桑泛素延伸蛋白基因片段的克隆及序列分析

目的:利用3’RACE技术克隆植物泛素基因,是进一步研究其功能的基础。方法:本研究从桑树(丰驰桑)(Morus bomby-cis)幼叶中提取总RNA,反转录成cDNA,根据已报道的泛素基因序列设计1条正向引物,利用3’RACE(Rapid Amplification of cDNAEnd)技术进行扩增。结果:扩增出1条690 bp的泛素基因片段。该片段5’端为编码156个氨基酸残基的阅读框,3’末端有219bp的非翻译区。结论:同源分析表明,此cDNA序列为泛素延伸蛋白基因(Genebank登录号为DQ839403)。用Genedoc软件对该片段编码的氨基酸序列进行同源性分析的结果表明:桑树泛素延伸蛋白与马铃薯、烟草、陆地棉、黄瓜的泛素延伸蛋白以及苜蓿的核糖体S27A蛋白的同源性都在96%以上。     

甜菜夜蛾泛素延伸蛋白基因cDNA的3′RACE-PCR扩增及其克隆和表达

根据已知的草地夜蛾Spodoptera frugiperda的泛素延伸基因 5'端核苷酸序列设计引物,应用3'RACE-PCR技术,从甜菜夜蛾S. exigua脂肪体组织总RNA中反转录扩增泛素基因的cDNA片段。扩增得到的片段全长513 bp,3'末端有123 bp的非翻译区,翻译区编码一个长为129个氨基酸残基的蛋白质,预测分子量为14.8 kD。同源分析表明,此cDNA序列为ubiquitin-53aa extension protein(ubi-53) 基因,在泛素蛋白后融合了一个核糖体L40蛋白（ribosomal L40 protein）。用MagAlign和Genedoc软件对cDNA编码的氨基酸序列进行了同源性分析,结果表明： 甜菜夜蛾的ubi-53基因与真核生物家蚕Bombyx mori、草地夜蛾、果蝇Drosophila melanogaster和人Homo sapienes泛素的同源性分别为96.9%、98.5%、95.3%和93.0%,与甜菜夜蛾核型多角体病毒(SeNPV)泛素的同源性为78.8%,说明真核生物的泛素基因与核型多角体病毒的泛素基因可能存在不同的分子进化途径。将甜菜夜蛾的ubI-53基因克隆到原核表达载体pET-28a上,转化至BL21（DE3）中,用IPTG进行诱导表达,用异源泛素单克隆抗体进行Western blot检测,证明原核表达蛋白是目的蛋白。     

The Tall Fescue Turf Grass Class I Chitinase Gene <Emphasis Type="Italic">FaChit1</Emphasis> Is Activated by Fungal Elicitors,Dehydration, Ethylene,and Mechanical Wounding

The cDNA, genomic DNA, and promoter sequence of FaChit1, a class I chitinase gene from Festuca arundinacea, were isolated and characterized in the present work. The deduced amino acid sequence of FaChit1 contains the chitin binding, catalytic, and proline and glycine-rich domains characteristic for most class I chitinases, but no C-terminal extension region. FaChit1 is induced effectively by fungal elicitors, dehydration, and ethylene, but only slightly by mechanical wounding. To identify potential stress-related cis-acting elements, 5′ sequences 935, 651, and 233 bp upstream of the FaChit1 start codon were fused to the GUS reporter gene and analyzed in transgenic tobacco. The results indicated that the 935 bp fragment closely mirrored endogenous gene expression and that the 651 bp fragment was sufficient to direct reporter the gene expression in response to fungal elicitors, ethylene, dehydration, or mechanical wounding due to both known and presently uncharacterized cis-acting elements. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cloning and characterization of ribonuclease T2 gene (RNHe30) from the basidiomycete,Hericium erinaceum

A gene encoding a ribonuclease T2 (RNase T2) family enzyme, RNHe30, was cloned from Hericium erinaceum by PCR. The deduced amino acid sequence from the complimentary DNA (cDNA) (1074 bp) encodes a 302-aa protein (RNase He30) that has the consensus amino acid sequences of RNase T2 family enzymes including the putative signal peptide. The presence of five introns in the genomic DNA was confirmed by comparison of the cDNA and genomic DNA sequences. The promoter region contains a putative CAAT box and a consensus TATA box. Genes coding homologous enzymes were also identified in various other basidiomycetes. A phylogenetic tree of RNase T2s from these fungi was constructed from a multiple alignment of the deduced amino acid sequences. The tree showed that the enzymes were divided into two main groups.     

