Transcriptomic analysis of succulent stem development of Chinese kale (Brassica oleracea var. alboglabra Bailey) and its synthetic allotetraploid via RNA sequencing.

Chinese kale (Brassica oleracea var. alboglabra Bailey, CC) is a succulent stem vegetable in the Brassica family. Its allotetraploid (AACC) vegetable germplasm, which was synthesized via distant hybridization with the colloquially named 'yellow turnip' (B. rapa L. ssp. rapifera Matzg., AA), has a swelling stem similar to CC. To address the molecular mechanism of stem development for CC and AACC, RNA sequencing (RNA-seq) was used to investigate transcriptional regulation of their stem development at three key stages including 28 days, 42 days and the bolting stage (BS) after sowing. As a result, 32,642, 32,665, 33,816, 32,147, 32,293 and 32,275 genes were identified in six corresponding cDNA libraries. Among them, 25,459 genes were co-expressed, while 7,183, 7,206, 8,357, 6,688, 6,834 and 6,814 genes were specifically expressed. Additionally, a total of 29,222 differentially expressed genes (DEGs) were found for functional enrichment as well as many genes involved in plant hormones including gibberellin (GA), abscisic acid (ABA), cytokinin (CTK) and auxin (AUX). Based on gene expression consistency between CC and AACC, the gene families including DELLA, GID, PYR/PYL, PP2C, A-ARR and AUX/IAA might be related to stem development. Among these, eight genes including Bo00834s040, Bo5g093140, Bo6g086770, Bo9g070200, Bo7g116570, Bo3g054410, Bo7g093470 and Bo5g136600 may play important roles in stem development based on their remarkable expression levels as confirmed by qRT-PCR. These findings provide a new theoretical basis for understanding the molecular mechanism of stem development in Brassica vegetable stem breeding.

Effect of short-term high temperature on the accumulation of glucosinolates in Brassica rapa.

Glucosinolates, an important class of secondary metabolites in cruciferous vegetables, play a crucial role in protecting plants from stress-related damage. The mechanism of glucosinolate synthesis under short-term high temperature stress has not been sufficiently studied. In this work, we investigated the changes in transcription factors, synthetic genes, and related metabolites involved in glucosinolate synthesis by pakchoi seedlings under short-term high temperature stress (40 °C for 8 h). Short-term high temperature stress inhibited the primary sulfur assimilation and the contents of methionine, cysteine and glutathione. The contents of aliphatic and indolic glucosinolates were increased by short-term high temperature stress, whereas the content of 4-methoxy-glucobrassicin increased significantly. During the stress period, the transcript level of glucosinolate related MYB transcription factors had been basically significantly up-regulated, whereas the transcript level of aliphatic and indolic glucosinolate synthetic genes were predominantly up-regulated and down-regulated respectively. In the early recovery period, primary sulfur assimilation up-regulated rapidly, and decreased during the late recovery process. The glucosinolate content and synthesis gene expression act similar to the primary sulfur assimilation, a short up-regulated in early recovery, then all go down at 40 and 48 h after short-term high temperature treatment.

One-Step Bioconversion of Fatty Acids into C8-C9 Volatile Aroma Compounds by a Multifunctional Lipoxygenase Cloned from Pyropia haitanensis.

The multifunctional lipoxygenase PhLOX cloned from Pyropia haitanensis was expressed in Escherichia coli with 24.4 mg·L-1 yield. PhLOX could catalyze the one-step bioconversion of C18-C22 fatty acids into C8-C9 volatile organic compounds (VOCs), displaying higher catalytic efficiency for eicosenoic and docosenoic acids than for octadecenoic acids. C20:5 was the most suitable substrate among the tested fatty acids. The C8-C9 VOCs were generated in good yields from fatty acids, e.g., 2E-nonenal from C20:4, and 2E,6Z-nonadienal from C20:5. Hydrolyzed oils were also tested as substrates. The reactions mainly generated 2E,4E-pentadienal, 2E-octenal, and 2E,4E-octadienal from hydrolyzed sunflower seed oil, corn oil, and fish oil, respectively. PhLOX showed good stability after storage at 4 °C for 2 weeks and broad tolerance to pH and temperature. These desirable properties of PhLOX make it a promising novel biocatalyst for the industrial production of volatile aroma compounds.

