Research progress of plant cytogenetics in Jiangsu province.

Cytogenetics was established based on the "Chromosome theory of inheritance", proposed by Boveri and Sutton and evidenced by Morgan's lab in early stage of the 20 th centrary. With rapid development of related research areas, especially molecular genetics, cytogenetics developed from traditional into a new era, molecular cytogenetics in late 1960s. Featured by an established technique named DNA in situ hybridization (ISH), molecular cytogenetics has been applied in various research areas. ISH provids vivid and straightforward figures showing the virtual presence of DNA, RNA or proteins. In combination with genomics and cell biology tools, ISH and derived techniques have been widely used in studies of the origin, evolution, domestication of human, animal and plant, as well as wide hybridization and chromosome engineering. The physical location and order of DNA sequences revealed by ISH enables the detection of chromosomal re-arrangments among related species and gaps of assembled genome sequences. In addition, ISH using RNA or protein probes can reveal the location and quantification of transcripted RNA or translated protein. Since the 1970s, scientists from universities or institutes belonging to the Jiangsu Society of Genetics have initiated cytogenetics researches using various plant species. In recent years, research platforms for molecular cytogenetics have also been well established in Nanjing Agricultural University, Yangzhou University, Nanjing Forestry University, Jiangsu Xuhuai Academy of Agricultural Sciences, and Jiangsu Normal University. The application of molecular cytogenetics in plant evolution, wide hybridization, chromosome engineering, chromosome biology, genomics has been successful. Significant progresses have been achieved, both in basic and applied researches. In this paper, we will review main research progresses of plant cytogenetics in Jiangsu province, and discuss the potential development of this research area.

[Application of QAMS for quality evaluation and control of Chinese patent medicines:taking Bufonis Venenum-contained preparations as examples].

As a new strategy capable of uncovering the characteristics of traditional Chinese medicines, the quantitative analysis of multi-components by single-marker(QAMS) has been widely employed for the quality evaluation of Chinese medicinal materials, slices, and extracts. However, its application in the assessment of Chinese patent medicines is yet to be explored. By referring to the determination of three bufogenins in Bufonis Venenum by QAMS described in Chinese Pharmacopoeia(2020 Edition), this paper selected seven representative preparations containing Bufonis Venenum and explored whether the relative correction factors(RCFs) of cinobufagin(CB) to bufalin(BF) and resibufogenin(RB) could be directly used for the quality control of Bufonis Venenum-contained preparations. Based on the qualitative analyses under the same chromatographic conditions as used for toad venom, combing specificity test, five preparations such as Yatong Yili Pills, Houzheng Pills, Xiongdan Jiuxin Pills, Liushen Pills and Niuhuang Xiaoyan Pills, were expected to use validated RCFs for the direct determination of three components. Taking Houzheng Pills as an example, the methodological validation of bufalin, cinobufagin and resibufogenin was carried out, and the recoveries of bufalin, cinobufagin and resibufogenin were 90.64%-106.1%. The obvious difference was not observed between the contents of bufalin and resibufogenin in 24 batches of preparation samples by QAMS and external reference method. In the tested samples, the content of bufalin, cinobufagin and resibufogenin were 1.27-2.61, 2.44-5.66 and 0.988-3.16 mg·g~(-1) in 10 batches of Liushen Pills samples. The contents of bufalin, cinobufagin and resibufogenin were 0.760-1.32, 1.35-2.39 and 0.600-1.55 mg·g~(-1) in 10 batches of Houzheng Pills samples from three manufacturers. The obtained data contribute to improving the quality standard of Bufonis Venenum-contained preparations, and they also provide some ideas for the application of QAMS in the quality evaluation and control of Chinese patent medicines.

[Study on quality control method of toad venom based on characteristic chromatogram and QAMS].

