洋紫荆(Bauhinia variegata L.)花的化学成分研究

民间用药洋紫荆(Bauhinia variegata L.)为豆科羊蹄甲属植物,广泛分布于我国南部省份。基于前人的研究,并未发现对洋紫荆花化学成分研究的相关报道,本文以采自云南德宏地区的洋紫荆(B.variegata)花为研究对象,运用各种柱色谱分离手段,从其乙酸乙酯部分中分离得到了8个化合物,经核磁共振波谱及质谱解析,分别鉴定为Isoliquiritigenin(1)、Naringenin(2)、Kaempferol(3)、Kaempferol-7-O-β-D-glucopyranoside(4)、Kaempferol-3-O-β-D-glucopyranoside(5)、Caffeic acid(6)、(-)-Catechin(7)及Kaempferol-3-O-L-rhamnopyranoside(8)。化合物1和4为首次从该种植物中分离得到。     

洋紫荆凝集素的分离纯化和性质

利用酸化处理的Sepharose 6B 亲和柱从洋紫荆种子中分离纯化出了洋紫荆凝集素(BVL),其比活性比抽提液提高了159倍,活力回收率为49.0%。BVL分子量为81 000,由两个相同的亚基组成。等电聚焦凝胶电泳测得其等电点为4.95。其紫外吸收高峰在276 nm处。BVL具一定的热稳定性和酸碱稳定性。N-乙酰-D-氨基半乳糖能强烈地抑制BVL对兔红细胞的凝集作用,乳糖、半乳糖也有较强的抑制作用。     

洋紫荆凝集素的分离纯化和性质

利用酸化处理的Ｓｅｐｈａｒｏｓｅ ６Ｂ亲和柱从洋紫荆种子中分离纯化出了洋紫荆凝集素（ＢＶＬ）,其比活性比抽提液提高了１５９倍,活力回收率为４９．０％。ＢＶＬ分子量为８１０００,由两个相同的亚基组成。等电聚焦凝胶电注测得其等电点为４．９５。其紫外吸收高峰在２７６ｎｍ处。ＢＶＬ具一定的的热稳定性和酸碱稳定性。Ｎ－乙酰－Ｄ氨基半乳能强烈地抑制ＢＶＬ对兔红细胞的凝集作用。乳糖、半乳糖也有较强的抑制定性。Ｎ     

Cloning, expression and characterization of Bauhinia variegata trypsin inhibitor BvTI

A Bauhinia variegata trypsin inhibitor (BvTI) cDNA fragment was cloned into the pCANTAB5E phagemid. The clone pAS 1.1.3 presented a cDNA fragment of 733 bp, including the coding region for a mature BvTI protein comprising 175 amino acid residues. The deduced amino acid sequence for BvTI confirmed it as a member of the Kunitz-type plant serine proteinase inhibitor family. The BvTI cDNA fragment encoding the mature form was cloned into the expression vector, pET-14b, and ex-pressed in E. coli BL21 (DE3) pLysS in an active form. In addition, a BvTI mutant form, r(mut)BvTI, with a Pro residue as the fifth amino acid in place of Leu, was produced. The recombinant proteins, rBvTI and r(mut)BvTI, were purified on a trypsin-Sepharose column, yielding 29 and 1.44 mg/l of active protein, respectively, and showed protein bands of approximately 21.5 kDa by SDS-PAGE. Trypsin inhibition activity was comparable for rBvTI (Ki=4 nM) and r(mut)BvTI (Ki=6 nM). Our data suggest that the Leu to Pro substitution at the fifth amino-terminal residue was not crucial for proteinase inhibition.     

Studies on a virus causing stem grooving and graft-union abnormalities in Virginia Crab apple*

A virus was transmitted from apple trees to Nicotiana glutinosa and Chenopodium spp. and back to a range of woody indicators in which it affected only Virginia Crab; symptoms were grooves in the xylem, and swelling and necrosis of the scion immediately above the union with the stock. The virus was distinct from that causing stem pitting in Virginia Crab, because although easily detectable in several apple varieties, it was not found in many trees infected with stem pitting virus. The stem grooving virus has flexous particles 600–700 m/μ long, a heat inactivation point of 67 °C, a dilution end-point of 10^-3 in N. glutinosa sap and remains infective for at least 2 days at 20 °C.     

Micropropagation of Bauhinia variegata and Parkinsonia aculeata from nodal explants of mature trees

In vitro propagation protocols were established for two leguminous trees, Bauhinia variegata and Parkinsonia aculeata. In each case axillary shoot proliferation was achieved from nodal explants from mature (6-2-8 years) trees using Murashige & Skoog's medium supplemented with 2.22–31.1 M of 6-benzyladenine. Subsequent rooting of the regenerated shoots was achieved on medium containing 2.46–14.8 M of indole-3-butyric acid. Successful transfer of the regenerants to soil has been accomplished.     

