草莓转基因研究进展

草莓是世界上重要的水果之一。现代生物技术的不断发展为草莓育种和种质创新提供了新的技术手段, 它可以直接将来自不同种属的异源目的基因插人到草莓基因组, 使草莓表达目标性状, 实现草莓品种的遗传改良。近年来, 国内外草莓转基因研究取得了重大进展。文章综述了草莓转基因研究在抗病毒、抗虫、抗除草剂、抗逆及品质改良等方面的最新进展, 分析了草莓转基因研究中存在的主要问题, 并对今后的研究方向和应用前景进行了讨论。     

Genetic Diversity of Pto-Like Serine/Threonine Kinase Disease Resistance Genes in Cultivated and Wild Strawberries

Degenerate oligonucleotide primers, designed based on conserved regions of several serine-threonine kinases (STK) previously cloned in tomato and Arabidopsis, were used to isolate STK candidates in wild and cultivated strawberries. Seven distinct classes of STKs were identified from three related wild species, i.e., Fragaria vesca, Fragaria chiloensis, and Potentilla tucumanensis, and seven different Fragaria x ananassa cultivars. Alignment of the deduced amino acid sequences and the Pto R protein from tomato revealed the presence of characteristic subdomains and conservation of the plant STK consensus and other residues that are crucial for Pto function. Based on identity scores and clustering in phylogenetic trees, five groups were recognized as Pto-like kinases. Strawberry Pto-like clones presented sequences that were clearly identified as the activation segments contained in the Pto, and some of them showed residues previously identified as being required for binding to AvrPto. Some of the non-Pto-like kinases presented a high degree of identity and grouped together with B-lectin receptor kinases that are also involved in disease resistance. Statistical studies carried out to evaluate departure from the neutral theory and nonsynonymous/synonymous substitutions suggest that the evolution of STK-encoding sequences in strawberries is subjected mainly to a purifying selection process. These results represent the first report of Pto-like STKs in strawberry.     

Arbuscular mycorrhizae formed by Penicillium pinophilum improve the growth, nutrient uptake and photosynthesis of strawberry with two inoculum-types

Penicillium pinophilum was isolated from the soil in a commercial strawberry field. The strain readily formed arbuscular mycorrhizae (AM) with the roots of strawberry 'Zoji' (Fragaria x ananassa Duch. CV.) when plants were inoculated with either fresh cultured hyphae or root/soil mixtures. Fresh hyphae, however, resulted in higher amounts of colonization than root/soil inoculum. Compared with uninoculated strawberries, inoculation increased plant dry weight by 31%, as well as nitrogen content (47%), phosphorus content (57%), and photosynthetic rate (71%). AM inoculation also shortened the blossom and ripening date by 3 and 4 days, respectively. This is the first report of a P. pinophilum strain resulting in mycorrhiza with strawberry roots. The significant advantages of this strain are that it is easy to culture and inoculation of plants results in significant growth benefits that may be useful in strawberry production.     

RESOLUTION OF STRAWBERRY VIRUS COMPLEXES

Strawberry virus 4 produces vein chlorosis and necrosis on strawberry (var. Royal Sovereign) and slight chlorotic spotting on wild strawberry ( Fragaria vesca L.). No vector is known. Virus 5 produces leaf curling and vein necrosis on Royal Sovereign and F. vesca. It is transmitted by strawberry aphids ( Pentatrichopus fragaefolii Cock.) which have fed on an infected plant for 1 hr. or more and persists for about 1 hr. in the vector.
The names strawberry mottle, mild yellow-edge, crinkle, vein chlorosis and leaf-curl virus are proposed for strawberry viruses 1, 2, 3, 4 and 5 respectively.     

Successful strategy for the selection of new strawberry-associated rhizobacteria antagonistic to Verticillium wilt

In order to isolate and characterize new strawberry-associated bacteria antagonistic to the soil-borne pathogenic fungus Verticillium dahliae Kleb., rhizobacterial populations from two different strawberry species, Greenish Strawberry (Fragaria viridis) and Garden Strawberry (F. x ananassa) obtained after plating onto King's B and glycerol-arginine agar, were screened for in vitro antagonism toward V. dahliae. The proportion of isolates with antifungal activity determined in in vitro assay against V. dahliae was higher for the Garden Strawberry than for the Greenish Strawberry. From 300 isolates, 20 isolates with strong antifungal activity were selected characterized by physiological profiling and molecular fingerprinting methods. Diversity among the isolates was characterized with molecular fingerprints using amplified ribosomal DNA restriction analysis (ARDRA) and the more discriminating BOX-PCR fingerprint method. The physiological profiles were well correlated with molecular fingerprinting pattern analysis. Significant reduction of Verticillium wilt by bacterial dipping bath treatment was shown in the greenhouse and in fields naturally infested by V. dahliae. The relative increase of yield ranged from 117% (Streptomyces albidoflavus S1) to 344% (Pseudomonas fluorescens P10) in greenhouse trials, and 113% (Streptomyces albidoflavus S1) to 247% (Pseudomonas fluorescens P6) in field trials. Evaluation resulted in the selection of three effective biocontrol agents (Pseudomonas fluorescens P6, P10, and Streptomyces diastatochromogenes S9) antagonistic to the Verticillium wilt pathogen.     