Toad venom (Chansu) is prepared from the dried secretion of parotid gland and skin gland from Bufo bufo gargarizans or B. melanostictus. Up to now, much attention shall be paid to the poor quality of commercial toad venom because of the adulteration. So, it is urgent to establish a scientific and perfect quality control method to improve the quality of toad venom and guarantee its safety and effectiveness in clinical application. The different batches of toad venom samples were assayed by high performance liquid chromatography (HPLC) and the quantitative analysis of multi-components by single marker (QAMS) was used to detect the contents of five bufagenins. As a result, the reference characteristic chromatogram was established, displaying serotonin, gamabufotalin, arenobufagin, hellebrigenin, telocinobufagin, bufotalin, cinobufotalin, bufalin, cinobufagin and resibufogenin as characteristic peaks. Taking cinobufagin as an internal reference substance, QAMS was verified for the determination of five bufagenins (gamabufotalin, bufotalin, bufalin, cinobufagin, resibufogenin) in toad venom samples. The durability and applicability of the relative correction factor (RCF) were also studied systematically. RCFs of cinobufagin to gamabufotalin, bufotalin, bufalin and resibufogenin were determined as 1.05, 0.895, 1.09 and 0.913, respectively. The characteristic chromatogram and QAMS established in this study could effectively control the quality of toad venom and provide scientific evidence for the improvement of the quality standard of the toad venom to be described in Chinese Pharmacopoeia (2020 edition).

[Advance on quality control of toad venom and its key influence factors].

Toad venom (Chansu) is prepared from the dried secretion of Bufo bufo gargarizans or B. melanostictus. It is not only one of the famous and expensive traditional Chinese medicines(TCMs) from animal origin, but also one of 28 kinds of toxic TCMs to be required for special management issued by the State Council of the People's Republic of China. Chansu contains the rich bufadienolides and indole alkaloids, and displays various bioactivity including cardiotonic, anti-tumor, analgesia, and local anesthesia. Based on the published references in the recent years, the advance on the identification of adulterants and quality evaluation as well as the influence factors on the quality of toad venom was summarized to improve the quality standards and promote the level of quality control of toad venom and its preparations.

Cytogenetic relationships among Citrullus species in comparison with some genera of the tribe Benincaseae (Cucurbitaceae) as inferred from rDNA distribution patterns.

BACKGROUND: Comparative mapping of 5S and 45S rDNA by fluorescent in situ hybridization (FISH) technique is an excellent tool to determine cytogenetic relationships among closely related species. RESULTS: In this study, the number and position of 5S and 45S rDNA loci in all Citrullus species and subspecies were determined. The cultivated watermelon (C. lanatus subsp. vulgaris), C. lanatus subsp. mucosospermus, C. colocynthis and C. naudinianus (or Acanthosicyos naudinianus) had two 45S rDNA loci and one 5S rDNA locus which was located syntenic to one of the 45S rDNA loci. C. ecirrhosus and C. lanatus subsp. lanatus had one 45S rDNA locus and two 5S rDNA loci, each located on a different chromosome. C. rehmii had one 5S and one 45S rDNA locus positioned on different chromosomes. The distribution of 5S and 45S rDNA in several species belonging to other genera in Benincaseae tribe was also investigated. The distribution pattern of rDNAs showed a great difference among these species. CONCLUSIONS: The present study confirmed evolutionary closeness among cultivated watermelon (C. lanatus subsp. vulgaris), C. lanatus subsp. mucosospermus and C. colocynthis. Our result also supported that C. lanatus subsp. lanatus was not a wild form of the cultivated watermelon instead was a separate crop species. In addition, present cytogenetic analysis suggested that A. naudinianus was more closely related to Cucumis than to Citrullus or Acanthosicyos, but with a unique position and may be a link bridge between the Citrullus and the Cucumis.

Bufadienolides and polyhydroxycholestane derivatives from Bufo bufo gargarizans.

