抗虫转基因马铃薯研究进展

马铃薯在整个生育期都易遭受虫害,从而导致马铃薯绝产或减产,同时,受到害虫危害的马铃薯品质也受到严重影响。当前马铃薯抗虫转基因的研究与应用是解决虫害的有效手段之一。总结了苏云金芽孢杆菌晶体蛋白基因、蛋白酶抑制剂基因、植物凝集素基因及RNAi的抗虫机制在马铃薯中的应用及其转基因植物研究中取得的进展,为马铃薯抗虫转基因研究和品种培育提供方法和对策。     

Production of dextran in transgenic potato plants

The production of dextran in potato tubers and its effect on starch biosynthesis were investigated. The mature dextransucrase (DsrS) gene from Leuconostoc mesenteroides was fused to the chloroplastic ferredoxin signal peptide (FD) enabling amyloplast entry, which was driven by the highly tuber-expressed patatin promoter. After transformation of two potato genotypes (cv. Kardal and the amylose-free (amf) mutant), dextrans were detected by enzyme-linked immunosorbent assay (ELISA) in tuber juices of Kardal and amf transformants. The dextran concentration appeared two times higher in the Kardal (about 1.7 mg/g FW) than in the amf transformants. No dextran was detected by ELISA inside the starch granule. Interestingly, starch granule morphology was affected, which might be explained by the accumulation of dextran in tuber juices. In spite of that, no significant changes of the physicochemical properties of the starches were detected. Furthermore, we have observed no clear changes in chain length distributions, despite the known high acceptor efficiency of DSRS.     

Induced phytophthora resistance in transgenic potato tubers

Resistance of transgenic cultivars based on the expression of one or more resistance genes is sooner or later broken by pathogens whose race-producing rates are high. Thus, combining transgenesis with elicitor-induced resistance is a promising approach. The elicitor-induced resistance is based on the expression of multiple resistance genes, which can prevent the adaptation of pathogens to transgenic races, maintain the stability of cultivars, and increase their lifespan. In this work, we used transgenic potato cultivars Temp and Superior transformed with Bacillus thuringiensis delta-endotoxin gene and Lukyanovskii transformed with leukocyte alpha-interferon gene. Arachidonic acid (10(-8) M) and soluble chitosan (5 kDa, 100 micrograms/ml) were used as elicitors for tuber treatment. Our data showed that pretreatment with elicitors causes a 15-25% increase in both the systemic prolonged resistance of potato tubers to Phytophthora infestans and their ability to repair mechanical damage.     

Cytokinins and cytokinin-binding sites in transgenic potato plants

The introduction of the gene for cytokinin biosynthesis into the potato genome led to a manifold increase in the level of cytokinins (zeatin and zeatin riboside) in transgenic plants grown in vitro. The high amount of endogenous cytokinins in 20-day-old plants of clone 1339-3A correlated with high cytokinin-binding capacity of ribosomes that is presumably attribute to a cytokinin receptor.     

Colorado potato beetles compensate for tomato cathepsin D inhibitor expressed in transgenic potato

We reported earlier the importance of digestive cathepsin D-like activity for initiating dietary protein hydrolysis in Colorado potato beetle, Leptinotarsa decemlineata Say [Brunelle et al. (1999) Arch. Insect Biochem. Physiol. 42:88-98]. We assessed here whether transgenic lines of potato (Solanum tuberosum L.) expressing a cathepsin D inhibitor (CDI) from tomato would show resistance to the beetle, or if the insect would compensate for the loss of cathepsin D activity after ingesting the recombinant inhibitor. Transgenic potato lines expressing tomato CDI were developed by Agrobacterium tumefaciens genetic transformation, and selected based on their relative amount of CDI. After confirming the absence of detectable visible effects of CDI on the plant's phenotype, diet assays with control and transgenic lines were carried out to assess the impact of the inhibitor on growth and development of the insect. Leaf consumption, relative growth rate, molting incidence, and digestive protease activity were monitored at 12-h intervals over 132 h for 3rd-instar larvae provided with transgenic potato foliage. Leaf consumption and relative growth rate were slightly reduced during the first 12 h for larvae fed CDI, but no significant differences were observed thereafter. In contrast, time for molting to the 4th larval stage was significantly longer for larvae fed modified plants, with developmental delays of approximately 10 h (0.5 day) compared to control larvae. Recombinant CDI also had an impact on the insect's digestive physiology, readily inducing overproduction of digestive proteases (rubiscases), followed by a gradual decrease of total and pepstatin-sensitive activity. Overall, these observations show the ability of Colorado potato beetle to compensate for the loss of cathepsin D activity by modulating its digestive protease complement in response to aspartate-type inhibitors in the diet. From a practical viewpoint, these data stress the importance of devising improved strategies for the effective inhibition of insect digestive proteinases in vivo, based on the use of hybrid inhibitors active against different protease classes.     

