Fine mapping of TFL, a major gene regulating fruit length in snake gourd (Trichosanthes anguina L).

Fruit length is a crucial agronomic trait of snake gourd (Trichosanthes anguina L); however, genes associated with fruit length have not been characterised. In this study, F2 snake gourd populations were generated by crossing the inbred lines, S1 and S2 (fruit lengths: 110 and 20 cm, respectively). Subsequently, bulk segregant analysis, sequencing, and fine-mapping were performed on the F2 population to identify target genes. Our findings suggest that the fruit length of snake gourd is regulated by a major-effect regulatory gene. Mining of genes regulating fruit length in snake gourd to provide a basis for subsequent selection and breeding of new varieties. Genotype-phenotype association analysis was performed on the segregating F2 population comprising 6,000 plants; the results indicate that the target gene is located on Chr4 (61,846,126-61,865,087 bp, 18.9-kb interval), which only carries the annotated candidate gene, Tan0010544 (designated TFL). TFL belongs to the MADS-box family, one of the largest transcription factor families. Sequence analysis revealed a non-synonymous mutation of base C to G at position 202 in the coding sequence of TFL, resulting in the substitution of amino acid Gln to Glu at position 68 in the protein sequence. Subsequently, an InDel marker was developed to aid the marker-assisted selection of TFL. The TFL in the expression parents within the same period was analysed using quantitative real-time PCR; the TFL expression was significantly higher in short fruits than long fruits. Therefore, TFL can be a candidate gene for determining the fruit length in snake gourd. Collectively, these findings improve our understanding of the genetic components associated with fruit length in snake gourds, which could aid the development of enhanced breeding strategies for plant species.

Molecular identification and development of an infectious cDNA clone of Trichosanthes kirilowii-infecting cucurbit mild mosaic virus.

Trichosanthes kirilowii has been mainly grown for use in traditional Chinese medicine. In this study, cucurbit mild mosaic virus (CuMMV) belonging to the genus Fabavirus was identified from T. kirilowii plants. CuMMV possesses a segmented, bipartite linear single-stranded RNA genome composed of RNA1 and RNA2. Sequence analysis showed that each genomic segment shares the highest sequence similarity with those of CuMMV isolated from pumpkin. A full-length infectious cDNA clone of CuMMV was further constructed and was found to induce typical symptoms in T. kirilowii, Cucumis sativus, C. melo, Citrullus lanatus, and Cucurbita pepo. The sap inoculum derived from the infectious cDNA clone of CuMMV could be mechanically transmitted and reproduce similar symptoms in the tested plants. This is the first report on the construction of a biologically active, full-length infectious cDNA clone of CuMMV, which will provide a useful tool in understanding CuMMV-encoded proteins and plant-CuMMV interactions.

Identification and analysis of miRNAs differentially expressed in male and female Trichosanthes kirilowii maxim.

Trichosanthes kirilowii Maxim. (TK) is a dioecious plant in the Cucurbitaceae family of which different sexes have separate medicinal uses. We used Illumina high-throughput sequencing technology to sequence miRNAs from male and female flower buds of TK. We performed bioinformatics analysis, miRNA identification, and target gene prediction on the data obtained from sequencing, and association analysis was performed in combination with the results of a previous transcriptome sequencing study. As a result, there were 80 differentially expressed miRNAs (DESs) between the female and male plants (48 upregulated and 32 downregulated in female plants). Moreover, 27 novel miRNAs in DESs were predicted to have 282 target genes, and 51 known miRNAs were predicted to have 3418 target genes. By establishing a regulatory network between miRNAs and target genes, 12 core genes were screened, including 7 miRNAs and 5 target genes. Among them, tkmiR157a-5p, tkmiR156c, tkmiR156_2, and tkmiR156k_2 jointly target the regulation of tkSPL18 and tkSPL13B. These two target genes are specifically expressed in male and female plants, respectively, and are involved in the biosynthesis process of BR, which is closely related to the sex differentiation process of TK. The identification of these miRNAs will provide a reference for the analysis of the sex differentiation mechanism of TK.

Molecular cloning and characterization of an alpha-amylase inhibitor (TkAAI) gene from Trichosanthes kirilowii Maxim.