Up- and down-regulation of Fragaria x ananassa O-methyltransferase: impacts on furanone and phenylpropanoid metabolism

A complex mixture of hundreds of substances determines strawberry (Fragaria x ananassa) aroma, but only approximately 15 volatiles are considered as key flavour compounds. Of these, 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) is regarded as the most important, but it is methylated further by FaOMT (Fragaria x ananassa O-methyltransferase) to 2,5-dimethyl-4-methoxy-3(2H)-furanone (DMMF) during the ripening process. It is shown here that transformation of strawberry with the FaOMT sequence in sense and antisense orientation, under the control of the cauliflower mosaic virus 35S promoter, resulted in a near total loss of DMMF, whereas the levels of the other volatiles remained unchanged. FaOMT repression also affected the ratio of feruloyl 1-O-beta-D-glucose and caffeoyl 1-O-beta-D-glucose, indicating a dual function of the enzyme in planta. Thus, FaOMT is involved in at least two different biochemical pathways in ripe strawberry fruit.     

Decaploidy in Fragaria iturupensis (Rosaceae)

The strawberry genus, Fragaria (Rosaceae), has a base chromosome number of x = 7. Cultivated strawberries (F. ×ananassa nothosubsp. ananassa) are octoploid (2n = 8x = 56) and first hybridized from F. chiloensis subsp. chiloensis forma chiloensis × F. virginiana subsp. virginiana. Europe has no known native octoploid species, and only one Asian octoploid species has been reported: F. iturupensis, from Iturup Island. Our objective was to examine the chromosomes of F. iturupensis. Ploidy levels of wild strawberry species, include diploid (2n = 2x = 14), tetraploid (2n = 4x = 28), pentaploid (2n = 5x = 35), hexaploid (2n = 6x = 42), octoploid (2n = 8x = 56), and nonaploid (2n = 9x = 63). Artificial triploid (2n = 3x = 21), tetraploid, pentaploid, octoploid, decaploid (2n = 10x = 70), 16-ploid, and 32-ploid plants have been constructed and cultivated. Surprisingly, chromosome counts and flow cytometry revealed that F. iturupensis includes natural decaploid genotypes with 2n = 10x = 70 chromosomes. This report is the first of a naturally occurring decaploid strawberry species. Further research on F. iturupensis and exploration on northern Pacific islands is warranted to ascertain the phylogeny and development of American octoploid species.     

The development of a bin mapping population and the selective mapping of 103 markers in the diploid Fragaria reference map.

We have identified a set of plants (the bin set) to permit "selective" or "bin" mapping using the diploid strawberry mapping population FV x FN, derived from the F2 cross F. vesca 815 x F. nubicola 601, which has been used to develop the Fragaria reference map. The bin set consists of 8 plants: the F. vesca 815 parent, the F1 hybrid individual, and 6 seedlings of the F2 population. This bin set divides the 578 cM of the diploid Fragaria genome into 46 bins, the largest mapping bin being 26 cM in length and the average bin size being 12.6 cM. To validate the FV x FN bin set, we used it to locate 103 loci into bins on the FV x FN map. These loci comprised 61 previously described SSRs, 38 new SSRs developed in this investigation from Fragaria x ananassa genomic DNA, EST and gene sequences, and 4 ripening-related genes developed for Prunus. The 103 markers were located to bins on all 7 linkage groups of the Fragaria map and a new mapping bin was identified with the novel markers, demonstrating that the map covers the majority of the diploid Fragaria genome and that the 6 bin-set seedlings selected were appropriate for bin mapping using this progeny.     

Development of microsatellite markers in Fragaria, their use in genetic diversity analysis, and their potential for genetic linkage mapping.

We have developed 21 new microsatellites in the model diploid perennial species Fragaria vesca from an enriched genomic library developed using F. vesca 'Ruegen'. The transferability of the primer pairs to other Fragaria species was high; all 31 primer pairs produced amplicons in 3 accessions of the octoploid strawberry Fragaria x ananassa, whereas 24 (77%) amplified a product in 7 other diploid Fragaria species. We analysed the allelic variation among 15 F. vesca accessions using the 21 microsatellites reported here and 10 F. vesca microsatellites described previously. The level of polymorphism detected at these microsatellite loci was high; five loci were monomorphic. Only two microsatellites were required to unambiguously discriminate among the 15 F. vesca accessions. A preliminary survey of segregation in an F2 progeny indicates that 20 of the 26 polymorphic loci (77%) could be mapped.     

