Increase in lipid content in potato tubers modified by 14-3-3 gene overexpression

Recently, transgenic potato plants were created with overexpression of the 14-3-3 protein derived from Cucurbita pepo. Detailed analysis of those plants suggested that the function of the isolated 14-3-3 isoform is in the control of carbohydrate and lipid metabolism in the plants. 14-3-3 protein overexpression gave rise to an increase in soluble sugar and catecholamine contents in both leaves and tubers. It is proposed that 14-3-3 protein affects carbohydrate metabolism in potato plants via regulation of catecholamine synthesis. Furthermore, genetically modified potato tubers with 14-3-3 protein overexpression showed changes in lipid content and composition. The transgenic potato tubers contained 69% more total fat compared to the wild-type plant. Separation of tuber lipids into polar and nonpolar fractions revealed that the transgenic potato tubers contained almost 3 times more nonpolar lipids than the control. Analysis of fatty acid composition, conducted by the means of gas chromatography, showed that linoleic acid was the main fatty acid present in the tubers of both modified and control potato plants. In the nonpolar fraction of the fat of the transgenic tubers the unsaturated fatty acids exhibited a higher participation in the sum of all fatty acids.     

Repression of the 14-3-3 gene affects the amino acid and mineral composition of potato tubers

Recently, transgenic potato plants were created showing underexpression of the 20R isoform of the 14-3-3 protein. The transgenic plants grown in tissue culture showed a significant increase in nitrate reductase activity and a decrease in nitrate level. The transgenic line with the lowest 14-3-3 quantity was field-trialed (1997-2000) and analyzed. The reduction in the 14-3-3 protein level consistently resulted in a starch content increase and in an increase in the ratio of soluble sugars to starch in the tubers, although the latter was only barely visible. The determination of amino acid composition in the tubers showed a significant increase in methionine, proline, and arginine content and a slight but consistent increase in hydrophobic amino acid and lysine content in the cells of the transgenic potato plants. We also observed an increase in the crude protein content, from 19 to 22.1% of the control value in consecutive years. It is proposed that all of these changes might have resulted from the downregulation of nitrate reductase and sucrose phosphate synthase activities by 14-3-3, although other potential mechanisms cannot be excluded (e.g., an increase in enzyme protein level). 14-3-3-repressed transgenic plants showed a significant increase in calcium content in their tubers. It is thus proposed that a function of the isolated 14-3-3 isoform is in the control of amino acid synthesis and calcium metabolism. However, the mechanism of this control is as yet unknown.     

14-3-3 protein down-regulates key enzyme activities of nitrate and carbohydrate metabolism in potato plants

The 14-3-3 protein is one of the best candidates for coordinating all plant metabolic pathways. To verify this suggestion transgenic potato plants with repression of one (J4 and J5 plants), two (G1 plants), and six (G3 plants) constitutive 14-3-3 protein isoforms as well as plants overexpressing the 14-3-3 protein were studied. Reduction in the 14-3-3 protein level in the J4 and J5 transformants, the G1 transformants, and the G3 transformants was close to 29, 41.5, 38, and 55%, respectively. In the case of the 14-3-3 overexpressing plants (J2), a 30% increase in protein content was detected. Changes in nitrate reductase (NR), sucrose phosphate synthase (SPS), and starch synthase (SS) activities in the transgenic plants perfectly reflect the overall 14-3-3 protein level. The highest increase in enzyme activities was observed for the G3 plants and the lowest for the J4 transformants. The same was detected for the measured metabolites. The highest increase in the protein, starch, and sucrose levels was detected in the tubers from the G3 transgenic plants. Because there was almost no change in the isoform ratio in the transgenic plants when compared to the control, it is suggested that it is the overall content of the 14-3-3 protein, rather than the content of particular isoforms, which plays a crucial role in the regulation of enzyme activities and thus in metabolite synthesis. The properties of the 14-3-3 overexpressing plants are very similar to those of the control ones, suggesting that the protein is in excess in the nontransformants and a further increase in its content is not recognized by cell metabolism. A considerable influence of the 14-3-3 protein level on potato plant metabolism was demonstrated. This effect was observed in key metabolic enzyme activities and metabolite content as well. A high variability between mean values, representing individual transgenes, with respect to nitrate reductase, sucrose phosphate synthase, and starch synthase activities in the examined genotypes was noted. These changes were closely correlated with metabolite levels, among them protein, starch, reducing sugars, and sucrose. The results obtained for the five types of transgenic potato plants in comparison with the control were statistically assessed using discriminate function and cluster analyses.     