Chromosomal-level genome assembly of the orchid tree Bauhinia variegata (Leguminosae; Cercidoideae) supports the allotetraploid origin hypothesis of Bauhinia

Cercidoideae, one of the six subfamilies of Leguminosae, contains one genus Cercis with its chromosome number 2n = 14 and all other genera with 2n = 28. An allotetraploid origin hypothesis for the common ancestor of non-Cercis genera in this subfamily has been proposed; however, no chromosome-level genomes from Cercidoideae have been available to test this hypothesis. Here, we conducted a chromosome-level genome assembly of Bauhinia variegata to test this hypothesis. The assembled genome is 326.4 Mb with the scaffold N50 of 22.1 Mb and contains 37,996 protein-coding genes. The Ks distribution between gene pairs in the syntenic regions indicates two whole-genome duplications (WGDs): one is B. variegata-specific, and the other is shared among core eudicots. Although Ks between gene pairs generated by the recent WGD in Bauhinia is greater than that between Bauhinia and Cercis, the WGD was not detected in Cercis, which can be explained by an accelerated evolutionary rate in Bauhinia after divergence from Cercis. Ks distribution and phylogenetic analysis for gene pairs generated by the recent WGD in Bauhinia and their corresponding orthologs in Cercis support the allopolyploidy origin hypothesis of Bauhinia. The genome of B. variegata also provides a genomic resource for dissecting genetic basis of its ornamental traits.     

Shoot tip culture and cryopreservation for eradication of Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from apple rootstocks ‘M9’ and ‘M26’

This study attempted to eradicate Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from virus‐infected in vitro shoots of apple rootstocks ‘M9’ and ‘M26’ using shoot tip culture and cryopreservation. In shoot tip culture, shoot tips (0.2 mm in length) containing two leaf primordia failed to show shoot regrowth. Although shoot regrowth rate was the highest in the largest shoot tips (1.0 mm in length) containing four leaf primordia, none of the regenerated shoots was virus‐free. Shoot tips (0.5 mm in length) containing two and three leaf primordia produced 100% and 10% of ASPV‐free shoots, respectively, while those (1.0 mm) containing four leaf primordia were not able to eradicate ASPV. ASGV could not be eradicated by shoot tip culture, regardless of the size of the shoot tips tested. In cryopreservation, shoot tips (0.5 mm in length) containing two leaf primordia did not resume shoot growth. Although 1.0‐mm and 1.5‐mm shoot tips gave similarly high ASPV‐free frequencies, the latter had much higher shoot regrowth rate than the former. Very similar results of shoot regrowth and virus eradication by shoot tip culture and cryopreservation were observed in both ‘M9’ and ‘M26’. Histological observations showed that only cells in upper part of apical dome and in leaf primordia 1–3 survived, while other cells were damaged or killed, in shoot tips following cryopreservation. Virus immunolocalization found ASPV was not detected in upper part of apical dome and leaf primordia 1 and 2, but was present in lower part of apical dome, and in leaf primordium 4 and more developed tissues in all samples tested. ASPV was also detected in leaf primordium 3 in about 16.7% and 13.3% samples tested in ‘M9’ and ‘M26’. ASGV was observed in apical dome and leaf primordia 1–6, leaving only a few top layers of cells in apical dome free of the virus. Different abilities of ASPV and ASGV to invade leaf petioles and shoot tips were also noted.     

In-vitro evaluation of the antimicrobial activity of Bauhinia variegata,locally known as koiralo

Bauhinia variegata, commonly known as Koiralo is considered as medicinal plant in Nepal and India. The alcoholic extract of this plant was found to have antimicrobial activity against Bacillus subtilis (ATCC 6635) Pseudomonas aeruginosa (ATCC 27853), Salmonella typhi, Shigella dysenteriae, Staphylococcus aureus (ATCC 29213) and Vibrio cholerae. The largest zone of inhibition (18 mm) was found to be exhibited against B. subtilis. For this organism the minimum bactericidal concentration (MBC) of the crude extract was 0.39 mg/ml. The extract was found to be more effective against gram-positive than gram-negative bacteria. The antimicrobial activity of the extract was found to be decreased during purification.     

Cytopathology of Apple stem pitting virus in Nicotiana occidentalis L.

Apple stem pitting virus (ASPV) is a causal agent of stem pitting associated disease in pomes fruit trees. The present report focuses on a cytopathological effect of ASPV infection in a herbaceous host Nicotiana occidentalis ‘37B’. A leaf dip preparation shows predominantly basic virus particles and aggregated particles 800 nm and 3200 nm long respectively. The main cytopathological effect observed in ASPV infected N. occidentalis includes fibrous aggregates of virus particles (massive/or few), formation of membranous vesicles and proliferation of the endoplasmic reticulum.     