Manipulation of strawberry fruit softening by antisense expression of a pectate lyase gene

Strawberry (Fragaria x ananassa, Duch., cv Chandler) is a soft fruit with a short postharvest life, mainly due to a rapid lost of firm texture. To control the strawberry fruit softening, we obtained transgenic plants that incorporate an antisense sequence of a strawberry pectate lyase gene under the control of the 35S promoter. Forty-one independent transgenic lines (Apel lines) were obtained, propagated in the greenhouse for agronomical analysis, and compared with control plants, non-transformed plants, and transgenic lines transformed with the pGUSINT plasmid. Total yield was significantly reduced in 33 of the 41 Apel lines. At the stage of full ripen, no differences in color, size, shape, and weight were observed between Apel and control fruit. However, in most of the Apel lines, ripened fruits were significantly firmer than controls. Six Apel lines were selected for further analysis. In all these lines, the pectate lyase gene expression in ripened fruit was 30% lower than in control, being totally suppressed in three of them. Cell wall material isolated from ripened Apel fruit showed a lower degree of in vitro swelling and a lower amount of ionically bound pectins than control fruit. An analysis of firmness at three different stages of fruit development (green, white, and red) showed that the highest reduction of softening in Apel fruit occurred during the transition from the white to the red stage. The postharvest softening of Apel fruit was also diminished. Our results indicate that pectate lyase gene is an excellent candidate for biotechnological improvement of fruit softening in strawberry.     

RNAi-induced silencing of gene expression in strawberry fruit (Fragaria x ananassa) by agroinfiltration: a rapid assay for gene function analysis

Intron-containing constructs encoding self-complementary 'hairpin' RNA (ihpRNA) have the potential to efficiently silence genes in a range of plant species. In this study we demonstrate the silencing of a ripening-related chalcone synthase (CHS) gene in strawberry fruits (Fragaria x ananassa cv. Elsanta) by a construct (ihpRNA) containing the partial sense and corresponding antisense sequences of CHS separated by an intron obtained from a F. x ananassa quinone oxidoreductase gene. An Agrobacterium strain carrying a T-DNA expressing the ihpRNA transgene was injected with a syringe into the receptacles of growing fruits still attached to the plant about 14 days after pollination. As a consequence of the reduced levels of CHS mRNA and enzymatic CHS activity, the levels of anthocyanins were downregulated and precursors of the flavonoid pathway were shunted to the phenylpropanoid pathway leading to a large increases in levels of (hydroxy) cinnamoyl glucose esters. We anticipate that this technique in combination with metabolite profiling analysis will be useful for studying the function of unknown genes during the development and ripening of strawberry fruit.     

Purification and identification of an IgE suppressor from strawberry in an in vitro immunization system

We purified and identified an IgE suppressor from the strawberry 'Toyonoka', based on the decrease of IgE production in in vitro immunization (IVI). Gel filtration experiment indicated that fractions in a 15-48 kDa range and <10 kDa have an IgE suppressive activity. Furthermore, the fraction in 15-48 kDa was subjected to chromatofocusing and found to have activities at isoelectric points, pI 6.0, 7.0, and 8.0-9.2. We focused on the active fractions of pI 8.0-9.2 and the purified a large amount of strawberry extracts by cation exchange resins in batch. A purified 39 kDa protein showed homology to plant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in N-terminal amino acid sequence and had GAPDH enzymatic activity. Nucleotide sequence and deduced amino acid sequence of the obtained cDNA clone of the protein matched with the sequence of Fragaria x ananassa GAPDH in the GenBank with >98% identical nucleotides and >99% identical amino acids, respectively. The purified strawberry GAPDH suppressed total IgE production in IVI in a dose-dependent manner. From these results, we identified GAPDH as IgE suppressor in the strawberry. Our study may be applicable to the development of new methods to relieve allergic conditions using GAPDH and the screening of other functional factors for human health.     

Occurrence of Tobacco Necrosis Virus in Strawberry Cultivars and Frageria vesca Indicators

Tobacco necrosis virus (TNV) was found occasionally in roots of indicator plants such as UC-4, UC-5 and alpine, commonly used for screening of strawberry virus diseases. The virus was purified and identified as TNV by its host-range, physical properties, electron microscopy and serological tests. The implication of the possible occurrence of TNV in indicator plants on reliable diagnosis of virus disease in strawberry is discussed.     

草莓开花期发生霜害的温度

用人工霜箱对草莓(Fragaria ananassa)叶片和花托进行模拟春霜实验。结果表明草莓叶片有忍耐胞间结冰的能力, 最低叶温-6.4 ℃以上, 结冰持续90分钟以内的叶片解冻后还能存活。花托不能忍耐胞间结冰, 是通过保持过冷却状态以回避结冰伤害。花托温度越低, 发生霜害的累积百分率越大, 半开花的花托温度降到-5.4 ℃时累计有50%发生结冰而造成霜害。盛开的花及鲜重大的花发生霜害的温度较高。