Cloning and characterization of an epoxide hydrolase-encoding gene from Rhodotorula glutinis

We cloned and characterized the epoxide hydrolase gene, EPH1, from Rhodotorula glutinis. The EPH1 open reading frame of 1230 bp was interrupted by nine introns and encoded a polypeptide of 409 amino acids with a calculated molecular mass of 46.3 kDa. The amino acid sequence was similar to that of microsomal epoxide hydrolase, which suggests that the epoxide hydrolase of R. glutinis also belongs to the α/β hydrolase fold family. EPH1 cDNA was expressed in Escherichia coli and resting cells showed a specific activity of 200 nmol min⁻¹ (mg protein)⁻¹ towards 1,2-epoxyhexane. Received: 2 August 1999 / Received revision: 4 October 1999 / Accepted: 10 October 1999     

Cloning of bovine LYST gene and identification of a missense mutation associated with Chediak-Higashi syndrome of cattle

An inheritable bleeding disorder with light coat color caused by an autosomal recessive gene has been reported in a population of Japanese black cattle. The disease has been diagnosed as Chediak-Higashi Syndrome (CHS) of cattle which correspond to a human inheritable disorder caused by mutation in LYST gene. To characterize the molecular lesion causing CHS in cattle, cDNAs encoding bovine LYST were isolated from a bovine brain cDNA library. The nucleotide and deduced amino acid sequences of bovine LYST had 89.6 and 90.2% identity with those of the human LYST gene, respectively. In order to identify the mutation within the LYST gene causing CHS in cattle, cDNA fragments of the LYST gene were amplified from an affected animal by RT-PCR and their nucleotide sequences were completely determined. Notably, a nucleotide substitution of A to G transition, resulting in an amino acid substitution of histidine to arginine (H2015R) was identified in the affected animal. The presence of the substitution was completely corresponding with the occurrence of the CHS phenotype among 105 members of pedigrees of the Japanese black cattle and no cattle of other populations had this substitution. These findings strongly suggested that H2015R is the causative mutation in CHS of Japanese black cattle. Received: 25 May 1999 / Accepted: 26 July 1999     

Cloning,expression and characterization of a gene which encodes a topoisomerase I with positive supercoiling activity in pea

We have isolated and sequenced the full length cDNA for topoisomerase I. Using degenerate primers, based on the conserved amino acid sequences of five eukaryotic topoisomerase I, a 386 bp fragment was PCR amplified using pea cDNA as template. This fragment was used as a probe to screen a pea cDNA library. Two partial cDNA clones were isolated which were truncated at the 5 end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of the gene was 3055 bp with an open reading frame of 2676 bp. The deduced structure of pea topoisomerase I contain 892 amino acids with a calculated molecular weight of 100 kDa and an estimated pI of 9.3. A comparison of the deduced amino acid sequences of the pea topo I with the other eukaryotic topoisomerases clearly suggested that they are all related. Pea topoisomerase I has been overexpressed in E. coli system and the recombinant topoisomerase purified to homogeneity. The purified protein relaxes both positive and negative supercoiled DNA in the absence of divalent cation Mg²⁺. In the presence of Mg²⁺ ions the purified enzyme introduces positive supercoils a unique property not reported in any other organism except in archaebacterial topoisomerase I. Polyclonal antibodies were raised against recombinant topoisomerase I and western blotting with sub-cellular fractions indicated the localization of this topoisomerase in pea nuclei.     

Molecular cloning of ribosomal protein L26 (RPL26) cDNA from Ailuropoda melanoleuca and its potential value in phylogenetic study

The cDNA fragment of ribosomal protein L26 (RPL26) was cloned from Ailuropoda melanoleuca using RT-PCR method. The cDNA fragment is composed of 475 bp, containing an open reading frame of 145 amino acids. Alignment analyses indicated that the nucleotide sequence and the deduced amino acid sequence showed high identity to other known RPL26 sequences from vertebrates and invertebrates. The cDNA sequence was used to construct phylogenetic trees with other known vertebrate and invertebrate RPL26 sequences, and the obtained trees demonstrated similar topology with the classical systematics, indicating the potential value of RPL26 gene in phylogenetic analysis.     