A new polyhydroxycholestane sulfate ester, 3α,12ß,25,26-tetrahydroxy-7-oxo-5ß-cholestane 26-O-sulfate (1), was isolated from dried skin of Bufo bufo gargarizans Cantor and its structure was elucidated on the basis of extensive 1D and 2D NMR as well as HR-ESI-MS analysis. A comparison of steroidal metabolite profiles, based on HPLC and LC-MS analyses, indicates that the chemical compositions of the various parts of toads, such as venom, skin and stratum corneum, are significantly different.

The novel antitumor compound clinopodiside A induces cytotoxicity via autophagy mediated by the signaling of BLK and RasGRP2 in T24 bladder cancer cells.

In the study, we investigated the anti-cancer effect of clinopodiside A and the underlying mechanisms using T24 bladder cancer cells as an experimental model. We found that the compound inhibited the growth of the bladder cancer cells in vitro and in vivo in a in a concentration- and dose-dependent manner, respectively, which showed a combinational effect when used together with cisplatin. In the bladder cancer cells, clinopodiside A caused autophagy, which was mediated by the signaling of BLK and RasGRP2, independently. Inhibition of the autophagy by chemical inhibitor 3-methyladenine or by the inhibition of the signaling molecules attenuated the cytotoxicity of clinopodiside A. Further analyses showed that clinopodiside A acted in synergism with cisplatin which itself could trigger both autophagy and apoptosis, which occurred with concomitant enhancements in autophagy and the cisplatin-evoked apoptosis. In conclusion, our results suggest that clinopodiside A inhibits the growth of the bladder cancer cells via BLK- and RasGRP2-mediated autophagy. The synergistic effect between clinopodiside A and cisplatin is attributed to the increases in autophagy and autophagy-promoted apoptosis. Clinopodiside A is a promising investigational drug for the treatment of cancer, at least blabber, which can be used alone or in combination with clinical drug(s).

Long-distance blue light signalling regulates phosphate deficiency-induced primary root growth inhibition.

Although roots are mainly embedded in the soil, recent studies revealed that light regulates mineral nutrient uptake by roots. However, it remains unclear whether the change in root system architecture in response to different rhizosphere nutrient statuses involves light signaling. Here, we report that blue light regulates primary root growth inhibition under phosphate-deficient conditions through the cryptochromes and their downstream signaling factors. We showed that the inhibition of root elongation by low phosphate requires blue light signal perception at the shoot and transduction to the root. In this process, SPA1 and COP1 play a negative role while HY5 plays a positive role. Further experiments revealed that HY5 is able to migrate from the shoot to root and that the shoot-derived HY5 autoactivates root HY5 and regulates primary root growth by directly activating the expression of LPR1, a suppressor of root growth under phosphate starvation. Taken together, our study reveals a regulatory mechanism by which blue light signaling regulates phosphate deficiency-induced primary root growth inhibition, providing new insights into the crosstalk between light and nutrient signaling.

Cerium caused life span shortening and oxidative stress resistance in Drosophila melanogaster.

To investigate the effects of the rare earth element cerium (Ce) on the life span and biomarkers of oxidative stress in the fruit fly (Drosophila melanogaster). Fruit flies were fed on media with different dose of ceric sulfate (1, 4, 16, 64, 256, 1024mg/L, corresponding to cerium concentrations of 0.45, 1.65, 6.91, 26.3, 104, and 429microg/g culture medium). Mean life span, maximum life span, and fertility were calculated. There was a significant decrease in mean life span and maximum life span with increasing doses of cerium. At some concentrations, there was a decrease in reproductive output, especially concentrations >6.91microg/g. We also measured superoxide dismutase (SOD) activity, catalase (CAT) activity, and lipid peroxidation product levels (malondialdehyde (MDA) content). Cerium caused a significant increase in MDA content and decrease in SOD and CAT activities at concentrations >6.91microg/g. These results suggest that cerium may result in oxidative toxicity to D. melanogaster.

[FISH analysis of chromosomes of sweet potato(Ipomoea batatas cv.Xushu No.18)].

