Cloning and prokaryotic expression of major ampullate spidroin gene of spider / 生物工程学报

RT-PCR was conducted with one degenerate primer designed according to repetitive regions' amino acid sequence of major ampullate spidroin (MaSp) in spiders and adaptor primer in the SMART cDNA Library Construction Kit. By cloning and sequencing of amplified products, one cDNA clone (GenBank Accession No. AY365017) of Argiope amoena MaSp gene was obtained. The deduced amino acid sequence can be distinctly divided into two regions: (1) Repetitive region that consists of an alternating alanine-rich and glycine-rich domain in which many prolines are present; and (2) C-terminal non-repetitive region. The region coding for 272 amino acids of MaSp gene was subcloned into prokaryotic expression vector pET28b(+) and an about 26kD recombinant protein was expressed at high levels in Escherichia coli BL21 (DE3) after induction of IPTG. After being purified with metal-affinity chromatography on Ni(2+) -IDA-Sepharose columns as well as gel filtration chromatography, the recombinant protein was confirmed to be predicted MaSp by means of amino acid composition analysis and N-terminal amino acid sequence analysis. The solubility behavior of recombinant MaSp with C-terminal non-repetitive region in the present study is similar to that of recombinant dragline silk proteins without C-terminal non-repetitive region expressed by bacteria and yeast in the other studies. The result shows that absence or presence of C-terminal non-repetitive region is not a crucial factor affecting the solubility of the recombinant MaSp.

Molecular identification of medicinal plants: Dendrobium chrysanthum, Dendrobium fimbriatum and their morphologically allied species by PCR-RFLP analyses / 药学学报

<p><b>AIM</b>To establish a simple method for molecular identification of original plants of D. chrysanthum and D. fimbriatum using molecular marker rDNA ITS region.</p><p><b>METHODS</b>Restriction patterns of ITS fragments were obtained using PCR-RFLP method. The PCR products of D. chrysanthum and its morphologically allied species were digested at 37 degrees C by Cla I and Apa LI, those of D. fimbriatum and its morphologically allied species were digested by Sph I.</p><p><b>RESULTS</b>D. chrysanthum, D. fimbriatum and their morphologically allied species could be identified by predicted restriction profiles of PCR-RFLP. The botanical origin of twenty-five fresh samples of "Shihu" collected in markets was identified by this method.</p><p><b>CONCLUSION</b>The results showed that PCR-RFLP analysis of the rDNA ITS region is a feasible, simple and inexpensive method for determining the botanical origin of the traditional Chinese medicine "Shihu".</p>

RbcL sequence analysis of Belamcanda chinensis and related medicinal plants of Iris / 药学学报

<p><b>AIM</b>To identify "Shegan" [Belamcanda chinensis (L.) DC.] and relative medicinal plants of Iris including Iris tectorum Maxim., I. dichotoma Pall., I. germanica L. and I. japonica Thunb. by ribulose 1,5-bisphosphate carboxylase Large Gene (rbcL) sequence analysis.</p><p><b>METHODS</b>General DNA was isolated from the fresh leaves of Belamcanda chinensis and 4 Iris spp. by CTAB. A pair of primers was designed to amplify the rbcL gene and PCR Preps DNA kit was used to purify the PCR products. The rbcL sequences were determined by ABI (Applied Biosystems Inco.) Prism 310 Genetic Analyzer.</p><p><b>RESULTS</b>A fragment of about 750 bp of rbcL gene from Belamcanda chinensis and 4 Iris spp. were amplified and sequenced. The rbcL sequences of Iris tectorum, I. dichotoma Pall. and I. japonica were reported for the first time. The rbcL sequences of 5 species of Iridaceae were aligned and analyzed using Clustal (Version 8.0) and MEGA (Version 2.0.) programs. The nucleotide number of difference is from 1.000 to 20.000. The tranversions is from 0.000 to 9.000 and the transitions is from 0.000 to 14.000. Phylogenetic tree based on rbcL partial sequence data indicated that the eleven samples of 5 species clustered separately.</p><p><b>CONCLUSION</b>The sequence variation of rbcL can be used to identify Belamcanda chinensis and 4 species of relative medicinal plants of Iris. The molecular phylogenetic tree accords with the classical taxonomy.</p>

