马铃薯块茎发育机理及其基因表达

马铃薯（Solanum tuberosum L.)块茎是有块茎马铃薯植物的地下变态器官,它由匍匐茎顶端膨大形成,对于马铃薯块茎形成的生理机制已有许多研究,这些研究表明,块茎发生受许多因素的影响,总体来讲短日 照,较低的温度以及离体条件下培养基较高的蔗糖浓度等有利于块茎形成,同时,块茎形成过程中内源激素亦发生一系列变化,然而,对于块茎形成中相关基因表达,进而调控块茎形成的系统研究目前还较滞后,已有研究显示,块茎形成与膨大涉及到一系列基因的表达与关闭,同时它也与淀粉合成和块茎储藏蛋白基因的表达有关,综述了这一领域现有的研究进展。     

Field evaluation of transgenic potato plants expressing an antisense granule-bound starch synthase gene: increase of the antisense effect during tuber growth

Transgenic plants of a tetraploid potato cultivar were obtained in which the amylose content of tuber starch was reduced via antisense RNA-mediated inhibition of the expression of the gene encoding granule-bound starch synthase (GBSS). GBSS is one of the key enzymes in the biosynthesis of starch and catalyses the formation of amylose. The antisense GBSS genes, based on the full-length GBSS cDNA driven by the 35S CaMV promoter or the potato GBSS promoter, were introduced into the potato genome by Agrobacterium tumefaciens-mediated transformation. Expression of each of these genes resulted in the complete inhibition of GBSS gene expression, and thus in the production of amylose-free tuber starch, in mature field-grown plants originating from rooted in vitro plantlets of 4 out of 66 transgenic clones. Clones in which the GBSS gene expression was incompletely inhibited showed an increase of the extent of inhibition during tuber growth. This is likely to be due to the increase of starch granule size during tuber growth and the specific distribution pattern of starch components in granules of clones with reduced GBSS activity. Expression of the antisense GBSS gene from the GBSS promoter resulted in a higher stability of inhibition in tubers of field-grown plants as compared to expression from the 35S CaMV promoter. Field analysis of the transgenic clones indicated that inhibition of GBSS gene expression could be achieved without significantly affecting the starch and sugar content of transgenic tubers, the expression level of other genes involved in starch and tuber metabolism and agronomic characteristics such as yield and dry matter content.     

马铃薯块茎发育机理及其基因表达

马铃薯(Solanum tuberosum L.)块茎是有块茎马铃薯植物的地下变态器官,它由匍匐茎顶端膨大形成。对于马铃薯块茎形成的生理机制已有许多研究,这些研究表明,块茎发生受许多因素的影响,总体来讲短日照、较低的温度以及离体条件下培养基较高的蔗糖浓度等有利于块茎形成,同时,块茎形成过程中内源激素亦发生一系列变化。然而,对于块茎形成中相关基因表达,进而调控块茎形成的系统研究目前还较滞后。已有研究显示,块茎形成与膨大涉及到一系列基因的表达与关闭,同时它也与淀粉合成和块茎储藏蛋白基因的表达有关。综述了这一领域现有的研究进展。     

HRE-type genes are regulated by growth-related changes in internal oxygen concentrations during the normal development of potato (Solanum tuberosum) tubers

The occurrence of hypoxic conditions in plants not only represents a stress condition but is also associated with the normal development and growth of many organs, leading to adaptive changes in metabolism and growth to prevent internal anoxia. Internal oxygen concentrations decrease inside growing potato tubers, due to their active metabolism and increased resistance to gas diffusion as tubers grow. In the present work, we identified three hypoxia-responsive ERF (StHRE) genes whose expression is regulated by the gradual decrease in oxygen tensions that occur when potato tubers grow larger. Increasing the external oxygen concentration counteracted the modification of StHRE expression during tuber growth, supporting the idea that the actual oxygen levels inside the organs, rather than development itself, are responsible for the regulation of StHRE genes. We identified several sugar metabolism-related genes co-regulated with StHRE genes during tuber development and possibly involved in starch accumulation. All together, our data suggest a possible role for low oxygen in the regulation of sugar metabolism in the potato tuber, similar to what happens in storage tissues during seed development.     