Ectopic expression of anthocyanin 5-o-glucosyltransferase in potato tuber causes increased resistance to bacteria

The principal goal of this paper was to investigate the significance of anthocyanin 5-O-glucosyltransferase (5-UGT) for potato tuber metabolism. The ectopic expression of a 5-UGT cDNA in the tuber improved the plant's defense against pathogen infection. The resistance of transgenic lines against Erwinia carotovora subsp. carotovora was about 2-fold higher than for nontransformed plants. In most cases the pathogen resistance was accompanied by a significant increase in tuber yield. To investigate the molecular basis of transgenic potato resistance, metabolic profiling of the plant was performed. In tuber extracts, the anthocyanin 3,5-O-substituted level was significantly increased when compared to that of the control plant. Of six anthocyanin compounds identified, the highest quantity for pelargonidin 3-rutinoside-5-glucoside acylated with p-coumaric acid and peonidin 3-rutinoside-5-glucoside acylated with p-coumaric acid was detected. A significant increase in starch and a decrease in sucrose level in transgenic tubers have been detected. The level of all other metabolites (amino acids, organic acids, polyamines, and fatty acids) was quite the same as in nontransformants. The plant resistance to bacterial infection correlates with anthocyanin content and sucrose level. The properties of recombinant glucosyltransferase were analyzed in in vitro experiments. The enzyme kinetics and its biochemical properties were similar to those from other sources.     

Genetic manipulation for enhancing calcium content in potato tuber

Increased calcium (Ca) in potatoes may increase the production rate by enhancing tuber quality and storability. Additionally, increased Ca levels in important agricultural crops may help ameliorate the incidence of osteoporosis. However, the capacity to alter Ca levels in potato tubers through genetic manipulations has not been previously addressed. Here we demonstrate that potato tubers expressing the Arabidopsis H+/Ca2+ transporter sCAX1 (N-terminal autoinhibitory domain truncated version of CAtion eXchanger 1) contain up to 3-fold more Ca than wild-type tubers. The increased Ca appears to be distributed throughout the tuber. The sCAX1-expressing potatoes have normally undergone the tuber/plant/tuber cycle for three generations; the trait appeared stable through successive generations. The expression of sCAX1 does not appear to alter potato growth and development. Furthermore, increased Ca levels in sCAX1-expressing tubers do not appear to alter tuber morphology or yield. Given the preponderance of potato consumption worldwide, these transgenic plants may be a means of marginally increasing Ca intake levels in the population. To our knowledge, this study represents the first attempts to use biotechnology to increase the Ca content of potatoes.     

Distribution of selenium in different biochemical fractions and raw darkening degree of potato (Solanum tuberosum L.) tubers supplemented with selenate

Effects of Se fertilization on potato processing quality, possible changes in Se concentration and form in tubers during storage, and retransfer of Se from seed tubers were examined. Potato plants were grown at five selenate (SeO4(2-)) concentrations. Tubers were harvested 16 weeks after planting and were stored at 3-4 degrees C prior to analysis. The results showed that the Se concentration did not decrease during storage for 1-12 months. In tubers, 49-65% of total Se was allocated in protein fraction, which is less than found in plant leaves in a previous study. The next-generation tubers produced by the Se-enriched seed tubers had increased Se concentrations, which evidenced the relocation of Se from the seed tubers. At low levels, Se improved the processing quality of potato tubers by diminishing and retarding their raw darkening. The value of Se-enriched potato tubers as a Se source in the human diet was discussed.     

patatin启动子调控烟草液泡转化酶抑制子在马铃薯抗低温糖化中的作用

为了降低马铃薯(Solanum tuberosum )块茎低温贮藏下还原糖的积累，实验构建了马铃薯块茎特异性启动子（CIPP）调控的烟草液泡转化酶抑制子Nt-VIF基因表达载体pBICNI，并转化马铃薯植株。PCR、Northern杂交和Southern杂交分析结果显示，CIPP调控的Nt-VIF基因全长cDNA成功地导入鄂马铃薯3号（E-3）植株。14个转基因株系块茎分别贮藏在4℃和20℃条件下，贮藏1个月后进行还原糖含量和液泡酸性转化酶（VI）活性测定。结果表明，在20℃条件下转基因株系块茎还原糖（RS）含量与对照相比差异不明显，在4℃条件下RS含量则显著下降，与对照相比下降幅度从34%（株系B-13）至76.8%（株系B-1），说明Nt-VIF cDNA在马铃薯中的表达，成功地抑制了液泡酸性转化酶的活性，导致还原糖含量降低。进一步分析表明，转基因块茎低温贮藏其液泡转化酶活性与还原糖含量呈显著的正直线相关（VI = 0.3084RS + 0.0673）。实验获得的B-1、B-2、B-6、B-9、B-14等5个转基因株系，块茎低温贮藏后能直接满足炸片加工对还原糖含量的要求。     

