Molecular cloning and characterization of novel isoforms of potato ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase) is one of the major enzymes involved in starch biosynthesis in higher plants. We report here the molecular cloning of two cDNAs encoding so far uncharacterized isoforms (AGP S2 and AGP S3) of the potato enzyme. Sequence analysis shows that the two polypeptides are more homologous to previously identified large subunit polypeptides from potato and other plant species than to small subunit isoforms. This observation suggests that AGP S2 and AGP S3 represent novel large subunit polypeptides. agpS2 is expressed in several tissues of the potato plant, including leaves and tubers. Expression was stronger in sink leaves than in source leaves, indicating developmental regulation. In leaves, agpS2 expression was induced 2- to 3-fold by exogenous sucrose; therefore, agpS2 represents a new sucrose-responsive gene of starch metabolism. Expression of agpS3 was restricted to tubers: no agpS3 expression could be seen in leaves of different developmental stages, or when leaves were incubated in sucrose. Therefore, agpS3 represents the only AGPase gene so far characterized from potato, which is not expressed in leaves. Conversely, all four AGPase isoforms known from potato are expressed in tubers.     

基于发根培养体系的甘薯品种抗线虫特性鉴定研究

利用发根农杆菌诱导的甘薯发根体系,鉴定甘薯品种抗线虫的特性。试验在甘薯品种徐薯18、栗子香和鲁78066诱导的发根体系上,接种马铃薯腐烂线虫,六周后调查发根繁殖线虫情况及线虫侵染发根情况,然后评价它们的抗线虫特性。结果表明:培养六周后,线虫在徐薯18、栗子香和鲁78066发根上繁殖倍数分别为8.82,0.76和0.70;在徐薯18发根上观察到多处线虫侵入位点,在栗子香和鲁78066发根上只观察到一处线虫侵入位点;基于以上结果,鉴定徐薯18为易感线虫病品种,栗子香和鲁78066为抗线虫病品种,徐薯18和鲁78066的鉴定结果和发病地自然诱发鉴定结果相一致,栗子香不同于发病地自然诱发鉴定结果。鉴定结果表明:用不同品种的甘薯发根作鉴定其抗线虫特性,具有体系简单、直观方便、重复性好以及不受自然环境影响等优点,进一步完善可以作为植物对线虫病抗性鉴定新的体系。     

Analysis of genes developmentally regulated during storage root formation of sweet potato

To identify the genes involved in storage root formation of sweet potato (Ipomoea batatas), we performed a simplified differential display analysis on adventitious roots at different developmental stages of the storage root. The expression patterns were confirmed by semiquantitative RT-PCR analyses. As a result, 10 genes were identified as being developmentally regulated and were named SRF1-SRF10. The expression of SRF1, SRF2, SRF3, SRF5, SRF6, SRF7, and SRF9 increased during storage root formation, whereas the expression of SRF4, SRF8, and SRF10 decreased. For further characterization, a full-length cDNA of SRF6 was isolated from the cDNA library of the storage root. SRF6 encoded a receptor-like kinase (RLK), which was structurally similar to the leucine-rich repeat (LRR) II RLK family of Arabidopsis thaliana. RNA gel blot analysis showed that the mRNA of SRF6 was most abundantly expressed in the storage roots, although a certain amount of expression was also observed in other vegetative organs. Tissue print mRNA blot analysis of the storage root showed that the mRNA of SRF6 was localized around the primary cambium and meristems in the xylem, which consist of actively dividing cells and cause the thickening of the storage root.     

Evidence that thaxtomin C is a pathogenicity determinant of streptomyces ipomoeae, the causative agent of streptomyces soil rot disease of sweet potato

Streptomyces ipomoeae is the causal agent of Streptomyces soil rot of sweet potato, a disease marked by highly necrotic destruction of adventitious roots, including the development of necrotic lesions on the fleshy storage roots. Streptomyces potato scab pathogens produce a phytotoxin (thaxtomin A) that appears to facilitate their entrance into host plants. S. ipomoeae produces a less-modified thaxtomin derivative (thaxtomin C) whose role in pathogenicity has not been examined. Here, we cloned and sequenced the thaxtomin gene cluster (txt) of S. ipomoeae, and we then constructed targeted txt mutants that no longer produced thaxtomin C. The mutants were unable to penetrate intact adventitious roots but still caused necrosis on storage-root tissue. These results, taken in context with previous histopathological study of S. ipomoeae infection, suggest that thaxtomin C plays an essential role in inter- and intracellular penetration of adventitious sweet potato roots by S. ipomoeae. Once inside the plant host, the pathogen uses one or more yet-to-be-determined factors to necrotize root tissue, including that of any storage roots it encounters.     