Ketocarotenoid Production in Soybean Seeds through Metabolic Engineering

The pink or red ketocarotenoids, canthaxanthin and astaxanthin, are used as feed additives in the poultry and aquaculture industries as a source of egg yolk and flesh pigmentation, as farmed animals do not have access to the carotenoid sources of their wild counterparts. Because soybean is already an important component in animal feed, production of these carotenoids in soybean could be a cost-effective means of delivery. In order to characterize the ability of soybean seed to produce carotenoids, soybean cv. Jack was transformed with the crtB gene from Pantoea ananatis, which codes for phytoene synthase, an enzyme which catalyzes the first committed step in the carotenoid pathway. The crtB gene was engineered together in combinations with ketolase genes (crtW from Brevundimonas sp. strain SD212 and bkt1 from Haematococcus pluvialis) to produce ketocarotenoids; all genes were placed under the control of seed-specific promoters. HPLC results showed that canthaxanthin is present in the transgenic seeds at levels up to 52 μg/g dry weight. Transgenic seeds also accumulated other compounds in the carotenoid pathway, such as astaxanthin, lutein, β-carotene, phytoene, α-carotene, lycopene, and β-cryptoxanthin, whereas lutein was the only one of these detected in non-transgenic seeds. The accumulation of astaxanthin, which requires a β-carotene hydroxylase in addition to a β-carotene ketolase, in the transgenic seeds suggests that an endogenous soybean enzyme is able to work in combination with the ketolase transgene. Soybean seeds that accumulate ketocarotenoids could potentially be used in animal feed to reduce or eliminate the need for the costly addition of these compounds.     

Synthesis of active oryzacystatin I in transgenic potato plants

Summary Transformation of potato (Solanum tuberosum L.) with cysteine proteinase inhibitor (PI) genes represents a potential way of controlling the major insect pest Colorado potato beetle (CPB; Leptinotarsa decemlineata Say). The present study describes the Agrobacterium-mediated transformation of potato (cv. Kennebec) with an oryzacystatin I (OCI) cDNA clone linked to a CaMV 35S promoter. The transgenic plants accumulated active OCI in potato leaves, as demonstrated by the papain-inhibitory activity of transgenic plant leaf extracts. In addition to their anti-papain activity, the extracts also caused a partial but significant inhibition of CPB digestive proteinases, similar to that observed with pure inhibitors. Recombinant OCI did not alter the activity of the major potato leaf endogenous proteinases, which seemed to be of the serine-type. Therefore we suggest that the OCI cDNA can be used for the production of CPB-resistant transgenic potato plants without interfering with endogenous proteinases of these plants.Abbreviations CPB Colorado potato beetle - E-64 trans-epoxy-succinyl-L-leucylamido (4-guanidino) butane - OCI oryzacystatin I - PI proteinase inhibitor - PMSF phenylmethylsulfonyl fluoride     

Ketocarotenoid biosynthesis by Haematococcus lacustris

Haematococcus lacustris incubated on a nutrient-depleted medium utilised acetate-[2-¹⁴C] from the medium and carbon fixed photosynthetically for the biosynthesis of ketocarotenoids. Conversion of β-carotene to astaxanthin occurs via the intermediates echinenone and canthaxanthin.     