Trichosanthes kirilowii Maxim taxonomically belongs to the Cucurbitaceae family and Trichosanthes genus. Its whole fruit, fruit peel, seed and root are widely used in traditional Chinese medicines. A ribosome-inactivating protein with RNA N-glycosidase activity called Trichosanthrip was isolated and purified from the seeds of T. kirilowii in our recent previous research. To further explore the biological functions of Trichosanthrip, the cDNA of T. kirilowii alpha-amylase inhibitor (TkAAI) was cloned through rapid-amplification of cDNA ends and its sequence was analyzed. Also, the heterologous protein was expressed in Escherichia coli and its alpha-amylase activity was further measured under optimized conditions. The full-length cDNA of TkAAI was 613 bp. The speculated open reading frame sequence encoded 141 amino acids with a molecular weight of 16.14 kDa. Phylogenetic analysis demonstrated that the Alpha-Amylase Inhibitors Seed Storage domain sequence of TkAAI revealed significant evolutionary homology with the 2S albumin derived from the other plants in the Cucurbitaceae group. In addition, TkAAI was assembled into pET28a with eGFP to generate a prokaryotic expression vector and was induced to express in E. coli. The TkAAI-eGFP infusion protein was proven to exhibit alpha-amylase inhibitory activity against porcine pancreatic amylase in a suitable reaction system. Analysis of gene expression patterns proved that the relative expression level of TkAAI in seeds is highest. The results presented here forecasted that the TkAAI might play a crucial role during the development of T. kirilowii seeds and provided fundamental insights into the possibility of T. kirilowii derived medicine to treat diabetes related diseases.

De novo transcriptome analysis and identification of candidate genes associated with triterpenoid biosynthesis in Trichosanthes cucumerina L.

KEY MESSAGE: De novo transcriptome analysis from callus, leaf, and fruit of Trichosanthes cucumerina L. for the identification of genes associated with triterpenoid biosynthesis, especially bryonolic acid and cucurbitacin B. Trichosanthes cucumerina L. (TC) has been used as a medicinal plant in Thailand with various potential functions. Two major triterpenoids found in this plant, bryonolic acid and cucurbitacin B, are receiving increased attention for their activities. Here, we provide TC transcriptome data to identify genes involved in the triterpenoid biosynthetic pathway through callus, where was previously suggested as a novel source for bryonolic acid production as opposed to leaf and fruit. A de novo assembly of approximately 290-thousand transcripts generated from these tissues led to two putative oxidosqualene cyclases: isomultiflorenol synthase (IMS) and cucurbitadienol synthase (CBS). TcIMS and TcCBS, genes that encode substrates for two characteristic triterpenoids in cucurbitaceous plants, were identified as isomultiflorenol synthase and cucurbitadienol synthase, respectively. These two genes were functionally characterised in mutant yeast Gil77 systems, which led to the productions of isomultiflorenol and cucurbitadienol. Moreover, the callus-specific gene expression profiles were also presented. These obtained information showed candidate cytochrome P450s with predicted full-length sequences, which were most likely associated with triterpenoid biosynthesis, especially bryonolic acid. Our study provides useful information and a valuable reference for the further studies on cucurbitaceous triterpenoids.

Transcriptome sequencing and screening of genes related to sex determination of Trichosanthes kirilowii Maxim.

Trichosanthes kirilowii Maxim. (TK) is a dioecious plant in the Cucurbitaceae for which different sexes have separate medicinal uses. In order to study the genes related to sex determination, transcriptome sequencing was performed on flower buds of male and female plants using the high-throughput sequencing technology. A total of 145,975 unigenes and 7110 DEGs were obtained. There were 6776 DEGs annotated to 1234 GO terms and enriched to 18 functional groups, including five biological processes related to sugar metabolism. KEGG pathway analysis indicated genes involved in hormone transduction, hormone synthesis and carbohydrate metabolism. Many DEGs of TK are involved in reproductive organ formation, hormone signal transduction and regulatory networks. Combining the results of GO, KEGG and qRT-PCR, 11 sex determining candidate genes of TK were selected, including MYB80, MYB108, CER1, CBL9, ABCB19, SERK1, HSP81-3, ACS9, SEP3, AUX1 and YUC6. The results provide a foundation for the study of sex differentiation in TK.

Cloning of a novel trypsin inhibitor from the Traditional Chinese medicine decoction pieces, Radix Trichosanthis.