Leaf and root glucosinolate profiles of Chinese cabbage (Brassica rapa ssp. pekinensis) as a systemic response to methyl jasmonate and salicylic acid elicitation.

Glucosinolates (GSs) are an important group of defensive phytochemicals mainly found in Brassicaceae. Plant hormones jasmonic acid (JA) and salicylic acid (SA) are major regulators of plant response to pathogen attack. However, there is little information about the interactive effect of both elicitors on inducing GS biosynthesis in Chinese cabbage (Brassica rapa ssp. pekinensis). In this study, we applied different concentrations of methyl jasmonate (MeJA) and/or SA onto the leaf and root of Chinese cabbage to investigate the time-course interactive profiles of GSs. Regardless of the site of the elicitation and the concentrations of the elicitors, the roots accumulated much more GSs and were more sensitive and more rapidly responsive to the elicitors than leaves. Irrespective of the elicitation site, MeJA had a greater inducing and longer lasting effect on GS accumulation than SA. All three components of indole GS (IGS) were detected along with aliphatic and aromatic GSs. However, IGS was a major component of total GSs that accumulated rapidly in both root and leaf tissues in response to MeJA and SA elicitation. Neoglucobrassicin (neoGBC) did not respond to SA but to MeJA in leaf tissue, while it responded to both SA and MeJA in root tissue. Conversion of glucobrassicin (GBC) to neoGBC occurred at a steady rate over 3 d of elicitation. Increased accumulation of 4-methoxy glucobrassicin (4-MGBC) occurred only in the root irrespective of the type of elicitors and the site of elicitation. Thus, accumulation of IGS is a major metabolic hallmark of SA- and MeJA-mediated systemic response systems. SA exerted an antagonistic effect on the MeJA-induced root GSs irrespective of the site of elicitation. However, SA showed synergistic and antagonistic effects on the MeJA-induced leaf GSs when roots and leaves are elicitated for 3 d, respectively.

A tomato endoplasmic reticulum (ER)-type omega-3 fatty acid desaturase (LeFAD3) functions in early seedling tolerance to salinity stress.

KEY MESSAGE: Transgenic tomato plants overexpressing LeFAD3 sense and antisense sequences were generated. Salt stress suppressed the growth of WT and antisense plants to a higher extent than that in sense plants. In this study, we investigated the role of the LeFAD3-encoding ER-type omega-3 fatty acid desaturase in salt tolerance in tomato plants. We created transgenic tomato plants by overexpressing its sense and antisense sequences under the control of the cauliflower mosaic virus 35S promoter. Based on the results of northern and western blotting as well as quantitative reverse transcription-polymerase chain reaction, sense plants expressed more desaturase than wild-type (WT) plants, whereas antisense plants expressed less desaturase than WT. Salt stress suppressed the growth of both WT and antisense plants to a higher extent than that in sense plants, which can be attributed to the fact that sense plants performed better in maintaining the integrity of the membrane system, as revealed by electron microscopy. The concomitant increase in superoxide dismutase (EC 1.15.1.1) and ascorbate peroxidase (EC 1.11.1.7) may have alleviated the photoinhibition caused by the increased level of ROS in sense plants. Our results suggest that LeFAD3 overexpression can enhance the tolerance of early seedlings to salinity stress.

Cloning and functional analysis of a novel ascorbate peroxidase (APX) gene from Anthurium andraeanum.