The Complete Amino Acid Sequence of a Trypsin Inhibitor from Bauhinia variegata var. candida Seeds

Trypsin inhibitors of two varieties of Bauhinia variegata seeds have been isolated and characterized. Bauhinia variegata candida trypsin inhibitor (BvcTI) and B. variegata lilac trypsin inhibitor (BvlTI) are proteins with M _r of about 20,000 without free sulfhydryl groups. Amino acid analysis shows a high content of aspartic acid, glutamic acid, serine, and glycine, and a low content of histidine, tyrosine, methionine, and lysine in both inhibitors. Isoelectric focusing for both varieties detected three isoforms (pI 4.85, 5.00, and 5.15), which were resolved by HPLC procedure. The trypsin inhibitors show K _i values of 6.9 and 1.2 nM for BvcTI and BvlTI, respectively. The N-terminal sequences of the three trypsin inhibitor isoforms from both varieties of Bauhinia variegata and the complete amino acid sequence of B. variegata var. candida L. trypsin inhibitor isoform 3 (BvcTI-3) are presented. The sequences have been determined by automated Edman degradation of the reduced and carboxymethylated proteins of the peptides resulting from Staphylococcus aureus protease and trypsin digestion. BvcTI-3 is composed of 167 residues and has a calculated molecular mass of 18,529. Homology studies with other trypsin inhibitors show that BvcTI-3 belongs to the Kunitz family. The putative active site encompasses Arg (63)–Ile (64).     

Elimination of apple stem grooving virus by chemotherapy and development of an immunocapture RT-PCR for rapid sensitive screening

Ombuin (7,4′-dimethyl quercetin) (10 μg ml^-1, for 12 wk), glycyrrhizin/quercetin (80 μg ml^-1and 10 μg ml^-1respectively, for 18 wk), ribavirin (10 μg ml^-1, for 12 wk) and quercetin/ribavirin (10 μg ml^-1each, for 9–12 wk) reduced the titre of apple stem grooving virus (ASGV) when applied in vitro to infected tissue cultures of Nicotiana occidentalis obliqua Wheeler, and/or Malus domestica. ASGV was not detectable in both plant species after the quercitin/ribavirin treatment when tested by ISEM, herbaceous host indexing, RT-PCR, and immunocapture RT-PCR. A sensitive immunocapture RT-PCR procedure for the detection of ASGV was developed for the screening of treated samples to assess antiviral activity.     

Structure Predictions of Two Bauhinia variegata Lectins Reveal Patterns of C-Terminal Properties in Single Chain Legume Lectins

Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and –II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins.     

Nucleotide sequences of a Korean isolate of apple stem grooving virus associated with black necrotic leaf spot disease on pear (Pyrus pyrifolia)

Pear black necrotic leaf spot (PBNLS) is a disease of pears caused by capillovirus-like particles, which can be observed under the electron microscope. The disease was analyzed by Western blot analysis with antisera raised against apple stem grooving virus (ASGV) coat protein. cDNAs covering the entire genome were synthesized by RT-PCR and RACE using RNA isolated from Chenopodium quinoa infected with sap extracted from pear leaves carrying black necrotic spot disease. The complete genome sequence of the putative pear virus, 6497 nucleotides in length excluding the poly (A) tail, was determined and analyzed. It contains two overlapping open reading frames (ORFs). ORF1, spans from nucleotide position 37 to 6354, producing a putative protein of 241 kDa. ORF2, which is in a different reading frame within ORF1, begins at nucleotide 4788 and terminates at 5750, and produces a putative protein of 36 kDa. The 241 kDa protein contains sequences related to the NTP-binding motifs of helicases and RNA-dependent RNA polymerases. The 36-kDa protein contains the consensus sequence GDSG found in the active sites of several cellular and viral serine proteases. Morphological and serological analysis, and sequence comparison between the putative pear virus, ASGV, citrus tatter leaf virus and cherry virus A of the capillovirus suggest that PBNLS may be caused by a Korean isolate of ASGV.     

Preparation of Antiserum to Apple stem pitting virus Using in Silico Prediction of Antigenic Epitopes

A sensitive antiserum is needed for the detection of Apple stem pitting virus (ASPV), one of the most important latent viruses that infect fruit trees. We have studied many properties of coat protein, such as the antigenic index, α‐helix, β‐sheet, β‐turn, coil structure, hydrophilicity, surface probability and flexibility and analysis with several software algorithms. Based on the rules for locating the antigenic epitopes in the regions including β‐turns and coil structures with the high hydrophilicity and surface probability, the predicted epitopes were located in the region of amino acid positions 4–18, 100–114, 400–414, respectively. Two linear synthetic peptides (CRGYEEGSRPNQRVLP and CTGGKIGPKPVLSIRK) were prepared and conjugated with carrier protein. The antisera, designated 1468 and 1469, were obtained by immunizing rabbits. The antibody produced a strong immuno‐reaction with the expression product of the ASPV coat protein gene in Escherichia coli. By testing ten apple samples, ASPV could be detected by Protein A Sandwich ELISA using antiserum 1468, but only some positive samples could be detected with antiserum 1469. To our knowledge, this is the first report of the preparation of antiserum to a pome fruit virus using the antigenic epitopes method.     

Natural occurrence of Bacillus thuringiensis on grass foliage

World Journal of Microbiology and Biotechnology -