Molecular cloning and nucleotide sequence of cDNA for sporamin,the major soluble protein of sweet potato tuberous roots

Summary Sporamin accounts for more than 80% of the total soluble proteins of tuberous roots of sweet potato, but very little, if any, in other tissues of the same plant. In vitro translation of RNA fractions from the tuberous roots in wheat germ extract and subsequent immunoprecipitation with the antibody to sporamin indicated that this protein is synthesized by membrane-bound polysomes as a precursor 4 000 daltons larger than the mature protein. A cDNA expression library was constructed from the total poly(A)⁺ RNA from the tuberous roots by a vector-primer method, and an essentially full-length cDNA clone for the sporamin mRNA was selected by direct immunological screening of the colonies. Northern blot analysis showed that sporamin mRNA is approximately 950 nucleotides in length and is specifically present in tuberous roots and very little, if any, in leaves, petioles and non-tuberous roots. Nucleotide sequence of the cDNA predicts a 37 amino acid extension in the precursor at the amino-terminus of the mature protein.     

马铃薯块茎中总RNA的快速提取

马铃薯块茎中由于含有大量的多糖和酚类物质,因此难以从中提取RNA。本实验采用CTAB法从马铃薯块茎中提取了总RNA,并进行了马铃薯病毒的检验。结果表明:得到的RNA完整性好,A260/A280在1.7～2.0范围内,纯度也比较高。用RT-PCR方法进行马铃薯Y病毒的检验,得到了一条与预计扩增长度相同的一条带,并检验出枣庄地区马铃薯Y病毒的感染率为75%。     

Wounding-induced Enhancement of the Activity of Chromatin-bound DNA-Dependent RNA Polymerases in Sweet Potato Root

Chromatin fractions were isolated from intact and wounded sweet potato root tissues. The synthesis of RNA by the chromatin fractions was dependent on four ribonucleoside triphosphates and a divalent cation such as Mg²⁺ and Mn²⁺, Mn²⁺ being most effective. Whereas phosphate did not interfere with the polymerase reaction, it was totally blocked by pyrophosphate. The reaction was inhibited by DNase and actinomycin D as well as RNase and trypsin. The RNA polymerases of sweet potato root needed SH-groups for catalysis. Activity of chromatin-bound RNA polymerases (EC 2.7.7.6) promptly increased in the 6 hr after wounding and then decreased gradually up to 24 hr. Under the present experimental conditions it was mostly due to the activity of RNA polymerase I. RNA polymerase II contributed only about 5 to 15% to the total activity. The increase in the activity after wounding was completely inhibited by cycloheximide. Plant hormones such as 2,4-dichlorophenoxyacetic acid, gibberellic acid and dibutyryl cyclic adenosine 3′,5′-monophosphate stimulated the increase in RNA polymerases three to four times after wounding. Ethylene partially suppressed the wound-induced increase of RNA polymerases.     

Potato virus Y mRNA expression knockdown mediated by siRNAs in cultured mammalian cell line

RNA interference (RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes. The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion. In our study, a 480bp fragment of the capsid protein gene of potato virus Y (CP-PVY) was used as a target to downregulate PVY mRNA expression in-vitro, as the CP gene interferes with viral uncoating, translation and replication. A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells. CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs. Six biological replicates were performed in this study. In our findings, one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%–90%.     

Phosphorus Efficiency of Cabbage (Brassica oleraceae L. var. capitata), Carrot (Daucus carotaL.), and Potato (Solanum tuberosumL.)

Plant species and genotypes within the same species may differ in phosphorus efficiency. The objective of this research was to study phosphorus efficiency of cabbage (Brassica oleraceae L.), carrot (Daucus carota L.), and potato (Solanum tuberosum L.) and to quantify the contribution of morphological root characteristics to P uptake of the plant species. An experiment was conducted in a glasshouse with six P levels: 0, 12, 27, 73, 124 and 234 mg P kg^–1 soil, and with six replications. Cabbage attained 80% of its maximum yield already at the level of no P supply, whereas carrot and potato reached only 4 and 16% of their highest yields respectively at this level of P supply. This indicated that cabbage was P-efficient compared to carrot and potato. Root/shoot ratio (cm root g^–1 shoot d. m.) increased in the order of cabbage < carrot < potato, and was enhanced at lower P levels. Root hair length was not affected by P level, and averaged 0.22, 0.03 and 0.18 mm for cabbage, carrot, and potato, respectively. Predicting P uptake by a mechanistic simulation model revealed that root hairs contributed about 50% to the total P uptake of cabbage and potato, but only 0.3% to that of carrot. The relationship between the observed P uptake and the predicted P uptake of the plants revealed that model parameters explained nearly 4/5th of the total P uptake of carrot and potato, but only 2/5th of that of cabbage. This showed that the P uptake of cabbage was strongly under-predicted, whereas that of carrot and potato was predicted well. Therefore, it was hypothesised that cabbage may have the ability to mobilise and take up soil P additionally by other root mechanisms such as exudation of organic acids.     