Most herbs of traditional Chinese medicine (TCM) are used as air-dried decoction pieces that are manufactured and kept at ambient temperature for long periods. Given the ability of some desiccation-tolerant plants to conserve RNA, it could be worthwhile to isolate mRNA from TCM decoction pieces as part of a transcriptomic strategy to identify new substances with potential pharmaceutical application. Here, we report the molecular cloning of a novel trypsin inhibitor (as the probable alleleic variants TKTI-2 and TKTI-3) from the decoction piece of Radix Trichosanthis, representing the dried root of Trichosanthes kirilowii. From this material, the total RNA was extracted and a cDNA library was constructed from the isolated mRNA from which the cDNAs of two precursors were successfully cloned and sequenced. TKTI-3 showed an amino-acid substitution in the otherwise highly-conserved P1-P1' reaction site of the mature peptide, which we confirmed to not be an artefact. Subsequent analysis using LC-MS/MS detected the presence of specific tryptic peptides expected from TKTI-3, confirming the presence and expression of this locus in Radix Trichosanthis. More generally, this study indicates that mRNA can persist in decoction pieces and so could present a viable option for the molecular cloning from other TCMs.

[Genetic Diversity and Genetic Relationship of Different Forms of Trichosanthes kirilowii from Shandong Province].

OBJECTIVE: To investigate the genetic diversity and genetic relationship of different forms of Trichosanthes kirilowii from Shandong Province. METHODS: Different forms of Trichosanthes kirilowii from the same planting base were detected by random amplified polymorphic DNA( RAPD) with eight random primers. The amplified bands were detected and the fingerprint was drawn by Biosens Gel Imaging System software. The Jaccard coefficient was worked out by using NTSYS-pc software, and a cluster dendrogram of different samples was established based on unweighted pair-group method with arithmetic means (UPGMA). RESULTS: The eight primers produced 56 bands, among which, 32 bands were polymorphic. The cluster dendrogram displayed there was some differences of genetic relationship of different samples. CONCLUSION: There is similar genetic material basis of Trichosanthes kirilowii from the same planting base, but also there is apparent individual differences.

Identification and validation of a new male sex-specific ISSR marker in pointed gourd (Trichosanthes dioica Roxb.).

The aim of the present study was to develop a genetic sex marker for the pointed gourd (Trichosanthes dioica Roxb.) to allow gender determination at any stage in the life cycle. Screening of genomic DNA with intersimple sequence repeat (ISSR) primers was used to discover sex-specific touch-down polymerase chain reaction (Td-PCR) amplification products. Using pooled DNA from male and female genotypes and 42 ISSR primers, a putative male specific marker (~550 bp) was identified. DNA marker specific to male is an indication of existence of nonepigenetic factors involved in gender development in pointed gourd. The ISSR technique has proved to be a reliable technique in gender determination of pointed gourd genotypes at the seedling phenophase. The sex marker developed here could also be used as a starting material towards sequence characterization of sex linked genes for better understanding the developmental as well as evolutionary pathways in sexual dimorphism.

A preliminary report on the genetic variation in pointed gourd (Trichosanthes dioica Roxb.) as assessed by random amplified polymorphic DNA.

Pointed gourd (Trichosanthes dioica Roxb.) is an economically important cucurbit and is extensively propagated through vegetative means, viz vine and root cuttings. As the accessions are poorly characterized it is important at the beginning of a breeding programme to discriminate among available genotypes to establish the level of genetic diversity. The genetic diversity of 10 pointed gourd races, referred to as accessions was evaluated. DNA profiling was generated using 10 sequence independent RAPD markers. A total of 58 scorable loci were observed out of which 18 (31.03%) loci were considered polymorphic. Genetic diversity parameters [average and effective number of alleles, Shannon's index, percent polymorphism, Nei's gene diversity, polymorphic information content (PIC)] for RAPD along with UPGMA clustering based on Jaccard's coefficient were estimated. The UPGMA dendogram constructed based on RAPD analysis in 10 pointed gourd accessions were found to be grouped in a single cluster and may represent members of one heterotic group. RAPD analysis showed promise as an effective tool in estimating genetic polymorphism in different accessions of pointed gourd.

Evolution and loss of long-fringed petals: a case study using a dated phylogeny of the snake gourds, Trichosanthes (Cucurbitaceae).