甘薯根腐病菌侵染对甘薯内源激素水平的影响

甘薯根腐病病原菌[Fusarium solani(Mart.)Sacc.f.sp.batatas McClure,简称FSB]侵染及其培养液滤液处理高敏感性甘薯品种‘胜利百号’后,引起甘薯叶片、茎尖和根部组织内源ABA含量大幅升高。其中在根部出现最早,但茎尖中积累浓度最高。侵染后甘薯叶片、茎尖和根部组织内源GA1/3含量显著低于对照。甘薯组培苗经FSB培养滤液处理9h后,ABA含量显著上升,处理15h,ABA含量呈下降趋势,而GA1/3含量在101和102稀释液处理15h(103稀释液处理12h)时出现显著上升。这些结果有助于解释甘薯根腐病株矮小不产生藤蔓,并在秋季大量现蕾开花的生理现象。     

甘薯MADS-box转录因子IbMADS11-Like的克隆及胁迫响应分析

MADS-box蛋白在植物生长发育及抗逆等过程中均发挥重要功能。本实验室根据甘薯近缘野生种I.trifida基因组序列,在甘薯栽培种徐薯22（Ipomoea batatas（L.） Xu22）中克隆到一个STMADS11亚家族MADS-box基因,命名为IbMADS11-Like。实时定量RT-PCR分析表明,IbMADS11-Like基因在甘薯根中大量表达,并且随着块根的形成和膨大表达量逐渐降低,表明该基因可能参与了甘薯块根的发育过程。胁迫处理分析表明,IbMADS11-Like基因的表达受干旱、盐和高温的诱导,而低温则抑制其表达。此外,IbMADS11-Like基因对ABA、IAA、ZT、BR、ACC、JA及GA等激素的处理也有不同程度的响应,暗示IbMADS11-Like基因可能参与了甘薯生长发育及胁迫的调控过程。这些结果为进一步分析IbMADS11-Like基因在甘薯块根发育和胁迫响应中的功能奠定了基础。     

The Effect of Gamma-Irradiation on the Sucrose Content in Sweet Potato Roots and Potato Tubers

The sucrose content in both potato tubers and sweet potato roots was considerably increased by gamma-irradiation. The maximum increase was achieved by an irradiation dose of 3 to 4 kGy for potatoes and 0.8 to 2 kGy for sweet potatoes. Cooling treatment (15°C, 2 weeks) for sweet potato roots also enhanced the sucrose content (almost 2 times) but was not additive to the irradiation treatment; the maximum sucrose content in irradiated sweet potato roots was in the range of 7 to 12% irrespective of the cooling treatment, depending on the variety of sweet potatoes. Irradiation made the sucrose content in the roots 2 to 4 times higher.     

Transgenic sweet potato expressing thionin from barley gives resistance to black rot disease caused by Ceratocystis fimbriata in leaves and storage roots

Black rot of sweet potato caused by pathogenic fungus Ceratocystis fimbriata severely deteriorates both growth of plants and post-harvest storage. Antimicrobial peptides from various organisms have broad range activities of killing bacteria, mycobacteria, and fungi. Plant thionin peptide exhibited anti-fungal activity against C. fimbriata. A gene for barley α-hordothionin (αHT) was placed downstream of a strong constitutive promoter of E12Ω or the promoter of a sweet potato gene for β-amylase of storage roots, and introduced into sweet potato commercial cultivar Kokei No. 14. Transgenic E12Ω:αHT plants showed high-level expression of αHT mRNA in both leaves and storage roots. Transgenic β-Amy:αHT plants showed sucrose-inducible expression of αHT mRNA in leaves, in addition to expression in storage roots. Leaves of E12Ω:αHT plants exhibited reduced yellowing upon infection by C. fimbriata compared to leaves of non-transgenic Kokei No. 14, although the level of resistance was weaker than resistance cultivar Tamayutaka. Storage roots of both E12Ω:αHT and β-Amy:αHT plants exhibited reduced lesion areas around the site inoculated with C. fimbriata spores compared to Kokei No. 14, and some of the transgenic lines showed resistance level similar to Tamayutaka. Growth of plants and production of storage roots of these transgenic plants were not significantly different from non-transgenic plants. These results highlight the usefulness of transgenic sweet potato expressing antimicrobial peptide to reduce damages of sweet potato from the black rot disease and to reduce the use of agricultural chemicals.