大豆疫霉菌多聚半乳糖醛酸酶pspg1基因的克隆及表达分析

大豆疫病严重影响我同及世界各国的农业生产,为探讨多聚半乳糖醛酸酶在大豆疫霉菌致病过程中的作用,采用PCR的方法从大豆疫霉菌中克隆了多聚半乳糖醛酸酶pspg1基因,并利用RT-PCR法对其在大豆中的表达进行了分析.结果表明:大豆疫霉菌pspg1基因开放阅读框长1236 bp,编码一个长412氨基酸的蛋白质.对其进化关系进行分析,发现该基因与其它卵菌的pg基因亲缘关系最近,形成一个独立的分支.RT-PCR分析表明:pspg1基因在接种大豆疫霉菌的大豆下胚轴中大量表达,而在健康大豆下胚轴中未检测到.克隆了大豆疫霉菌pspg1基因,并发现该基因在大豆疫霉菌侵染大豆过程中发挥重要作用.     

颗粒剂农药防治辣椒疫病的研究

选择了５种杀菌剂，将其制成具有缓释性能的颗粒剂，施在辣椒根茎基部防治辣椒疫病。室内接种试验，有４种颗粒剂施药７ｄ后防效１００％，２１ｄ后防效９５％以上。田间试验防效平均在８４％以上，连续３年田间应用，防效为８０％左右。     

大豆根特异性GmPR1-9启动子的鉴定及其在根腐病抗性中的应用

【目的】 鉴定大豆根特异性启动子及其最小调控片段,并利用启动子工程技术构建时空特异人工启动子并评价其在根腐病抗性中的应用价值,为大豆抗疫霉根腐病的遗传改良提供遗传元件。【方法】 通过分析大豆根、茎和叶片转录组数据,筛选在根中特异高水平表达的基因,克隆获得其启动子序列。根据顺式元件的分布位置构建截短载体,并驱动GUS报告基因在大豆发状根组织中超表达,筛选控制根特异性表达的核心片段。将获得的核心启动子片段与疫霉菌诱导启动子元件p4XD串联构建人工启动子驱动疫霉抗性相关基因GmNDR1在大豆发状根中超表达,分析转基因组织对疫霉菌抗性水平及目的基因在病原菌侵染过程中的表达水平。利用转基因本氏烟草从整株水平评价转基因材料对疫霉菌的抗性水平。【结果】 通过筛选发现6个大豆根特异性表达的PR1同源基因,其中,pGmPR1-9具有最高的启动子表达活性。PLACE在线预测发现其启动子区域含有大量的根特异表达相关顺式元件。对pGmPR1-9启动子进行截短试验,发现5′端截短片段L1、L2、L3、L4和L5均具有启动GUS表达活性,长度为166 bp的L5（-166—-1）片段具有全长启动子80%的活性,并可驱动GUS在转基因烟草根中特异表达;3个3′端截短片段R1、R2、R3和1个双端截短片段M1几乎检测不到GUS酶活性。p4XD-L5融合片段驱动GmNDR1在大豆发状根中超表达后可显著提高大豆发状根对疫霉菌的抗性,超表达发状根接种病原菌后发病程度和病斑长度显著低于对照,疫霉菌丝积累量在接种48 h时减少66.5%。GmNDR1在超表达组织中始终维持在高表达水平,在接种前,表达量是对照组织的39.2倍,接种后,表达量受疫霉菌侵染诱导进一步上调,并在36 h达到最高。GmNDR1在p4XD-L5::NDR1转基因本氏烟草根中的表达量显著高于茎和叶片,表现出明显的根部表达偏好性。超表达株系接种辣椒疫霉菌15 d后的株高、根长和鲜重显著高于对照,同时叶片萎蔫率和病斑长度显著低于对照植株。【结论】 鉴定获得一个大豆根特异性表达启动子及其核心序列,融合诱导性和组织特异性启动子核心元件的人工启动子p4XD-L5驱动抗性基因GmNDR1超表达,可显著增强转基因大豆发状根和本氏烟草对疫霉菌的抗病性。     

黄瓜疫霉菌拮抗内生细菌的筛选与鉴定

【目的】从黄瓜叶片中筛选抗疫霉菌效果好的内生细菌并进行鉴定,为进一步研究黄瓜疫病的理 论探索及防治应用提供实验材料。【方法】以黄瓜叶片为实验材料筛选内生细菌,根据待检菌株的镜检特征、 革兰氏染色反应、菌落排列方式和菌落形态特征等进行初步筛选。采用平板对峙法和发酵液拮抗法实验对目标 内生细菌进行拮抗作用测定。通过 16S rDNA 序列测定及同源性分析,并构建系统发育树确定目标菌株种属。 【结果】初步筛选出 32 株内生细菌将其编号为 CL1~CL32,其中革兰氏阴性杆菌 10 株、革兰氏阳性杆菌 13 株、革兰氏阳性球菌 9 株,利用平板对峙法发现 8 株对掘氏疫霉菌有较强抑制作用的拮抗菌株,菌丝抑制率为 10.3%~54.3%,抑菌半径为 1~7 mm,对其中抑菌效果较好的 3 株菌株（CL7、CL9、CL15）进行发酵液拮抗实验, 结果显示其抑菌率分别为 54.4%、72.2%、63.3%。【结论】对掘氏疫霉菌具有较强抑制作用的 3 株内生细菌（CL7, CL9、CL15）分别属于泛菌属 Pantoea sp.、假单胞菌属 Pseudomonas sp. 和葡萄球菌属 Staphylococcus sp.,其中 抑制效果最明显的为 CL9,即假单胞菌属 Pseudomonas sp.。     