An 888-bp full-length ascorbate peroxidase (APX) complementary DNA (cDNA) gene was cloned from Anthurium andraeanum, and designated as AnAPX. It contains a 110-bp 5'-noncoding region, a 28-bp 3'-noncoding region, and a 750-bp open reading frame (ORF). This protein is hydrophilic with an aliphatic index of 81.64 and its structure consisting of α-helixes, ß-turns, and random coils. The AnAPX protein showed 93%, 87%, 87%, 87%, and 86% similarities to the APX homologs from Zantedeschia aethiopica, Vitis pseudoreticulata, Gossypium hirsutum, Elaeis guineensis, and Zea mays, respectively. AnAPX gene transcript was measured non-significantly in roots, stems, leaves, spathes, and spadices by real-time polymerase chain reaction (RT-PCR) analysis. Interestingly, this gene expression was remarkably up-regulated in response to a cold stress under 6 °C, implying that AnAPX might play an important role in A. andraeanum tolerance to cold stress. To confirm this function we overexpressed AnAPX in tobacco plants by transformation with an AnAPX expression construct driven by CaMV 35S promoter. The transformed tobacco seedlings under 4 °C showed less electrolyte leakage (EL) and malondialdehyde (MDA) content than the control. The content of MDA was correlated with chilling tolerance in these transgenic plants. These results show that AnAPX can prevent the chilling challenged plant from cell membrane damage and ultimately enhance the plant cold tolerance.

Variation in glucosinolates in pak choi cultivars and various organs at different stages of vegetative growth during the harvest period.

Glucosinolates (GSs) play an important role in plant defense systems and human nutrition. We investigated the content and composition of GSs in the shoots and roots of seven cultivars of pak choi. We found that 'Si Yue Man' had the highest total and aliphatic GS contents in the shoots and the highest benzenic GS content in the roots, 'Shanghai Qing' contained the highest amounts of benzenic and total GS contents in the roots, while 'Nanjing Zhong Gan Bai' had the lowest benzenic, indole, and total GS contents in both the shoots and roots. Therefore, the 'Si Yue Man' cultivar appears to be a good candidate for future breeding. Variation between the shoots and roots was also examined, and a significant correlation among the total, aliphatic, and some individual GSs was found, which is of value in agricultural breeding. GS concentrations of the leaf, petiole, and root increased dramatically during the period of rapid growth of the dry matter of the plant 10 to 20 d after transplantation, reaching peak values on Day 20 and decreasing on Day 25. We conclude that the pak choi should be harvested and consumed from 20 to 25 d after transplantation to take advantages of the high GS content in the plant.

Antisense-mediated depletion of GMPase gene expression in tobacco decreases plant tolerance to temperature stresses and alters plant development.

In our previous work [1] we investigated the role of tomato GDP-mannose pyrophosphorylase (EC 2.7.7.22) in plants by overexpressing its gene in tobacco leaves and showed its function in AsA metabolism and detoxification of reactive oxygen species under temperature stresses. In this study, we use the antisense technique to block the endogenous GMPase gene expression in tobacco in order to further investigate its function. Northern and western blot analysis confirmed that the expression of endogenous tobacco GMPase mRNA and protein was inhibited by this antisense expression. Consequently, the activity of GMPase and the content of AsA in the leaves of antisense transgenic plants were markedly decreased. This was also the case for the activities of both chloroplastic SOD (superoxide dismutase EC 1.15.1.1), APX (ascorbate peroxidase EC 1.11.1.7) and the content of AsA in leaves of the transgenic plants. On the contrary, the contents of H(2)O(2) and O(2) (-•) were increased. Meanwhile, the net photosynthetic rate (Pn) and the maximal photochemical efficiency of PSII (Fv/Fm) also declined in the leaves of antisense plants. Under high or low temperature stresses, the seed germination rate of the antisense transgenic plants was significantly decreased in comparison with that of the wild-type tobacco. Interestingly, the antisense plants had smaller leaves and an earlier onset of flowering. In conclusion, the depletion of GMPase decreased the content of AsA, resulting in the plants susceptible to the oxidative damage caused by temperature stresses and subjected to developmental alternations.

An assessment of psychological noise reduction by landscape plants.