In order to understand the chromosome structure of sweet potato (Ipomoea batatas cv. Xushu 18), molecular cytogenetic analyses were carried out on I. batatas. by using 45S rDNA fluorescence in situ hybridization (45S rDNA-FISH), self genomic in situ hybridization (self-GISH), and silver staining techniques. Twelve, sixteen, and eighteen regions were silver stained in the interphase nucleus of I. batatas. The results of FISH analysis demonstrated 16 or 18 signals with different intensity on chromosomes of I. batatas. Self-GISH analysis showed that the intensive signals on I. batatas mitotic chromosomes were distributed along the chromosomes. However, the signals located in centromeric, subcentromeric, and telomeric regions were stronger and denser than those in other regions.

A novel glutathione S-transferase gene from sweetpotato, IbGSTF4, is involved in anthocyanin sequestration.

Anthocyanins are synthesized by multi-enzyme complexes localized at the cytoplasmic surface of the endoplasmic reticulum (synthesis site), and transported to the destination site, the vacuole. Three mechanisms for the vacuolar accumulation of anthocyanin in plant species have been proposed. Previous studies have indicated that glutathione S-transferase (GST) genes from model and ornamental plants are involved in anthocyanin transportation. In the present study, an anthocyanin-related GST, IbGSTF4, was identified and characterized based on transcriptome results. Phylogenetic analysis revealed that IbGSTF4 was most closely correlated to PhAN9 and CkmGST3, the anthocyanin-related GST of Petunia hybrida and Cyclamen. Furthermore, the expression analysis revealed that IbGSTF4 is strongly expressed in pigmented tissues, when compared to green organs, which is in agreement to the ability to correlate with anthocyanin accumulation. A GST activity assay uncovered that the IbGST4 protein owned similar activities with the GST family. Furthermore, the molecular functional complementation of Arabidopsis thaliana mutant tt19 demonstrated that IbGSTF4 might play a vital role in the vacuole sequestration of anthocyanin in sweetpotato. Moreover, the dual luciferase assay revealed that the LUC driven by the promoter of IbGSTF4 could not be directly activated by IbMYB1, suggesting that the regulatory mechanism of anthocyanin accumulation and sequestration in sweetpotato was intricate.

Sucrose Signaling Regulates Anthocyanin Biosynthesis Through a MAPK Cascade in Arabidopsis thaliana.

Anthocyanin accumulation specifically depends on sucrose (Suc) signaling. However, the molecular basis of this process remains unknown. In this study, in vitro pull-down assays identified ETHYLENE-INSENSITIVE3 (EIN3), a component of both sugar signaling or/and metabolism. This protein interacted with YDA, and the physiological relevance of this interaction was confirmed by in planta co-immunoprecipitation, yeast two-hybrid (Y2H) assay, and bimolecular fluorescence complementation. Ethylene insensitive3-like 1 (eil1) ein3 double-mutant seedlings, but not ein3-1 seedlings, showed anthocyanin accumulation. Furthermore, ein3-1 suppressed anthocyanin accumulation in yda-1 plants. Thus, EMB71/YDA-EIN3-EIL1 may form a sugar-mediated gene cascade integral to the regulation of anthocyanin accumulation. Moreover, the EMB71/YDA-EIN3-EIL1 gene cascade module directly targeted the promoter of Transparent Testa 8 (TT8) by direct EIN3 binding. Collectively, our data inferred a molecular model where the signaling cascade of the YDA-EIN3-TT8 appeared to target TT8 via EIN3, thereby modulating Suc signaling-mediated anthocyanin accumulation.

Antifungal and eliciting properties of chitosan against Ceratocystis fimbriata in sweet potato.