草铵膦对转基因抗草铵膦马铃薯田间杂草的防效及安全性评价

为了解草铵膦对转基因抗草铵膦马铃薯田间杂草的防效及对马铃薯和环境的安全性,本研究在转基因抗草铵膦马铃薯苗期向田间杂草和马铃薯茎叶定向喷施有效成分分别为0(G0)、847.5(G1)、1 271.25(G2)和1 695 g·hm^-2(G3)的草铵膦,系统比较了药后1、4、11和20 d时马铃薯叶片丙二醛(MDA)和脯氨酸(Pro)含量、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性,药后30和45 d时马铃薯植株的平均株高、茎直径、根茎叶鲜干重和杂草株数,以及成熟期马铃薯块茎营养品质、单株产量、土壤和块茎中草铵膦残留在各处理间的差异。结果表明,药后不同时期,各处理间的马铃薯叶片MDA和Pro含量、SOD和CAT活性均无显著差异。药后30和45 d时,除各草铵膦处理区的根茎叶鲜干重显著高于清水对照外,各处理间的马铃薯株高、茎直径和成熟期块茎营养品质均无显著差异,说明试验剂量的草铵膦对马铃薯生长发育及品质无显著影响。与清水对照相比,各剂量的草铵膦对杂草均有明显的防除效果,且可以明显地提高马铃薯的单株产量,杂草的株、鲜重防效和成熟期马铃薯单株产量在各处理间的差异均依次表现为G3>G2>G1,但在土壤和块茎中均未检测到草铵膦残留。综上,采用有效成分1 695 g·hm^-2草铵膦可以有效防除杂草,对马铃薯安全,且在土壤和马铃薯块茎中未检出草铵膦残留。本研究结果为草铵膦的科学使用提供了理论依据。     

Phosphorus and silicon effects on growth,yield, and phosphorus forms in potato plants

Silicon (Si) can increase phosphorus (P) use efficiency (PUE) by increasing P availability in the soil and altering P metabolism in the plant, thus resulting in improved yield under low soil P conditions. The objective was to evaluate the effect of P (10, 50, 100, and 200?mg dm^?3) and Si (0, 50, and 200?mg dm^?3) interrelationship on P and Si uptake by plants, forms of P in leaves and tubers, plant growth, and tuber yield of potato plants. Silicon supply increased organic and inorganic P in the leaves. At low soil P rates, Si also increased organic P in the tubers. However, alterations in the P metabolism of potato plants with Si supply did not promote significant changes in dry matter (DM) production and tuber yield. Phosphate fertilization increased growth and yield of plants. Silicon uptake by the plants were also increased by phosphate fertilization with soil Si supply.     

利用Cre/lox特异位点重组系统获得无选择标记转AtMYB12基因的马铃薯

类黄酮是植物产生的一类次级代谢产物的总称,长期食用含类黄酮的果实和蔬菜有易于人体健康,目前广泛种植的马铃薯品种中仅含有痕量的类黄酮。AtMYB12是拟南芥中鉴定的调控类黄酮生物合成的特异性转录因子,并在烟草和番茄中得到了功能验证。为了增加马铃薯中类黄酮的含量,本研究构建pX6-patatin：：AtMYB12无选择标记...     

Effect of potato plants expressing snowdrop lectin (GNA) on the performance and colonization behaviour of the peach-potato aphid Myzus persicae

Abstract

Transgenic potato plants expressing snowdrop lectin (GNA potatoes) are developed to increase resistance against sap-feeding insects. When expressing GNA at relatively high levels such potatoes may have a negative effect on the fecundity and development of the first generation of the important pest, the peach-potato aphid Myzus persicae (Sulzer) (Hemiptera: Aphididae). However, the effects on M. persicae over several generations, and how such plants affect the alate aphids’ colonization behaviour have not been reported. In this laboratory study, the performance of single M. persicae on potatoes with low GNA expression, measured as developmental time, fecundity, size and survival, was compared with the control, following two successive generations of single apterous aphids. Aphid population growth on the two plant lines was also studied. In addition, colonization behaviour was investigated in a choice experiment where the alate aphids could choose between the GNA and an isogenic control potato plant in a cage. The present study showed that the apterous aphid performance was not significantly different on the two potato lines, although the aphids tended to perform slightly poorer on the transgenic potato. However, the transgenic potato was less likely to be colonized by alate aphids. It is concluded that such transgenic potato plants expressing the lectin at a relatively low level, maximum 0.2% of the soluble protein, have no significant impact on the performance of apterous M. persicae once on the plant, but may have a potential in controlling the aphids by altering the colonization behaviour of alates.     