The emphasis in the term 'Green Transportation' is on the word 'green'. Green transportation focuses on the construction of a slow transport system with a visually pleasing, easy and secure trip environment composed of urban parks, green roadside spaces and some other space that is full of landscape plants. This trip environment encourages residents to make trip choices that reduce fuel consumption and pollution and is one of the most important ways of popularizing green transportation. To study the psychological benefits provided by urban parks and other landscape environments, we combined a subjective approach (a questionnaire) with an objective quantitative approach (emotional tests using an electroencephalogram; EEG). Using a questionnaire survey, we found that 90% of the subjects believed that landscape plants contribute to noise reduction and that 55% overrated the plants' actual ability to attenuate noise. Two videos (showing a traffic scene and a plant scene) were shown to 40 participants on video glasses. We detected and recorded EEG values with a portable electroencephalograph, and a comparison between the results of the two groups revealed that there was a highly significant asymmetry between the EEG activity of the vegetation scene and traffic scene groups. The results suggest that the emotions aroused by noise and visual stimuli are manifested in the synchronization of beta frequency band and the desynchronization of alpha frequency band, indicating that landscape plants can moderate or buffer the effects of noise. These findings indicate that landscape plants provide excess noise attenuating effects through subjects' emotional processing, which we term 'psychological noise reduction'.

Overexpression in tobacco of a tomato GMPase gene improves tolerance to both low and high temperature stress by enhancing antioxidation capacity.

GDP-mannose pyrophosphorylase (GMPase: EC 2.7.7.22) plays a crucial role in the synthesis of L-ascorbate (AsA) and the consequent detoxification of reactive oxygen species (ROS). Herein, a GMPase (accession ID DQ449030) was identified and cloned from tomato. The full-length cDNA sequence of this gene contains 1,498 bp nucleotides encoding a putative protein with 361 amino acid residues of approximate molecular weight 43 kDa. Northern blot analysis revealed that the GMPase was expressed in all examined tomato tissues, but its expression level was up-regulated in tomato plants subjected to abnormal temperatures. We then overexpressed this tomato GMPase in tobacco plants and observed that the activity of GMPase and the content of AsA were significantly increased by two- to fourfold in the leaves of transgenic tobacco plants. The effect of this gene overexpression was superimposed by the treatments of high or low temperature in tobacco, since the activities of both chloroplastic SOD (superoxide dismutase EC 1.15.1.1), APX (ascorbate peroxidase EC 1.11.1.7) and the content of AsA in leaves were significantly higher in transgenic plants than those of WT, while the contents of H(2)O(2) and O(2)(-·) were reduced. Meanwhile, relative electric conductivity increased less in transgenic plants than that in WT, and the net photosynthetic rate (P(n)) and the maximal photochemical efficiency of PSII (F(v)/F(m)) of transgenic plants were notably higher than those of WT under temperature stresses. In conclusion, the overexpression of GMPase increased the content of AsA, thereby leading to the increase in tolerance to temperature stress in transgenic plants.

Effects of osmotic stress on antioxidant enzymes activities in leaf discs of PSAG12-IPT modified Gerbera.

Leaf senescence is often caused by water deficit and the chimeric gene P(SAG12)-IPT is an auto-regulated gene delaying leaf senescence. Using in vitro leaf discs culture system, the changes of contents of chlorophylls, carotenoids, soluble protein and thiobarbituric acid reactive substance (TBARS) and antioxidant enzymes activities were investigated during leaf senescence of P(SAGl2)-IPT modified gerbera induced by osmotic stress compared with the control plant (wild type). Leaf discs were incubated in 20%, 40% (w/v) polyethylene glycol (PEG) 6000 nutrient solution for 20 h under continuous light [130 micromol/(m(2) x s)]. The results showed that the contents of chlorophylls, carotenoids and soluble protein were decreased by osmotic stress with the decrease being more pronounced at 40% PEG, but that, at the same PEG concentration the decrease in the transgenic plants was significantly lower than that in the control plant. The activities of superoxide dismutase (SOD), catalases (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and dehydroascorbate reductase (DHAR) were stimulated by PEG treatment. However, the increases were higher in P(SAG12)-IPT transgenic plants than in the control plants, particularly at 40% PEG treatment. Lipid peroxidation (TBARS content) was increased by PEG treatment with the increase being much lower in transgenic plant than in the control plant. It could be concluded that the increases in the activities of antioxidant enzymes including SOD, CAT, APX, GPX and DHAR were responsible for the delay of leaf senescence induced by osmotic stress.