BACKGROUND: The Cucurbitaceae genus Trichosanthes comprises 90-100 species that occur from India to Japan and southeast to Australia and Fiji. Most species have large white or pale yellow petals with conspicuously fringed margins, the fringes sometimes several cm long. Pollination is usually by hawkmoths. Previous molecular data for a small number of species suggested that a monophyletic Trichosanthes might include the Asian genera Gymnopetalum (four species, lacking long petal fringes) and Hodgsonia (two species with petals fringed). Here we test these groups' relationships using a species sampling of c. 60% and 4759 nucleotides of nuclear and plastid DNA. To infer the time and direction of the geographic expansion of the Trichosanthes clade we employ molecular clock dating and statistical biogeographic reconstruction, and we also address the gain or loss of petal fringes. RESULTS: Trichosanthes is monophyletic as long as it includes Gymnopetalum, which itself is polyphyletic. The closest relative of Trichosanthes appears to be the sponge gourds, Luffa, while Hodgsonia is more distantly related. Of six morphology-based sections in Trichosanthes with more than one species, three are supported by the molecular results; two new sections appear warranted. Molecular dating and biogeographic analyses suggest an Oligocene origin of Trichosanthes in Eurasia or East Asia, followed by diversification and spread throughout the Malesian biogeographic region and into the Australian continent. CONCLUSIONS: Long-fringed corollas evolved independently in Hodgsonia and Trichosanthes, followed by two losses in the latter coincident with shifts to other pollinators but not with long-distance dispersal events. Together with the Caribbean Linnaeosicyos, the Madagascan Ampelosicyos and the tropical African Telfairia, these cucurbit lineages represent an ideal system for more detailed studies of the evolution and function of petal fringes in plant-pollinator mutualisms.

[Construction and expression of trichosanthin mutant gene TCS(RL28-29CG) and purification of expressed product].

AIM: To construct and express a trichosanthin(TCS)gene mutant and purify the expressed product. METHODS: Predict the potential antigenic determinant on TCS molecule by computer modeling and induce site-directed mutation. Amplify gene mutant TCS(RL28-29CG); by PCR using the genomic DNA of Trichosanthes kirilowii as a template and insert into expression vector pRSET-A, then transform to E.coli BL21(DE3)for expression under induction of IPTG. Purify the expressed product by Ni-NTA afinity column chromatography. RESULTS: The target protein in a soluble form was successfully expressed in E.coli. Homogenous TCS mutant protein was obtained after purification of expressed product. CONCLUSION: TCS mutant gene TCS(RL28-29CG); is succ-essfully constructed and expressed.

Triton, a novel family of miniature inverted-repeat transposable elements (MITEs) in Trichosanthes kirilowii Maximowicz and its effect on gene regulation.

Miniature inverted-repeat transposable elements (MITEs) have a broad impact on genome structure and function. Although MITEs are found associated to genes, little is known about their effect on gene regulation. We have identified a novel MITE family, named Triton, whilst analyzing two independent trichosanthin (TCS) gene promoters (TP9 and TP12) cloned from Trichosanthes kirilowii Maximowicz. Triton1 and Triton2 are nested in TP9, and Triton3 (with 93% sequence similarity to Triton2) is in TP12. To assess the effect of MITE insertion on TCS promoters, we excised Triton1 from TP9 and inserted it into TP12. GUS activity analysis revealed that nested Triton1 is required for effective repression of promoter activity. Detailed analyses of a series of 5'-truncated promoters concerning Triton1 showed that a dark-specific repressor and some constitutive elements endow Triton1 with ability to response to light conditions. These results suggest that Triton1 MITE, which contains cis-regulatory elements, could mediate gene expression.

[A new molecular identification method: anchored primer amplification polymorphism DNA].

To build up a stable and easy doing method for molecular identification in traditional Chinese medicine, on basis of RAPD, the new method mainly changed the primer length and PCR annealing temperature. Panax ginseng, Panax quinquefolius and its nine adulterants were used to establish the method and test it using MARMS primers published in 2004. The new method also used to authenticate Chinese Materia Medica of Tian-hua-fen (Radix Trichosanthes) and Bai-zhi (Radix Angelica). Primer Pg-q36F obtained polymorphic bands of P. Ginseng, P. quinquefolius and its adulterants. The identification result is identical to that published before and more stable. Primer TkS1-64F obtained polymorphic bands of Tian-hua-fen and its nine adulterants. Primer AfS1-100F obtained polymorphic bands of Bai-zhi and its three adulterants. The method has good stability and reproducibility and can easily identify authertic medicines from their adulterants. It was a potential molecular method to identify other Chinese Materia Medica. The method was named as anchored primer amplification polymorphism DNA (APAPD).