Control of enzymatic browning in potato (Solanum tuberosum L.) by sense and antisense RNA from tomato polyphenol oxidase

Polyphenol oxidase (PPO) activity of Russet Burbank potato was inhibited by sense and antisense PPO RNAs expressed from a tomato PPO cDNA under the control of the 35S promoter from the cauliflower mosaic virus. Transgenic Russet Burbank potato plants from 37 different lines were grown in the field. PPO activity and the level of enzymatic browning were measured in the tubers harvested from the field. Of the tubers from 28 transgenic lines that were sampled, tubers from 5 lines exhibited reduced browning. The level of PPO activity correlated with the reduction in enzymatic browning in these lines. These results indicate that expression of tomato PPO RNA in sense or antisense orientation inhibits PPO activity and enzymatic browning in the major commercial potato cultivar. Expression of tomato PPO RNA in sense orientation led to the greatest decrease in PPO activity and enzymatic browning, possibly due to cosuppression. These results suggest that expression of closely related heterologous genes can be used to prevent enzymatic browning in a wide variety of food crops without the application of various food additives.     

Compositional analysis of tubers from insect and virus resistant potato plants

Genetically modified potato plants that are resistant to the Colorado potato beetle, plus either the potato leaf roll virus or potato virus Y, have recently been commercialized. As part of the safety assessment for plants produced by modern biotechnology, the composition of the food/feed must be compared to that of the food/feed produced by an equivalent plant variety from a conventional source. The composition of important nutritional and antinutritional factors in tubers produced by virus- and insect-resistant potato plants were compared to tubers produced by conventional potato plants. Key nutritional, quality, and antinutritional components measured were total solids, vitamin C, dextrose, sucrose, soluble protein, and glycoalkaloids. Proximate analyses included fat, ash, calories, total protein, and crude fiber. Minor nutrients measured were vitamin B6, niacin, copper, magnesium, potassium, and amino acids. The results from these analyses confirm that tubers produced by insect- and virus-protected varieties are substantially equivalent to tubers produced by conventional potato varieties.     

Seasonal changes and the effect of nitrogen- and potash-fertilization on the solanine and α-chaconine content in various parts of the potato plant

The concentration of solanine and α-chaconine in leaves and tubers decreased with the progress of the potato plant towards maturity. In stems an increase of these alcaloids up to the first part of the second stage of the vegetation period could be observed. With a higher level of nitrogen the concentration of both glycoalcaloids in leaves and stems increased, while in tubers a depression of the glycoalcaloid content was estimated. An increased level of potassium did not change markedly the concentration of glycoalcaloids in leaves and stems of the potato plant but decreased both alcaloids in the tuber.     

西红柿原系统素基因转化水稻的抗虫性研究

构建了两个含西红柿原系统素基因的双元载体pNAR304（UbiI5’+Prosystemin+NOS3’）和pNAR305 (UbiI5’+Prosystemin+NOS3’+ PinⅡ5’+PinⅡ+PinⅡ3’)，并用农杆菌介导方法将其转入水稻品种秀水63、合江19和日本晴。经潮霉素抗性、PCR和Southern blot确证，共获得转基因水稻14株。Northern blot检测表明，原系统素基因(Prosystemin)和马铃薯蛋白酶抑制剂基因(PinⅡ)在这些转基因植株中都能转录表达。然而，植株的二化螟和褐飞虱抗虫性鉴定表明：单独转入Prosystemin（pNAR304）不能提高转基因水稻的抗虫能力；Prosystemin和PinⅡ双价（pNAR305）转基因植株能明显提高水稻的抗虫性，但其抗性水平与PinⅡ单个基因的转基因水稻植株间并无显著差异。 这表明，Prosystemin基因转入水稻并不能有效调节转基因水稻PinII基因的表达量。据此试验结果推测，水稻中可能不存在类似于西红柿系统素的信号途径，很可能水稻的伤害信号转导是经由与双子叶植物系统素体系不同的其它途径来实现的。     

Antioxidant capacity manipulation in transgenic potato tuber by changes in phenolic compounds content

The main goal of this study was to generate potato tubers with increased levels of flavonoids and thus modified antioxidant capacities. To accomplish this, the vector carrying multigene construct was prepared and several transgenic plants were generated, all overexpressing key biosynthesis pathway enzymes. The single-gene overexpression or simultaneous expression of genes encoding chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR) resulted in a significant increase of measured phenolic acids and anthocyanins. The increase in phenolic compounds synthesis is accompanied by decreases in starch and glucose levels in transgenic plants. The flavonoids-enriched plants showed improved antioxidant capacity; however, there is a complex relationship between antioxidant capacity and flavonoids content, suggesting the great participation of other compounds in the antioxidant potential of the plants. These other compounds are not yet recognized.     

