奠定了基础。     

转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响

【目的】为探究转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响。【方法】以转Cry1Ac/1Ab基因棉与其亲本常规棉为实验材料,利用取食不同棉花品种叶片的棉铃虫饲喂异色瓢虫幼虫。【结果】与常规亲本棉相比,取食饲喂转基因棉花叶片的初孵棉铃虫幼虫的异色瓢虫幼虫从1龄发育至化蛹期时间延长0.77 d,但差异不显著;除1龄幼虫体重增加(0.0773 mg)外,其余各龄期幼虫体重均有所下降,但差异均不显著;异色瓢虫1、2、3、4龄幼虫对初孵棉铃虫捕食量均随棉铃虫密度的增加而增加,捕食功能反应均符合HollingⅡ圆盘方程。【结论】转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育无显著影响,饲喂取食转Cry1Ac/1Ab基因棉花的棉铃虫对异色瓢虫捕食功能无显著差异。     

巴西橡胶树HbSUT3和HbSUT5 mRNA在嫩茎和中脉中的原位杂交分析

为研究巴西橡胶树(Hevea brasiliensis)中HbSUT3和HbSUT5基因的功能,采用地高辛标记的RNA探针与橡胶树嫩茎和中脉两种组织切片分别进行RNA原位杂交,对这2种SUT基因在组织中的表达区域与表达特点进行了分析。结果表明,在橡胶树嫩茎中,两个SUT基因主要在树皮的韧皮部和皮层细胞中表达;在中脉中,两个SUT基因在除木质部导管系统外的其它部位均有表达;HbSUT3基因在嫩茎和中脉中的表达量相近,而HbSUT5基因在嫩茎中的表达量远高于中脉。这些揭示HbSUT3和HbSUT5基因可能广泛参与韧皮部装载、蔗糖运输与库细胞供给等活动,同时两个SUT基因也存在功能分化。     

基因CfATG6和CfATG14参与调控果生刺盘孢细胞自噬和致病力

【目的】炭疽病是油茶的主要病害,由刺盘孢属的多种真菌引起,其中果生刺盘孢分布范围最广、分离率最高,是油茶炭疽病的主要致病菌。研究自噬相关蛋白CfAtg6和CfAtg14的生物学功能,为进一步揭示果生刺盘孢通过细胞自噬调控致病的分子机制,并为油茶炭疽病的防治提供理论基础。【方法】根据同源重组原理,通过聚乙二醇(polyethylene glycol, PEG)介导的方法,在果生刺盘孢中敲除基因CfATG6和CfATG14,并进一步获得回补菌株ΔCfatg6-C和ΔCfatg14-C。【结果】酵母双杂交试验结果显示,果生刺盘孢蛋白CfAtg6和CfAtg14可能存在互作关系。生物学表型测定结果表明,相较于野生型和回补菌株,突变体ΔCfatg6和ΔCfatg14均表现出营养生长速率显著减慢,附着胞形成率分别只有野生型的5%和18%;突变体ΔCfatg6和ΔCfatg14致病力均极显著减弱,造成的油茶叶片病斑面积少于野生型和回补菌株的1/3;CfATG6和CfATG14基因缺失突变体均丧失转运和降解CfAtg8蛋白的能力,并对细胞壁胁迫更敏感。突变体ΔCfatg6的分生孢子产量显著降低,仅为野生型的20%左右;氧化胁迫试验结果表明,相较于野生型和回补菌株,过氧化氢对突变体的生长抑制率升高10%左右。内质网压力胁迫试验表明,ΔCfatg14对二硫苏糖醇抑制率升高5%以上。【结论】自噬相关基因CfATG6和CfATG14参与调控了果生刺盘孢生长发育、细胞自噬和致病力。