圆滑番荔枝叶乙醇提取物不同萃取组分对胡椒瘟病菌的抑菌活性测定

采用液-液分配法对圆滑番荔枝叶乙醇提取物进行萃取分离,得到石油醚、三氯甲烷、乙酸乙酯等不同萃取组分。采用生长速率法测定不同萃取组分对胡椒瘟病菌的抑制作用。结果表明:乙酸乙酯萃取组分对胡椒瘟病菌的抑制作用最强,其中20 mg/mL乙酸乙酯萃取组分对胡椒瘟病菌菌丝生长的抑制率达到74.43%,具有较好的抑菌效果。     

Oligochitosan inhibits Phytophthora capsici by penetrating the cell membrane and putative binding to intracellular targets

The mechanism of action of oligochitosan, which has shown great antifungal activity against Phytophthora capsiciin vitro, was studied using 2-aminoacridone-labeled oligochitosan (2-AMAC-oligochitosan) and a gel-retardation experiment. Internalization of 2-AMAC-oligochitosan in cysts, germtubes and sporangia of P. capsici was confirmed by confocal laser scan microscopy (CLSM), and the degree of uptake depended on the incubation concentration. 2-AMAC-oligochitosan localized mainly in the cytoplasm and showed no binding to both cell wall and cell membrane. Mannose, an inhibitor for oligochitosan uptake by macrophages, could not inhibit the internalization of oligochitosan in P. capsici. The gel-retardation experiment showed that oligochitosan bound strongly to DNA and RNA of P. capsici. These results indicate that oligochitosan exerts its antifungal activity by penetrating the cell membrane and putative binding to intracellular targets such as DNA and RNA.     

番茄对晚疫病的株龄相关抗性及Ph-3的抗性机制

目前已明确番茄抗晚疫病R基因表现明显的株龄相关抗性(Age-related Resistance,ARR),但抗晚疫病QTL的抗性规律尚不明确。以携带抗晚疫病基因Ph-3的栽培种番茄(Solanum lycopersicum)‘CLN2037B’和野生种醋栗番茄(S.pimpinellifolium)‘L3708’以及易感病材料栽培种番茄(S.lycopersicum)‘LA2818’为对照,对含有抗晚疫病QTL的多毛番茄(S.habrochaites)‘LA2099’、‘LA1033’和‘LA1777’是否也存在株龄相关抗性进行了分析。结果表明,携带抗晚疫病QTL的3个材料的6叶期和9叶期植株病情级数均比3叶期明显降低,表明QTL抗性与株龄相关。利用乙烯、水杨酸和茉莉酸素合成或缺失的突变体和病毒诱导基因沉默(Virus Induced Gene Silencing,VIGS)技术,初步明确了乙烯和水杨酸参与6叶期Ph-3基因介导的对晚疫病的抗性,而茉莉酸不参与。     

Multi-trait selection in a diallel crossing scheme of cocoa

Cocoa breeding seeks to increase the yield by area unit. In this context, one must consider the resistance to Phytophthora disease, the most serious one in cocoa culture. Thus, herein two traits were used: yield, i.e. number of healthy pods obtained in 7 years and Phytophthora resistance, estimated by pod rot rate. A combined individual‐family selection is proposed on the basis of the total genetic value in order to spread clonal material. This selection method was applied in a 6 × 6 diallel scheme in Cameroon. The heritability of Phytophthora resistance (near 0.2) was higher than for yield (0.13). A selection index was constructed to obtain good genetic gains on each of the two traits (yield and Phytophthora resistance). To improve prediction of genetic values, a third trait, potential yield, was used. With the chosen weight, the expected genetic gain was about 35% for resistance at 1% level of selection. This selection was applied and a confirmation trial is being established with the selected trees.     

Differentiation of the high molecular weight glutenin subunit D t x 2.1 of Aegilops tauschii indicated by partial sequences of its encoding gene and SSR markers

The devastating late blight pathogen Phytophthora infestans infects foliage as well as tubers of potato. To date, resistance breeding has often focused on foliage blight resistance, but tuber blight resistance is becoming more and more important in cultivated potatoes. In this study, a reliable tuber assay for resistance assessment was developed and foliage and tuber blight resistance (R) was compared in four mapping populations. In the RH4X-103 population, tuber blight resistance inherited independently from foliage blight resistance. Three specific R genes against P. infestans were segregating. The Rpi-abpt and R3a genes function as foliage-specific R genes, whereas the R1 gene acts on both foliage and tuber. In the segregating populations SHRH and RH94-076, tuber and foliage blight resistance correlated significantly, which suggests that resistance in foliage and tuber is conferred by the same gene (could be R3b) and quantitative trait loci (QTL), respectively. In the CE population neither tuber nor foliage resistance was observed.