The inhibitive effects of chitosan on black rot disease caused by Ceratocystis fimbriata in sweet potato tuber root (TR) were evaluated. The results demonstrated that chitosan effectively inhibited the mycelial growth and spore germination of C. fimbriata and directly led to the cell necrosis. Chitosan altered the chitin deposition and influenced the fatty acid composition of C. fimbriata. The application of chitosan effectively controlled the C. fimbriata development in sweet potato TRs 17 days of storage 25 °C. Phenylalanine ammonia lyase (PAL) and superoxide dismutase (SOD) activity were clearly enhanced by the chitosan treatment, while the malondialdehyde (MDA) production was not increased. These findings suggest that chitosan effectively controlled the infection of C. fimbriata in sweet potato TRs owing to its antifungal and eliciting properties, which induced some defense responses during storage.

Visualization of the ribosomal DNA (45S rDNA) of Indica rice with FISH on some phases of cell cycle and extended DNA fibers.

The ribosomal DNA (45S rDNA) behaviors during the cell cycle were analyzed on interphase nuclei, prophases, metaphases, pachytene chromosomes and extended DNA fibers in rice (Oryza, sativa ssp. indica cv. Guangluai No.4) by using high-resolution fluorescent in situ hybridization (FISH). The results show that 45S rDNA is located at the ends of short arms of chromosomes 9 and 10. But the signals are much more intense on chromosome 9 than on chromosome 10 in metaphase. Pachytene chromosome has rDNA signal arrays on chromosome 9. Different phases are described and discussed. These results indicate that the activity ofrDNA at individual loci may also vary through the cell cycle in rice. On extended DNA fibers, 45S rDNA signals appear as strings of numerous red spots, but some signals are missed in some regions, probably result from weak signals or intergenic spacers.

[The progress and research of chromosome territories (CTs)].

Chromosomes are non-randomly distributed in interphase nucleus, and each chromosome occupies a distinct domain in the chromosome territories (CTs), the positioning and arrangement of CTs are well-organized, at the same time, the chromosome territories have definite dynamics characteristics, the strict positioning and spatial organization of chromosome territories are closely related with gene expression regulation. This paper reviewed progress in these respects.

FISH analysis of pachytene chromosome and DNA fiber of telomere sequence in rice (Oryza sativa L. indica).

Telomere sequences were analyzed by using pachytene chromosome and extended DNA fiber FISH in rice (Oryza, sativa ssp. indica cv. -Guangluai No.4). Pachytene FISH results showed that most of chromosome ends possess the telomere tandem repeats, but the signals on different chromosomes were not the same in intensity. Fiber FISH results indicated that the longest string of beads was 6.55 microm, while the shortest one was 1.82 microm long,which were equal to 16.44 and 4.56 kb correspondingly based on a stretching factor of 2.51 kb/microm. The average value of signal length was 3.62 +/- 1.32 microm, i.e. 9.09 +/- 3.31 kb. It could be estimated that the longest and the shortest as well as the average value were equal to 2,349,651 and 1298 +/- 473 of copy number respectively.

Fluorescence in situ hybridization analysis of alien genes in Agrobacterium-mediated Cry1A(b)-transformed rice.

The transgene in Agrobacterium-mediated Cry1A(b)-transgenic rice plants has been detected and its chromosomal location determined by fluorescence in situ hybridization (FISH). Eight of the nine transgenic lines tested showed hybridization signals. Signals were located on regions of the chromosome in which fraction length (FL) values varied from 26.2 (near the centromere) to 95.2 (distal regions). No signal was found on regions where the fraction length was less than 26.2, while six of the nine signals detected were located on regions with FL values of 75.3 or over. This demonstrates that Agrobacterium-mediated genes can integrate into multiple sites distributed in different parts of the chromosome, but that distal regions are the preferred sites and regions near the centromeres are colder for T-DNA integration. The donor DNA of the transformation was divided into two parts, labelled separately as probes for two-colour FISH. Results show that the transformed DNA sequences remained linked in the recipient genome. The relationship between integration position and transgene silencing, known as the 'position effect', is discussed.