高海拔生境下怀玉山高山马铃薯和本土农家薯的全基因组重测序分析

为了探究怀玉山高山马铃薯只限于高海拔生境下的适应机制,本研究以高海拔生境下怀玉山高山马铃薯和怀玉山本土农家薯采后块茎萌发的芽为材料,进行了全基因组重测序分析。结果表明:HAP的总SNP数量为2 835 211,SNP的密度为3.925 212,编码区nsSNP总数为104 456;LAP的总SNP数量为3 968 618,SNP的密度为5.427 855,编码区nsSNP总数为141 060。经CV分析后,LAP和HAP共有319个nsSNP关联了315个基因,其中76个基因具有超过1个的nsSNP。nsSNP的防御反应、蛋白氨基酸磷酸化、蛋白激酶等GOterms显著富集。HAP的总Indel数量为159 797,编码区的Indel数量为1 850;LAP的总Indel数量为154 291,编码区的Indel数量为1 714;两组的总Indel数量远远小于两组的总SNP数。经差异分析后,共有1 629个Indel关联到了726个基因。Indel的防御反应、胁迫应答、细胞凋亡等GOterms显著富集。nsSNP和Indel的GOterms大多与高海拔生境和光合作用相关。nsSNP和Indel分布在端粒附近相对于中心粒具有更高密度的变异。LAP和HAP共获得了1 490个CNV,其中缺失(Deletion)为610个,中性(Neutral)为548个,扩增(Amplification)为332个。本研究结果可为高海拔生境下怀玉山高山马铃薯的SNP和Indel相关标记的开发、优异基因的挖掘提供重要理论依据。     

Identification and Nucleotide Sequence of a Tobacco rattle virus RNA-1 Variant that Causes Spraing Disease in Potato cv. Bintje

An isolate of Tobacco rattle virus (TRV) obtained from a site in the Netherlands induced symptoms of spraing disease in tubers of the potato variety Bintje, which is generally considered to be resistant to infection by TRV. The isolate contained two phenotypically distinguishable RNA‐1 variants, one of which was shown to carry the determinant for the ability to cause spraing in cv. Bintje. The nucleotide sequence of the coding region of this RNA‐1 was determined and found to differ at 5.2–5.4% of positions from other TRV RNA‐1 sequences in the database. The amino acid sequences of the predicted translation products were between 92 and 99% identical to those of a TRV RNA‐1 that did not cause spraing in cv. Bintje, with P1b being the most divergent and the replicase read‐through domain the least.     

Ribonucleic Acid Changes in Germinating Potato Tubers

Abstract

The changes of the total RNA occurring during germination in potato tubers have been investigated. During germination the development of the new sprouts is accompanied by a progressive decrease of RNA. A preferential breakdown of high molecular weight RNA has been observed. Tubers deprived of the buds mantain the level of RNA of the quiescent tuber. Gibberellic acid treatment promotes the development of the sprouts, but is ineffective in inducing changes in the RNA level when supplied to isolated explants of potato tuber both germinating or deprived of buds.     

Novel Potato Micro-Tuber-Inducing Compound, (3R,6S)-6-Hydroxylasiodiplodin,from a Strain of Lasiodiplodia theobromae

A novel potato micro-tuber-inducing compound was isolated from the culture broth of Lasiodiplodia theobromae Shimokita 2. The structure of the isolated compound was determined as (3R,6S)-6-hydroxylasiodiplodin by means of spectroscopic analyses, the modified Mosher method, and chemical conversion. The compound showed potato micro-tuber-inducing activity at a concentration of 10^?4 M, using the culture of single-node segments of potato stems in vitro.     

