Increased abundance of patatins,lipoxygenase and miraculins in a thaxtomin A-habituated potato Russet Burbank somaclone with enhanced resistance to common scab

Potato common scab caused by the actinobacterium Streptomyces scabiei is characterized by the formation of corky lesions on tubers that reduce their marketability. Management of common scab is very complex and often ineffective under various environmental conditions. Using potato varieties that are more resistant to common scab remains one of the most efficient strategies to control this disease. However, very little is known about the factors associated with resistance to common scab. Somaclone RB9 regenerated from thaxtomin A-habituated potato Russet Burbank calli produced tubers more resistant to common scab than the original variety. Comparison of the RB9 tuber proteome with that of Russet Burbank using label-free quantitative proteomic analysis revealed changes in the accumulation of defence-related proteins from the patatin and lipoxygenase (LOX) families, which are involved in the metabolism of lipids, and of two miraculins of the Kunitz-type protease inhibitors family. The implication of LOX during common scab infection was studied using synchronized minitubers developed from leaf-bud cuttings. S. scabiei infection stimulated the accumulation of LOX in both Russet Burbank and RB9 minitubers, but this accumulation was intensified in RB9 minitubers. Infection also increased LOX activity in Russet Burbank and RB9 minitubers. However, LOX activity measured in noninfected RB9 minitubers was similar to that of infected Russet Burbank minitubers, indicating endogenous activation of LOX activity in RB9 minitubers. We discuss how increased LOX abundance and activity in the somaclone RB9 may contribute to improving tuber defence against common scab.     

农杆菌介导的马铃薯试管薯遗转化体系的优化及反义class Ⅰ patatin基因的导入

用两个马铃薯栽培品种“鄂马铃薯3号”和“甘农薯2号”的试管薯为供体材料，建立了一种农杆菌介导的简单、快速和高效的遗传转化系统。在含有75mg／L卡那霉素的选择培养基上，2～3周可产生抗性芽，4～5周获得完整的转基因植株。筛选出了试管薯遗传转化的优化条件，特别是在再生培养基中加入2mg／L玉米素，两个品种的转化频率分别高达45．5％和43．9％。周期短(4～5周)、一步培养和转化频率高，使该转化体系能够广泛用于马铃薯转基因的研究。用含有反义class Ⅰ patatin基因的表达载体pBSAP转化两个品种，共获得120株卡那霉素抗性植株。PCR、PCR-Southern和Northern杂交分析证明，此反义基因已整合到马铃薯基因组中并在转基因植株中正常转录。反义基因的表达导致部分转基因植株的试管结薯株率和单株结薯数降低。结果表明，该class Ⅰ patatin基因可能参与了块茎形成的调控。     

Analysis of potato microtuber proteins by sodium dodecylsulfate polyacrylamide gel electrophoresis

Summary Protein banding patterns of 119 lines (85 Japanese potato lines and introductions, 22 dihaploids, 1 wild source (Solanum bulbocastanum Dun.), 10 transformants and 1 somaclonal variant) were compared by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Microtubers formed in vitro were used as the source of plant materials. There were more than 15 bands in the 35–20 kDa molecular weight range and less than 10 bands in the 65–40 kDa range. The bands in the 65–40 kDa range which corresponded to the potato tuber storage protein patatin were focussed to about 40 kDa after heat treatment. The results indicate a distinct difference in the banding patterns among potato lines.     

马铃薯classⅠpatatin基因在试管块茎形成中的功能

将正反义classⅠpatatin基因导入马铃薯品种甘农薯2号中,有2个转正义基因株系试管块茎的可溶性蛋白含量和LAH活性与对照相比有不同程度的增加,有3个转反义基因株系的可溶性蛋白质含量下降,并且所有转反义基因植株的LAH活性都不同程度地降低。试管块茎的诱导结果表明,有1个转正义基因株系的结薯株率和单株结薯数比其对照明     

Solanum phureja genes are expressed in the leaves and tubers of aneusomatic potato dihaploids

Summary The expression of leaf isozymes and tuber patatin in dihaploids derived from the Solanum tuberosum cv. Pentland Crown was investigated. Seven of the dihaploids were aneusomatic containing additional chromosomes from the S. phureja dihaploid inducer. Of these, four genotypes expressed leaf isozymes characteristic of the S. phureja dihaploid inducer, and the tubers of three aneusomatic dihaploids contained a S. phureja form of patatin. Aneusomatic dihaploids in which the proportion of cells containing additional S. phureja chromosomes was relatively small (i.e. 1–15%) did not express leaf isozyme markers or patatin bands characteristic of the dihaploid inducer or showed only faint expression of one or two markers. However, those with a high proportion of cells containing additional chromosomes (50–55%) had a range of strongly expressed leaf isozymes that were characteristic of the dihaploid inducer and also expressed the S. phureja tuber patatin.One dihaploid genotype was exclusively euploid (2n<24), yet is expressed a S. phureja leaf isozyme marker and S. phureja tuber patatin, suggesting recombination or chromosome substitution between the genome of the S. phureja dihaploid inducer and the cultivar Pentland Crown.     

农杆菌介导的马铃薯试管薯遗传转化体系的优化及反义class Ⅰ patatin基因的导入

用两个马铃薯栽培品种"鄂马铃薯3号"和"甘农薯2号"的试管薯为供体材料,建立了一种农杆菌介导的简单、快速和高效的遗传转化系统.在含有75 mg/L卡那霉素的选择培养基上,2～3周可产生抗性芽,4～5周获得完整的转基因植株.筛选出了试管薯遗传转化的优化条件,特别是在再生培养基中加入2 mg/L玉米素,两个品种的转化频率分别高达45.5     

Patatin、Wun1启动子驱动的PPO反义基因植物表达载体的构建

以植物表达载体PC3-ASPPOty为基础,分别构建了由Patatin、Wun1启动子调控的PPO反义基因植物表达载体pCP-ASPPOty、pCW-ASPPOty,并通过直接导入法将重组质粒分别导入根癌农杆菌LBA4404中,经PCR鉴定证明,此基因已整合到农杆菌Ti上。表达载体的建立为获得地上部分PPO活性正常、块茎损伤低褐化的马铃薯加工型改良品种奠定了基础。     

Patatin启动子驱动的人胰岛素原基因的植物表达载体的构建

使用马铃薯的偏爱密码子设计合成人胰岛素原基因,以植物表达载体pCAMBIA1301为基础,构建由patatin启动子调控的人胰岛素原基因植物表达载体p1301Ph,并将导入根癌农杆菌LBA4404中,经PCR鉴定证明,此基因已整合到农杆菌Ti上。表达载体的建立为日后转基因工作奠定了基础。     

The use of dihaploids in increasing the homozygosity of tetraploid potatoes

Summary Electrophoresis banding patterns of the tuber proteins of 12 dihaploids of the cultivar Pentland Crown showed that four had types of patatin unlike that of the parent. The patatin types of somatically chromosome-doubled clones derived from three of the dihaploids were identical to those of the dihaploid progenitors. Fourteen dihaploids produced from the chromosome-doubled derivative (PDH40X2) of one dihaploid, which had a variant patatin, had the patatin type of the parent dihaploid.The experiment showed that dihaploids and somatic chromosome doubling could be used to fix variation found in heterozygous tetraploid potatoes. Used together, haploidisation and chromosome doubling can generate highly homozygous tetraploids for use by plant breeders.