[Effects of high concentration manganese on active oxygen production and antioxidant enzyme activities in cucumber leaves under different irradiations].

Effects of high concentration manganese on active oxygen production and antioxidant enzymes in cucumber leaves under natural irradiation and 1/2 natural irradiation were studied. High concentration manganese increased the H2O2 content and O2*- producing rate of cucumber leaves (Fig.1A, B) and caused lipid peroxidation (Fig.1C). Compare to natural irradiation, 1/2 natural irradiation significantly decreased active oxygen production and lipid peroxidation (Fig.1). CAT activities in cytosols and chloroplasts were inhibited by high concentration manganese (Fig.2B), and other antioxidant enzyme activities were enhanced by high concentration manganese (Fig.2A, C-F). Especially activities of APX, DHAR and GR in chloroplasts were greatly stimulated by high concentration manganese under natural irradiation compared to 1/2 irradiation, which may play important roles in scavenging active oxygen species. Antioxidant enzymes in mitochondria showed higher activities under high concentration manganese under natural irradiation, but the differences were not significant under 1/2 natural irradiation (Fig.2A, C-F).

[Effects of exogenous silicon on active oxygen scavenging systems in chloroplasts of cucumber (Cucumis sativus L.) seedlings under salt stress].

With K(2)SiO(4) (1.0 mmol/L) treatment, the effects of Si on the distribution of Na(+), K(+) to chloroplasts and antioxidant system of cucumber leaves under 50 mmol/L NaCl stress were studied. The results showed that there was a selective transport of K(+) into the chloroplasts so that Na(+) content of chloroplasts was lower under Si treatment (Table 1); H(2)O(2) and MDA contents in chloroplasts were significantly decreased (Fig.1), and the activities of SOD, APX, GR and DHAR were increased simultaneity (Fig.2), and AsA, GSH contents were also increased in chloroplasts of salt-stressed cucumber by additional Si treatment (Fig.3). It may be concluded that Si could decrease absorption of Na(+) and increase ability of active oxygen scavenging in chloroplasts, therefore the injury of chloroplast membrane under salinity stress in cucumber was alleviated.

[Effects of iso-osmotic salt stress on the activities of antioxidative enzymes, H+-ATPase and H+-PPase in tomato plants].

Seedlings of tomato (Lycopersicon esculentum Mill.) were grown in nutrient solution to investigate the changes in malondialdehyde (MDA) and proline content, and activities of antioxidative enzymes, plasmalemma H(+)-ATPase, and tonoplast H(+)-ATPase and H(+)-PPase to iso-osmotic stress of Ca(NO(3))(2) (80 mmol/L) and NaCl (120 mmol/L). The results indicated that both Ca(NO(3))(2) and NaCl stress significantly increased superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activities in leaf cytosols and chloroplasts. The extent of increase caused by NaCl was higher than that by Ca(NO(3))(2). The activities of SOD, CAT and APX in mitochondria were increased under Ca(NO(3))(2) stress and decreased under NaCl stress. Salt stress enhanced the activities of plasmalemma H(+)-ATPase, tonoplast H(+)-ATPase and H(+)-PPase. The increases in tonoplast H(+)-ATPase and H(+)-PPase activity were much higher under Ca(NO(3))(2) stress than under NaCl stress. The MDA and proline content increased under salt stress, especially under NaCl stress. From the results, it can be concluded that both salt inhibited the plant growth, the effect of NaCl being much heavier.

Genotypic differences in effects of cadmium exposure on plant growth and contents of cadmium and elements in 14 cultivars of bai cai.