Development of field-grown potato plants derived from meristem plants multiplied with different methods

Abstract

In the current multiplication technique first-generation seed tubers produced in the field by transplanting plants raised on peat in plastic rolls from plants cultured by repetitive multiplication using tip- and stem-cuttings and truncated plants are compared with in vitro micro-plants from the aspect of obtaining optimal-sized, disease-free seed tubers.

The objective of the study is to compare the dynamics of total plant dry mass and tuber dry mass of field-grown potato plants, and analyse the effect of the year and variety. Two local late-maturing potato varieties, Ants and Vigri, were used in the study. The field experiments were carried out in 2005–2007.

Significant impact of the multiplication method and experimental year on total plant dry mass and tuber dry mass was observed. The plants multiplied in vitro from micro-cuttings produced lower total dry mass and also lower tuber dry mass per m². The plants multiplied by tip- and stem-cuttings as well as truncated plants proved to be more adaptable to unfavourable weather conditions than in vitro plants; in a favourable year, however, the differences were insignificant. In the early phase of growth the ratio of the tuber dry mass to total plant dry mass increased more rapidly in the case of in vitro plants, whereas by the end of the growing season the relevant ratio was similar for all multiplication methods and years.

All developed multiplication methods are suitable for practical seed potato production in the field and ensure a reasonable potato crop.     

Cultivation of transgenic cyanophycin-producing potatoes does not negatively affect growth,reproduction and activity of the earthworm Lumbricus terrestris (L.)

A microcosm experiment was performed to investigate the effects of post-harvest potato tubers from transgenic cyanophycin-producing potatoes on Lumbricus terrestris (L.) activity and biomass, number of cocoons and their hatchability as well as the remaining cyanophycin content in soil and cast samples during a period of 80 days. Potato tubers from four transgenic potato events with different cyanophycin content in a range from 0.8 to 7.5% were compared to the near isogenic, non-transgenic control (Solanum tuberosum L. cv. Albatros) and a comparative potato cultivar (S. tuberosum L. cv. Désirée). One treatment with transgenic tuber residue but without earthworms was prepared as an additional control. Potato tuber loss from the surface of the microcosms was significantly higher in the treatments with transgenic potato tubers compared with non-transgenic treatments. It can be estimated that the earthworm contribution to potato tuber loss from the soil surface was approximately 61%. Mean number of cocoons in addition to the number of hatched cocoons varied from 2.6 to 6.2 and from 7 to 15 accounting for 45.2–83.35% hatchability, respectively, but no significant differences between the treatments were found. The same was true for the development of earthworm biomass in the various treatments. The cyanophycin content in soil samples was significantly higher when earthworms were present indicating that the cyanophycin content in the upper soil layer might have been enhanced through earthworm burrowing activity. Overall, it is concluded that tubers from transgenic cyanophycin potatoes are easily degradable and neither inhibit nor stimulate earthworm growth, reproduction, and activity.     

Expression Pattern of Ａ Recombinant Phloem-specific Promoter from Pumpkin in Transgenic Potato Plants

以本实验室构建的重组南瓜(Cucurbita moschata)韧皮部特异启动子dENP构建了植物表达载体pBdENP。利用根癌农杆菌(Agrobacterium tumefaciens)LBA44O4介导转化马铃薯(Solanum tuberosum)品种Favorita，经过抗生素筛选，共获得转pBdENP和对照pBI121的抗卡那霉素马铃薯再生植株106株。通过PCR初步筛查，筛选出65株为转基因阳性植株。通过Southern blot对部分植株进一步分析,确证外源gus基因已经插入到转基因马铃薯植株的基因组中，插入拷贝数在1个或2个以上。对这些转基因马铃薯植株进行GUS染色结果表明, dENP和CaMV35S启动子一样均能驱动gus基因的表达，前者仅在马铃薯的韧皮部内特异表达，而CaMV35S启动子驱动的gus基因为组成型性表达。GUS酶活力测定结果进一步表明dENP和CaMV35S启动子驱动gus基因表达水平没有明显区别。以上结果证明dENP启动子驱动的外源基因在马铃薯中也具有韧皮部特异而高效表达的特征，从而可用于马铃薯抗病、抗蚜虫转基因研究。