Effective transgenic resistance to Globodera pallida in potato field trials

A cysteine proteinase inhibitor expressed in potato plants provides the first demonstration that transgenic resistance to nematodes such as the potato cyst nematode Globodera pallida can be effective under field conditions. The highest level of resistance obtained in the field for one of the four transformed lines of the normally fully susceptible Solanum tuberosum tuberosum cv. Désirée was 70±9%. The partially resistant cv. Sante that is currently of commercial use in the UK showed a resistance of 85±3%. In containment experiments a change in G. pallida population to one known to be virulent against cv. Sante caused a significant loss of its resistance from 80±4% to only 51±6%. In contrast, the resistance of transgenic Désirée was similar for challenge by the avirulent and virulent populations with values of 68±6% and 71±4%, respectively. Constitutive expression of the cystatin had no detrimental effect on either number or weight of tubers and the weight of the haulm for 3 of the 4 lines in the field. The results establish that transgenic field resistance against G. pallida can be achieved. Clearly food and environmental safety must be assured before commercialisation can be contemplated. However, a prima facie case can be made that the technology is benign and can reduce the use of environmentally hazardous nematicides.     

Transport and metabolism of raffinose family oligosaccharides in transgenic potato

Raffinose family oligosaccharides (RFOs) are involved in the storage and transport of carbon and serve as compatible solutes for protection against abiotic stresses like drought or cold. RFOs are usually transported in plant species that load sugars symplastically into the phloem. Loading probably occurs by a polymer trapping mechanism which establishes a concentration gradient of assimilates between the mesophyll and the vasculature. Transgenic approaches have demonstrated phloem transport of small molecules produced in the companion cells of apoplastic loading species, but these molecules have been non-native transport substances to plants. In this study, transgenic potato plants with constitutive or companion cell specific overexpression of galactinol synthase (GS) or GS plus raffinose synthase (RS) are characterized, which together provide new insights into the metabolism and transport of RFOs in plants. It is demonstrated that raffinose and galactinol are both transported in the phloem and that, whilst the effect of GS overexpression is promoter-independent, that of RS is dependent on the promoter used. The presence of significant amounts of galactinol in the phloem is shown and also that transgenic potato is unable to transport large amounts of raffinose despite high RS expression and substrate concentrations. These data indicate that there may be additional features of intermediary cells, the specialized companion cells of RFO transporting plants, required for significant RFO synthesis and transport that are currently not well-understood.     

禽流感病毒血凝素疫苗在转基因马铃薯中的表达

利用转基因马铃薯表达禽流感病毒血凝素疫苗,将含有禽流感病毒血凝素序列的表达载体导入农杆菌,再感染马铃薯的幼茎外植体。转化植株的再生及温室栽培,Western blot分析表明,83％的转化植株在其块茎组织中表达了重组血凝素,表达量占总蛋白量的0．03－0．04％,结果显示用马铃薯生产口服禽流感疫苗是可行的。     

RNAi-mediated tocopherol deficiency impairs photoassimilate export in transgenic potato plants

Tocopherols (vitamin E) are lipophilic antioxidants presumed to play a key role in protecting chloroplast membranes and the photosynthetic apparatus from photooxidative damage. Additional nonantioxidant functions of tocopherols have been proposed after the recent finding that the Suc export defective1 maize (Zea mays) mutant (sxd1) carries a defect in tocopherol cyclase (TC) and thus is devoid of tocopherols. However, the corresponding vitamin E deficient1 Arabidopsis mutant (vte1) lacks a phenotype analogous to sxd1, suggesting differences in tocopherol function between C4 and C3 plants. Therefore, in this study, the potato (Solanum tuberosum) ortholog of SXD1 was isolated and functionally characterized. StSXD1 encoded a protein with high TC activity in vitro, and chloroplastic localization was demonstrated by transient expression of green fluorescent protein-tagged fusion constructs. RNAi-mediated silencing of StSXD1 in transgenic potato plants resulted in the disruption of TC activity and severe tocopherol deficiency similar to the orthologous sxd1 and vte1 mutants. The nearly complete absence of tocopherols caused a characteristic photoassimilate export-defective phenotype comparable to sxd1, which appeared to be a consequence of vascular-specific callose deposition observed in source leaves. CO2 assimilation rates and photosynthetic gene expression were decreased in source leaves in close correlation with excess sugar accumulation, suggesting a carbohydrate-mediated feedback inhibition rather than a direct impact of tocopherol deficiency on photosynthetic capacity. This conclusion is further supported by an increased photosynthetic capacity of young leaves regardless of decreased tocopherol levels. Our data provide evidence that tocopherol deficiency leads to impaired photoassimilate export from source leaves in both monocot and dicot plant species and suggest significant differences among C3 plants in response to tocopherol reduction.     