Fourteen cultivars of bai cai (Brassica campestris L. ssp. chinensis var. communis) were grown in the nutrient solutions containing 0-0.5 microg mL(-1) of cadmium (Cd) to investigate genotypic differences in the effects of Cd exposure on the plant growth and uptake and distribution of Cd in bai cai plants. The Cd exposure significantly reduced the dry and fresh weights of roots and shoots, the dry weight ratio of shoot/root (S/R), total biomass, and chlorophyll content (SPAD value). Cd concentrations in bai cai ranged from 13.3 to 74.9 microg g(-1) DW in shoots and from 163.1 to 574.7 microg g(-1) DW in roots under Cd exposure, respectively. The considerable genotypic differences of Cd concentrations and accumulations in both shoots and roots were observed among 14 bai cai cultivars. Moreover, Cd mainly accumulated in the roots. Cd also caused the changes of uptake and distribution of nutrients in bai cai and under the influence of cadmium, the concentration of potassium (K) decreased in shoot and increased in root. However, the concentrations of magnesium (Mg), phosphorus (P), manganese (Mn), boron (B), and iron (Fe) increased in shoots and decreased in roots. In addition, Cd exposure resulted in an increase in calcium (Ca), sulphur (S), and zinc (Zn) concentrations in both shoots and roots but had no significant effects on the whole uptake of the examined mineral nutrients except for S.

[Transformation of wml1 5' promoter region into tomato plants and studies on its transcriptional regulation role].

According to its restriction sites, fragments of 1573 bp, 1197 bp, 896 bp and 795 bp were obtained from the 5' promoter region of wml1 and fused with the coding sequence of the GUS gene. Constructs containing these fragments were introduced into tomato plants via Agrobacterium-mediated transformation. Histochemical assay of GUS expression in transgenic tomato plants revealed that fragments of 1573 bp, 1197 bp, 896 bp were able to direct GUS expression in fruits of 15, 30, 45 days after anthesis with the expression level of GUS increasing with fruit development, but not in leaves, stems and roots. While no GUS expression was observed in tomato plants transformed by construct containing fragment of 795 bp. It was determined that the region from 857 bp to 957 bp contains the elements necessary for directing fruit-specific expression.

Expression of CycD3 is transiently increased by pollination and N-(2-chloro-4-pyridyl)-N'-phenylurea in ovaries of Lagenaria leucantha.

Lagenaria leucantha is an important vegetable crop and a potential model for the study of fruit development. To study the function of D cyclins in fruit development, full-length cDNA clones for two D cyclin genes were isolated from young ovaries of Lagenaria leucantha. They were classified as D3 cyclins by sequence similarities and phylogenetic analysis, and nominated LlCycD3;1 and LlCycD3;2, respectively. The deduced amino acid sequence of both LlCycD3 genes contained a retinoblastoma-binding motif and a PEST-destruction motif. Unpollinated ovaries failed to develop and eventually aborted. N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU) induced parthenocarpic fruit significantly larger than pollinated ones. In unpollinated ovaries, the expression of both LlCycD3 genes was abundant at anthesis and then suddenly decreased, concomitant with the cessation of cell division. Pollination/fertilization induced an activation of the cell cycle accompanied by a large increase in the transcript levels of LlCycD3;1 and LlCycD3;2 in young fruits. Treating ovaries with CPPU also reactivated cell division and transcription of CycD3 genes and the effect was more rapid and pronounced than after pollination/fertilization.

[Primary targeting of functional regions involved in transcriptional regulation on watermelon fruit-specific promoter WSP].