The Fate of Patatin and Its mRNA in Slices of Potato Tuber

The fate of patatin mRNA was investigated in slices of potatotuber since this mRNA appeared to be a potential example ofa preexisting mRNA that is involved in the rapid formation ofpolysomes which occurs in such slices. Levels of patatin, whichis the major storage protein in mature potato tubers, decreasedslightly during the first 4 h after slicing but remained constantfor the next 44 h. Analysis of products of in vitro translationshowed that patatin mRNA was present and stable in the tubercells even after several months of storage. The translationalactivity of patatin mRNA relative to total translational activityin total cellular RNA fraction increased transiently duringthe first hour and then decreased rapidly to undetectable levelswithin 6 h. By contrast, the activity in polysomal RNA fractiondecreased immediately after slicing. The difference betweenthe relative activities of patatin mRNA in total and polysomalRNA fractions during the first hour suggests that the extentof incorporation of patatin mRNA into polysomes was not in directproportion to its abundance in the cells of the slices. Additionof actinomycin D to the slices did not prevent the transientincrease in the translational activity of patatin mRNA in totalRNA fraction at 1 h, indicating that the transient increasewas not due to synthesis of patatin mRNA de novo after slicing.However, the inhibitor prevented the degradation of patatinmRNA in the slices. This result indicates that the synthesisof new mRNAs is necessary for the degradation of patatin mRNA. ¹Present address: Aburahi Laboratories, Shionogi and Co., Ltd.,Koka-cho, Shiga, 520-34 Japan (Received June 30, 1989; Accepted May 9, 1990)     

Characterization and Sensitivity to Fungicides of Rhizoctonia spp. Recovered from Potato Plants in Bolu,Turkey

Isolates of Rhizoctonia spp. associated with stem canker and black scurf disease of potato were examined for their anastomosis group, sequence variations in the ITS‐5.8S rDNA region, pathogenicity and sensitivity to fungicides. A total of 92 isolates were obtained from diseased tuber, stolon and sprouts of the potato plants, collected from five districts of Bolu province, Turkey. Based on the anastomosis group and the similarity of the nucleotide sequence of the ITS‐5.8S rDNA, most of the isolates (81.5%) were identified as AG 3 PT. Other isolates belonged to AG 2‐1 (1.08%), AG 2‐2 IV (1.08%), AG 4 HG II (8.07%), AG 5 (2.17%), binucleate Rhizoctonia AG A (1.08%) and AG K (4.35%). Pathogenicity tests showed that isolates of AG 3 PT, AG 4 HG II and AG 5 caused similar degrees of disease severity on 45‐day‐old potato seedlings, whereas AG 2‐1 was moderately virulent. AG 2‐2 IV and binucleate Rhizoctonia spp. were weakly pathogenic or non‐pathogenic on potato seedlings. In this study, anastomosis groups of Rhizoctonia spp. isolates associated with potato in Turkey were characterized for the first time using molecular techniques and classified at the level of subgroups. Furthermore, the effect of selected fungicides was evaluated on disease development caused by soil‐borne inoculums of different anastomosis groups (AGs). Flutolanil and Bacillus subtilis QST 713 were found to be most effective against the Rhizoctonia isolates tested. These results revealed significant differences among the fungicides on disease development resulted from the different AGs.     

Expression of Potato Virus Y Coat Protein Gene in Transgenic Potato

Potato virus Y (PVY) N coat protein (CP) coding sequence was cloned into a plant expression vector pMON316 under the CaMV 35S promoter. Leaf discs of potato (Solanum tuberosum) were used to Agrobacterium-mediated gene transfer. A large number of regenerated putative transgenic plants were obtained based on kanamycin resistance. Using total DNA purified from transgenic plants as templates and two oligonucleotides synthesized from 5' and 3' of the PVY coat protein gene as primers, the authors carried out polymerase chain reaction (PCR) to check the presence of this gene and obtained a 0. 8 kb specific DNA fragment after 35 cycles of amplification. Southern blot indicated that the PCR product was indeed PVY CP gene which had been integrated into the potato genome. Enzyme-linked immunosorbent assay (ELISA) of our transgenic plants showed that CP gene was expressed in at least some transgenic potato plants.     

Increase in Catalase mRNA in Wounded Sweet Potato Tuberous Root Tissue

Catalase protein, as well as its activity, increases in woundedsweet potato tuberous root tissue [Esaka et al. (1983) PlantCell Physiol. 24: 615]. Whether catalase mRNA increases in woundedtissue was examined with a hybridization probe of a cDNA forsweet potato catalase mRNA. The content of catalase mRNA inthe tissue increased after a lag phase of 10 h to reach a maximumat 30 h after wounding, whereas total RNA content increasedwithout a lag phase. The increase in the mRNA content afterthe lag phase preceded that in catalase activity. In the earlystages after wounding, catalase mRNA in polysomes increasedin spite of no increase in the total content of mRNA in thetissue. We propose that an increase in catalase mRNA is responsiblefor the increase in catalase protein in wounded sweet potatotuberous root tissue. In the early stage after wounding, however,the translation of catalase mRNA seems to be activated throughincreased availability of ribosomes. (Received January 28, 1987; Accepted May 6, 1987)