Expression profiling of potato germplasm differentiated in quality traits leads to the identification of candidate flavour and texture genes

Quality traits such as flavour and texture are assuming a greater importance in crop breeding programmes. This study takes advantage of potato germplasm differentiated in tuber flavour and texture traits. A recently developed 44,000-element potato microarray was used to identify tuber gene expression profiles that correspond to differences in tuber flavour and texture as well as carotenoid content and dormancy characteristics. Gene expression was compared in two Solanum tuberosum group Phureja cultivars and two S. tuberosum group Tuberosum cultivars; 309 genes were significantly and consistently up-regulated in Phureja, whereas 555 genes were down-regulated. Approximately 46% of the genes in these lists can be identified from their annotation and amongst these are candidates that may underpin the Phureja/Tuberosum trait differences. For example, a clear difference in the cooked tuber volatile profile is the higher level of the sesquiterpene alpha-copaene in Phureja compared with Tuberosum. A sesquiterpene synthase gene was identified as being more highly expressed in Phureja tubers and its corresponding full-length cDNA was demonstrated to encode alpha-copaene synthase. Other potential 'flavour genes', identified from their differential expression profiles, include those encoding branched-chain amino acid aminotransferase and a ribonuclease suggesting a mechanism for 5'-ribonucleotide formation in potato tubers on cooking. Major differences in the expression levels of genes involved in cell wall biosynthesis (and potentially texture) were also identified, including genes encoding pectin acetylesterase, xyloglucan endotransglycosylase and pectin methylesterase. Other gene expression differences that may impact tuber carotenoid content and tuber life-cycle phenotypes are discussed.     

Molecular Cloning and Sequence of Potato Granule-Bound Starch Synthase Gene

It is known that tuber-specific expressions of many genes exist in the process of tuber development from stolon in potato (Solanum tuberosum). Study on the regulation of those gene expression will share light on the mechanism of organ-specific gene expression. Potato GBSS (granule-bound starch synthase) gene, which is solely responsive for the pres- ence of amylose in potato tuber, expression is tuber-specific. The paper describes the construction of a genomic library of a Chinese potato cultivar "Dongnong 303" in which 20 clones were isolated using partial GBSS gene sequence ampified by PCR. 5428 bp DNA sequence of one clone (GBSS17-1) was determined, including 1823 bp 5' flanking region. 2964 bp structure gene, and 641 bp 3' flanking region. It is highly homologious with reported GBSS gene sequence. In addition, the 730 bp most upstream sequence of 5' flanking region which was not reported previously contained stem and loop structures. The present result may provide some important information for further study in the molecular mechanism of organ specific gene expression.     

Cold sweetening in diploid potato: mapping quantitative trait loci and candidate genes

A candidate gene approach has been used as a first step to identify the molecular basis of quantitative trait variation in potato. Sugar content of tubers upon cold storage was the model trait chosen because the metabolic pathways involved in starch and sugar metabolism are well known and many of the genes have been cloned. Tubers of two F(1) populations of diploid potato grown in six environments were evaluated for sugar content after cold storage. The populations were genotyped with RFLP, AFLP, and candidate gene markers. QTL analysis revealed that QTL for glucose, fructose, and sucrose content were located on all potato chromosomes. Most QTL for glucose content mapped to the same positions as QTL for fructose content. QTL explaining >10% of the variability for reducing sugars were located on linkage groups I, III, VII, VIII, IX, and XI. QTL consistent across populations and/or environments were identified. QTL were linked to genes encoding invertase, sucrose synthase 3, sucrose phosphate synthase, ADP-glucose pyrophosphorylase, sucrose transporter 1, and a putative sucrose sensor. The results suggest that allelic variants of enzymes operating in carbohydrate metabolic pathways contribute to the genetic variation in cold sweetening.     

A potato molecular-function map for carbohydrate metabolism and transport

Molecular-linkage maps based on functional gene markers (molecular-function maps) are the prerequisite for a candidate-gene approach to identify genes responsible for quantitative traits at the molecular level. Genetic linkage between a quantitative trait locus (QTL) and a candidate-gene locus is observed when there is a causal relationship between alleles of the candidate gene and the QTL effect. Functional gene markers can also be used for marker-assisted selection and as anchors for structural and functional comparisons between distantly related plant species sharing the same metabolic pathways. A first molecular-function map with 85 loci was constructed in potato based on 69 genes. Priority was given to genes operating in carbohydrate metabolism and transport. Public databases were searched for genes of interest from potato, tomato, or other plant species. DNA sequence information was used to develop PCR-based marker assays that allowed the localization of corresponding potato genes on existing RFLP linkage maps. Comparing the molecular-function map for genes operating in carbohydrate metabolism and transport with a QTL map for tuber starch content indicates a number of putative candidate genes for this important agronomic trait. Received: 19 March 2000 / Accepted: 16 May 2000     

Statistical epistasis between candidate gene alleles for complex tuber traits in an association mapping population of tetraploid potato