Bacterial expression of a Trichosanthes kirilowii defensin (TDEF1) and its antifungal activity on Fusarium oxysporum.

The gene encoding Trichosanthes kirilowii defensin (TDEF1) was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR). The newly discovered TDEF1 cDNA contains 231 bp (Genbank accession number DQ526373) and encodes a 76-amino acid protein, which consists of a 29-amino acid signal peptide and a 47-amino acid mature peptide. The partial cDNA, corresponding to the mature peptide coding region of TDEF1, was inserted into bacterial expression vector pET32a(+). Subsequent expression showed that TDEF1 was produced as a 26-kDa fusion protein in the form of inclusion body in Escherichia coli BL21 (DE3). After protein refolding and purification, the fusion TDEF1 displayed an inhibitive activity against the fungal pathogen, Fusarium oxysporum, with EC(50) of 247 microg/ml by means of fungal growth inhibition method.

[A new molecular method to authenticate radix trichosanthis as well as its adulterants and substitutes].

OBJECTIVE: To explore a new molecular method to authenticate Radix Trichosanthis. METHOD: Three 20 mer primers based on the ITS sequence was designed. The PCR reaction system was optimized and applied to nineteen different sources of Radix Trichosanthis and nine adulterants and substitutes. RESULT: Polymorphic map of Radix Trichosanthis and its adulterants was obtained from primer TKS1-64. 560 bp and 960 bp bands were authentic markers for Radix Trichosanthis. CONCLUSION: Primer TKS1-64F possesses the advantages of good stability and reproducibility. This new method is named as anchored primer amplification polymorphism DNA(APAPD). It was a potential method to used in molecular identification of other meteria medica.

Effect of N- and C-terminal deletions on the RNA N-glycosidase activity and the antigenicity of karasurin-A, a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica.

Karasurin-A, from root tubers of Trichosanthes kirilowii var. japonica, is a type I ribosome-inactivating protein (RIP) that displays activity of RNA N-glycosidase to remove an adenine in the conserved sarcin/ricin loop of the largest RNA in the ribosome. We expressed recombinant proteins of karasurin-A and its various mutants with N- or C-terminal deletions in Escherichia coli as fusion proteins with maltose-binding protein (MBP), and compared their enzymatic activities and antigenicities. Muteins of karasurin-A generated by deleting either the first 100 N-terminal or the last 30 C-terminal amino acid residues lost activity of RNA N-glycosidase. The mutant proteins whose 80 N-terminal or 20 C-terminal amino acids were deleted could depurinate rRNA although the activities were decreased drastically. The antigenicities of the recombinant proteins were considerably reduced by deleting 20 amino acid residues from either N- or C-terminal regions.

[Genetic transformation of hairy roots in Trichosanthes kirilowii Maxim. by Ti and Ri plasmids].

OBJECTIVE: To establish a hairy root culture system by double transformation for Trichosanthes kirilowii. METHOD: 1. Crown galls were induced by direct infection of sterile seedlings of T. kirilonii with Agrobacterium tumefaciens C58, and then the hairy roots were obtained from the regenerated plants by infection with A. rhzogenes 15834; 2. Transformation of Ti and Ri plasmids was inspected by high-pressure-paper electrophoresis; 3. The protein contents in the tissues of T. kirilowii were inspected by spectrophotometer and SDS-PAGE. RESULT: A hairy root culture system has been established successfully by double transformation with Ti and Ri plasmids in T. kirilowii. CONCLUSION: Compared with the ordinary hairy roots, the double transformed hairy roots grow faster but retain similar protein contents.

[Analysis of the genetic relationship among the three types of Trichosanthes kirilowii Maxim. by random amplified polymorphic DNA (RAPD)].

OBJECTIVE: Discussing the discordance in purchasing and utilizing a new type of Trichosanthes kirilowii called "Niuxin-gualou" from Shandong Province. METHOD: The genetic relationship among the three types of T. kirilowii including "Ren-gualou", "Tang-gualou" and "Niuxin-gualou" from Shandong was analyzed by random amplified polymorphic DNA(RAPD). RESULTS: Among the three types studied, the genetic distance is the smallest between "Niuxin-gualou" and "Ren-gualou", and the largest between "Niuxin-gualou" and "Tang-gualou". CONCLUSION: "Niuxingualou" is genetically more similar to "Ren-gualou".