Fruit ripening is associated with a number of physiological and biochemical changes. They include degradation of chlorophyll, synthesis of flavor compounds, carotenoid biosynthesis, conversion of starch to sugars, cell wall solublisation and fruit softening. These changes are brought about by the expression of specific genes. People are interested in the molecular mechanism involved in the regulation of gene transcription during fruit ripening. Many fruit-specific promoters such as PG, E4, E8, and 2A11 have been characterized and shown to direct ripening-specific expression of reporter genes. AGPase plays the key role in catalyzing the biosynthesis of starch in plants. It is a heterotetrameric enzyme with two small subunits and two large subunits, which are encoded by different genes. In higher plants, small subunits are highly conserved among plant species and expressed in all tissues. And the large subunits are present at multiple isoforms and expressed in a tissue-specific pattern. In fruits, the expression pattern of the large subunits varies with plant species. That made it important to study the transcriptional regulation of the large subunits of AGPase in different plant species. Northern-blot analysis indicates in watermelon, an isoform of the large subunits Wml1 expressed specifically in fruits, not in leaves. The 5' flanking region of Wml1, which covers 1573bp, has been isolated through the method of uneven PCR. And transient expression assay has shown that the 1573bp (named WSP) can direct fruit-specific expression of GUS gene. Our goal in this study was to scan the promoter region for main regulatory regions involved in fruit-specific expression. A chimaeric gene was constructed containing the WSP promoter, the beta-glucuronidase (GUS) structural sequence as a reporter gene and the nopaline synthase polyadenylation site (NOS-ter). The plasmid pSPA was digested with Hind III + Hinc II and promoter fragment of 1573bp (from 180bp to 1752bp) was cut out and cloned into Sma I sites of pBluescript SK(-), to produce pBSPA-16. The same insert was then cut out with Hind III + BamH I, and ligated with transient expression vector pBI426 digested by HindIII + Bgl II to produce pISPA-16. Three 5'-end deletions of the promoter were obtained and fused to GUS gene in plant transient expression vector pBI426: the 1201bp fragment (from 551bp to 1752bp) was generated by digestion of pBSPA-16 with BamH I + SnaB I, the 898bp fragment (from 854bp to 1752bp) by BamH I + EcoRV. Both fragments were ligated with pBluescript SK(-) digested by BamH I + Sma I, to produce pBSPA-12 and pBS-PA-9. The inserts were cut out with HindmIII + BamH I and ligated with pBI426 digested by Hind III + Bgl II, to produce pISPA-12 and pISPA-9. The 795bp fragment (from 957bp to 1752bp) was generated by digestion of pSPA with Hinc II + EcoR I, promoter fragment was cut out and cloned into Sma I sites of pBluescript SK(-), to produce pBSPA-8. The same insert were cut out with Hind III + BamH I, and ligated with transient expression vector pBI426 digested by Hind III + Bgl II. The 1573bp fragment and three 5'-end deletions were delivered into watermelon leaf, stem, flower and fruit of different development stages (5, 10, 20 days after pollination) via particle bombardment using a biolistic PDS-1000/He particle gun. Bombardment parameters were as follows: a helium pressure of 1200 psi, vacuum of 91432.23Pa, 7 cm between the stopping screen and the plate. Histochemical assay were done on all the tissues bombarded after incubation for 2 days. The 1573bp fragment had the strongest promoter activity, and can induce GUS expression in fruits of 5 and 20 days after anthesis and flowers, but not in fruits of 10 days after anthesis, leaves and stems. Fragments of 1201bp and 898bp can induce GUS expression only in fruits of 20 days after anthesis, and with lower expression levels than 1573bp. Fragment of 795bp was not able to direct GUS expression in any of the tissues bombarded (data not shown). It can be concluded that of the 1573bp, 1201 bp, 898bp Wml1 5'flanking regions include the necessary information directing fruit-specific expression. Deletion from 180bp to 551bp doesn't affect the fruit-specificity of the promoter, but lowered the expression level. There may be some cis-acting elements located in this region, which can enhance external gene expression in later stages of fruit development. Deletion from 854bp and 958bp led to loss of GUS expression. This region includes the necessary information needed for gene expression as well as the regulatory elements for fruit-specific transcription.

[Transformation of Bt-CpTi fusion protein gene to cabbage (Brassica oleracea var. capitata) mediated by Agrobacterium tumefaciens and particle bombardment].

Bt-CpTI fusion protein gene was transferred to the explants of hypocotyl, cotyledon with petiole and shoot apex of cabbage (Brassica oleracea L. var. capitata) variety "Zhonggan No 8" via Agrobacterium tumefaciens and particle bombardment, and 13 kanamycin-resistant plants were obtained. PCR and Southern blotting hybridization verified that all these plants of the kanr plants of type I regenerated from hypocotyl and cotyledon with petiole mediated by A. tumefaciens were transgenic plants, 2 karr plants of type II stemed from shoot apex mediated by A. tumefaciens were "false-positive"plants and one of 2 kanr plants of type III regenerated from shoot apex via particle bombardment was non-transformed plants. It was showed that part of transgenic plants had high activity of trypsin inhibitor and strong resistance to resist common cabbage worm through the analysis of CpTI relative capacity and insect-resistant test.