Pleiotropic effect of flavonoid biosynthesis manipulation in transgenic potato plants

Three approaches were successfully used to manipulate content of flavonoids in transgenic plants. Overexpressing either the adaptor 14-3-3 protein or genes coding the key enzymes of the flavonoid biosynthesis pathway resulted in a significant increase in the compound content in potato tuber epidermis. The opposite effect was observed in transgenic plants in which these proteins were repressed; this strongly supports the view that the gene construct determines transgenic plant features. The most effective construct was, however, the one containing single dihydroflavonol reductase (DFR) gene in sense orientation. In all cases the increase in flavonoid content resulted in the expected enhancement of the antioxidant capacity of tuber extract. At the biochemical level a decrease in the starch content in transgenic plant overexpressing proteins regulating flavonoid biosynthesis was detected. In the case of glucosyl transferase (GT) gene overexpression, the content of phenolic compounds remained at the control level, however, the antioxidant capacity of tuber extracts significantly decreased. The GT plants grew faster and were more resistant to pathogen attacks, the tuber yield was significantly higher than that of nontransformants. Thus it is speculated that it is the chemical structure and degree of glucosylation of flavonoids rather than their quantity which determines transgenic plant features.     

Gel formation in mixtures of high amylopectin potato starch and potato starch

The effects of starch granules on the rheological behaviour of gels of native potato and high amylopectin potato (HAPP) starches have been studied with small deformation oscillatory rheometry. The influence of granule remnants on the rheological properties of samples treated at 90 °C was evident when compared with samples treated at 140 °C, where no granule remnants were found. The presence of amylose in native potato starch gave to stronger network formation since potato starch gave higher moduli values than HAPP, after both 90 and 140 °C treatments. In addition, amylose may have strengthened the network of HAPP because higher moduli values were obtained when native potato starch was added to the system. The moduli values of the mixtures also increased with increasing polysaccharide concentration in the system, which is due to an increment in the polysaccharide chain contacts and entanglements. Finally, it was found that a mixture of commercial amylose from potato starch and HAPP resulted in lower values of G′ compared to native potato starch. This indicates that the source of amylose is important for the properties in a blend with native amylopectin.     

The catecholamine potentiates starch mobilization in transgenic potato tubers.

In human and animal cells, the catecholamines are involved in glycogen mobilization. Since the compounds are found in a potato, their function in starch mobilization was hypothesized. In order to verify this hypothesis, the transgenic potato plants Solanum tuberosum L. cv. Desiree overexpressing tyrosine decarboxylase (TD EC 4.1.1.25) cDNA from parsley has been generated. The cDNA expression was judged by the northern blot analysis and the enzyme activity measurements. Four independent transgenic lines with the highest TD mRNA expression were selected and analyzed. The expected substantial decrease in tyrosine content was followed by significant increase in tyramine and dramatic enhancement of norepinephrine synthesis was detected. The level of L-3,4-dihydroxyphenylalanin (L-Dopa) was only slightly increased and dopamine significantly decreased in most cases in these plants. The increase in norepinephrine was accompanied by changes in carbohydrate metabolism. The significant increase in glucose and sucrose and the decrease in starch content were characteristic features of TD overexpressed transgenic potato tubers. The features mentioned above indicate that catecholamines potentiate starch mobilization in potato plants in common with animal cells. The decrease in tyrosine content in transgenic plants is also compensated by significant increase in chlorogenic acid synthesis thus potentially increasing the antioxidant capacity of transgenic tubers. The glycoalkaloids content is changed in the transformants. This may originate from glucose accumulation and glycolysis activation. The obtained transgenic potato provides material for further detailed studies of the physiological function of catecholamines in plants.     

Expression of the potato leafroll luteovirus coat protein gene in transgenic potato plants inhibits viral infection

Transgenic potato plants, cultivar Désirée, were produced that contained the coat protein gene of potato leafroll luteovirus (PLRV). The transformed potato plants expressed the PLRV coat protein (CP) RNA sequences but accumulation of coat protein in transgenic tissues could not be detected. Upon inoculation with PLRV, the PLRV CP RNA expressing potato plants showed a reduced rate of virus multiplication.