Association mapping using DNA-based markers is a novel tool in plant genetics for the analysis of complex traits. Potato tuber yield, starch content, starch yield and chip color are complex traits of agronomic relevance, for which carbohydrate metabolism plays an important role. At the functional level, the genes and biochemical pathways involved in carbohydrate metabolism are among the best studied in plants. Quantitative traits such as tuber starch and sugar content are therefore models for association genetics in potato based on candidate genes. In an association mapping experiment conducted with a population of 243 tetraploid potato varieties and breeding clones, we previously identified associations between individual candidate gene alleles and tuber starch content, starch yield and chip quality. In the present paper, we tested 190 DNA markers at 36 loci scored in the same association mapping population for pairwise statistical epistatic interactions. Fifty marker pairs were associated mainly with tuber starch content and/or starch yield, at a cut-off value of q ≤ 0.20 for the experiment-wide false discovery rate (FDR). Thirteen marker pairs had an FDR of q ≤ 0.10. Alleles at loci encoding ribulose-bisphosphate carboxylase/oxygenase activase (Rca), sucrose phosphate synthase (Sps) and vacuolar invertase (Pain1) were most frequently involved in statistical epistatic interactions. The largest effect on tuber starch content and starch yield was observed for the paired alleles Pain1-8c and Rca-1a, explaining 9 and 10% of the total variance, respectively. The combination of these two alleles increased the means of tuber starch content and starch yield. Biological models to explain the observed statistical epistatic interactions are discussed.     

Characterization of gene expression during potato tuber development in individuals and populations using the luciferase reporter system

Analysis of gene expression and enzyme activity in pooled tuber samples has previously indicated different developmental events occurring in a fixed sequential order during tuber development, starting with the up-regulation of starch synthesis then induction of protein storage followed by cell division and cell enlargement. In this report we analysed in vivo promoter activity of genes related to cell division and storage of reserves during tuber development in individual in vitro tubers, using the non invasive firefly luciferase reporter system. The average activity of the storage related promoters (AGPaseS and Pat21) was up-regulated prior to visible swelling, while the average activity of both cell cycle genes (cycB1;1 and CDC2a) showed an up-regulation after the onset of swelling. However, this novel system allowed expression analysis in individual tubers, which showed a variable up-regulation of both storage genes in relation to the moment of swelling, from 4 days before to 10 days after the onset of swelling. We conclude that during the first stages of tuber development, the moment of storage gene induction is independent from swelling. These results indicate that the developmental program of potato tubers does not consist of a fixed sequential order of events, but consists of independent developmental programs (storage and swelling), together resulting in the formation of a potato tuber. It is concluded that analysis of developmental programs by studying individuals may result in new insights, possibly obscured when using pooled samples.     

Isolation,culture, and regeneration of plants from potato protoplasts

A technique is described for the routine isolation of protoplasts from storage parenchyma cells of potato tubers grown in vitro. The protoplasts typically contained many starch grains. On culture, most of the starch grains were metabolised during the first 7 days, after which the cells began to divide. Following further culture, protoplast-derived colonies and calli were obtained, from which shoots and intact plants were regenerated. Cytological study of regenerated plants showed that the majority were octaploid or aneuploid at the octaploid level. This aspect is compared with plants regenerated from mesophyll protoplasts of potato. The use of tuber protoplasts for studies on tissue-specific transient gene expression of chimeric gene constructs, following their introduction into the protoplasts by electroporation, is discussed, together with the uses of tuber protoplasts in fundamental physiological and biochemical studies.     

Comparative analysis of expressed sequence tags from Sesamum indicum and Arabidopsis thaliana developing seeds

Sesame (Sesamum indicum) is an important oilseed crop which produces seeds with 50% oil that have a distinct flavor and contains antioxidant lignans. Because sesame lignans are known to have antioxidant and health-protecting properties, metabolic pathways for lignans have been of interest in developing sesame seeds. As an initial approach to identify genes involved in accumulation of storage products and in the biosynthesis of antioxidant lignans, 3328 expressed sequence tags (ESTs) were obtained from a cDNA library of immature seeds 5-25 days old. ESTs were clustered and analyzed by the BLASTX or FASTAX program against the GenBank NR and Arabidopsis proteome databases. To compare gene expression profiles during development of green and non-green seeds, a comparative analysis was carried out between developing sesame and Arabidopsis seed ESTs. Analyses of these two seed EST sets have helped to identify similar and different gene expression profiles during seed development, and to identify a large number of sesame seed-specific genes. In particular, we have identified EST candidates for genes possibly involved in biosynthesis of sesame lignans, sesamin and sesamolin, and also suggest a possible metabolic pathway for the generation of cofactors required for synthesis of storage lipid in non-green oilseeds. Seed-specific expression of several candidate genes has been confirmed by northern blot analysis.