Expression of a novel bHLH-Zip gene in human testis / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To identify specifically expressed genes in the adult and fetal testes.</p><p><b>METHODS</b>A human testis cDNA microarray was established. Then the mRNA of adult and fetal testis was purified and probes were prepared by a reverse transcription reaction with the testis mRNA as template. The microarray was hybridized with probes of adult and fetal testes. The nucleic sequences of differentially expressed genes were determined and homologies were searched in the databases of the GenBank.</p><p><b>RESULTS</b>When hybridized with adult or fetal testis probes, the positive clones were 96.8 % and 95.4 %, respectively. Among these genes, one was a new testis-specific gene, which was named TSP1. TSP1 was highly expressed in human adult testis. The cDNA of TSP1 was 1,484 bp in length. The cDNA sequence of this clone was deposited in the Genbank (AF333098). TSP1 was also determined as Interim Gen Symbol (Unigene, No. Hs.98266). Protein analysis showed that TSP1 contained two functional domains: an N-terminal basic helix-loop-helix (bHLH) and a C-terminal leucine zipper (Zip). Homologous analysis showed that the 430 amino acid sequences deduced from the 1293 bp open reading frame (ORF) had a homology with the human gene FLJ2509 (AK098575). TSP1 had also a sequence homology with Spz 1 protein of mouse. Expression profiles showed that TSP1 was specifically and strongly expressed in the testis.</p><p><b>CONCLUSION</b>TSP1 is a gene highly expressed in adult testis. It may play an important role in spermatogenesis in the humans.</p>

Database establishment of the whole rDNA ITS region of Dendrobium species of "fengdou" and authentication by analysis of their sequences / 药学学报

<p><b>AIM</b>To establish the whole rDNA ITS region sequence database of various Dendrobium species of "Fengdou" and to authenticate exactly the inspected species of "Fengdou".</p><p><b>METHODS</b>The rDNA ITS regions of various Dendrobium species of "Fengdou" were amplified and sequenced. The database of their rDNA ITS regions was established in order to authenticate the inspected species by means of the softwares of CLUSTRAL and MEGA which were used to analyze the rDNA ITS region.</p><p><b>RESULTS</b>A database of the rDNA ITS sequences of 21 species of Dendrobium has been established. The notable and stable differences of the interspecies of the rDNA ITS regions have been demonstrated. The numbers of transitions and transversions among 21 species are 11-122. The variable sites are 341 while the informative sites are 195. The ITS sequence differences between the outgroup species (Pholidota yunnanensis) and species of "Fengdou" are obvious. The numbers of transitions and transversions are 131-161. The population differences of the rDNA ITS region of various species of "Fengdou" are very small (0-6).</p><p><b>CONCLUSION</b>On the basis of the database of various Dendrobium species of "Fengdou" and two genetics software, the botanical origin of the inspected species of "Fengdou" has been authenticated successfully by sequencing the rDNA ITS regions.</p>

Study on sequence difference and SNP pheomenon of rDNA ITS region in F type and H type population of Dendrobium officinale / 中国中药杂志

<p><b>OBJECTIVE</b>To study rDNA ITS sequence differences between F type and that of H type of Dendrobium officinale in main habitat of China.</p><p><b>METHOD</b>The population differences of the rDNA ITS region (including ITS1, ITS2, 5.8S) sequences of D. officinale were studied by the method of DNA sequences analysis.</p><p><b>RESULT</b>There were two different sites between the rDNA ITS sequence of F type and that of H type. One was in ITS1 region, and the other was in 5.8S region. It was proved that there was some relativity between the character of rDNA ITS region and the life type of the populations. The phenomenon of single nucleotide polymorphism (SNP) existed in 5.8S region of rDNA ITS region between F type and H type. The sequences of rDNA ITS region of D. officinale were reported for the first time, and the sequences of ITS region ranged 634 bp (ITS1 231 bp, ITS2 240 bp, 5.8S 163 bp).</p><p><b>CONCLUSION</b>The analysis of rDNA ITS of D. officinale deeply reveal the population differences of D. officinale of F type and H type.</p>

Molecular authentication of Dendrobium chrysanthum from its allied species of Dendrobium / 中国中药杂志

<p><b>OBJECTIVE</b>To define molecular characters to distinguish D. chrysanthum from its allied species D. primulinum, D. lituiflorum, D. aphyllum, D. crepidatum.</p><p><b>METHOD</b>The molecular characteristics of D. chrysanthum and its allied species were compared. The sequences of rDNA ITS regions were exploited to explore the evidence for authentication D. chrysanthum and its allied species.</p><p><b>RESULT</b>Although the morphological difference was slight, the sequence difference of ITS regions among five rDNAs was obvious and stable. Fifteen sites of ITS region were defined as DNA character to identify D. chrysanthum from the other four allied species.</p><p><b>CONCLUSION</b>The difference of rDNA ITS sequences can be used to authenticate accurately D. chrysanthum from three allied species of Dendrobium.</p>