Characterization of IS1676 from Rhodococcus erythropolis SQ1

To develop a transposable element-based system for mutagenesis in Rhodococcus, we used the sacB gene from Bacillus subtilis to isolate a novel transposable element, IS1676, from R. erythropolis SQ1. This 1693 bp insertion sequence is bounded by imperfect (10 out of 13 bp) inverted repeats and it creates 4 bp direct repeats upon insertion. Comparison of multiple insertion sites reveals a preference for the sequence 5′-(C/T)TA(A/G)-3′ in the target site. IS1676 contains a single, large (1446 bp) open reading frame with coding potential for a protein of 482 amino acids. IS1676 may be similar to an ancestral transposable element that gave rise to repetitive sequences identified in clinical isolates of Mycobacteriumkansasii. Derivatives of IS1676 should be useful for analysis of Rhodococcus strains, for which few other genetic tools are currently available. Received: 1 April 1999 / Received revision: 6 July 1999 / Accepted: 1 August 1999     

Characterization,expression, and functional analysis of testis-specific serine/threonine kinase 1 (Tssk1) in the pen shell Atrina pectinata

Abstract

Testis-specific serine/threonine kinase 1 (Tssk1) was named since its function was determined in mouse spermiogenesis. In our research, we cloned a homolog of the pen shell Atrina pectinata Tssk1, ApTssk1, and determined its expression characteristics at mRNA. The full length of ApTssk1 cDNA was 1517 bp, with an open reading frame of 1089 bp (58–1146), which encodes a peptide including 362 amino acids. The homologous analysis indicated that the deduced amino acid sequences of ApTssk1 shared a close identity with other reported Tssk1. In addition, the highly conserved fragment containing a serine/threonine protein kinase catalytic (S-TKc) domain was predicted to exist in ApTssk1. Results from the RT-qPCR display that ApTssk1 was only transcribed in the male gonad of A. pectinata, and was highest in the mature testis. Therefore, this study indicated that ApTssk1 might play a functional role during spermatogenesis in A. pectinata.     

Molecular cloning and characterization of a calreticulin cDNA from the pinewood nematode Bursaphelenchus xylophilus

The cloning and characterization of a cDNA encoding a calreticulin from the pinewood nematode Bursaphelenchus xylophilus is described herein. The full-length cDNA (Bx-crt-1) contained a 1200 bp open reading frame that could be translated to a 399 amino acid polypeptide. The deduced protein contained highly conserved regions of a calreticulin gene and had 66.2–70.1% amino acid sequence identity to other calreticulin sequences from nematodes. RNAi, RT-PCR amplification, and southern blot suggest that Bx-crt-1 may be important for the development of B. xylophilus.     

Characterization of Geranium (Pelargonium graveolens) Chloroplast EF-Tu cDNA

A geranium (Pelargonium graveolens) chloroplast translational elongation factor EF-Tu (tufA) cDNA was isolated. The geranium tufA cDNA is 1,584 bp long with 20 bp of 5 untranslated region (UTR) and 139 bp of 3 UTR. It encodes 474 amino acids including a putative chloroplast transit peptide of 65 amino acids. The deduced polypeptides of the geranium tufA cDNA contains four GTP binding sequences in its N-terminal region and two chloroplast EF-Tu signature regions in the C-terminal region. The predicted molecular weight of the mature geranium chloroplast EF-Tu protein was about 45,000 and its amino acid sequence identity with the chloroplast EF-Tu proteins of tobacco, pea, Arabidopsis, rice, and soybean ranges from 85% to 91%. The geranium tufA appears to exist as a single copy gene like Arabidopsis and rice, whereas other known dicot plants have more than one copy in their nuclear genomes.     

Molecular Characterization of Mx1 Gene in Native Indian Breeds of Chicken

The genetic polymorphism of Mx1 gene was explored in Indian chicken breeds. PCR-RFLP analysis in 102?bp fragment of partial intron 13 and partial exon 14 of Mx1 gene revealed two genotypes viz. RS and SS with two alleles viz. R and S both in Naked Neck and Tellicherry breeds of chicken. The homozygous genotype RR was not identified. When deduced amino acid sequences were compared, the asparagine amino acid was found to be substituted in “R” allele for serine in “S” allele. PCR-SSCP analysis of 284?bp fragment in 5′-UTR and partial promoter region revealed three genotypes viz. CC, CG, and CH with three different alleles viz. C, G, and H in Naked Neck breed of chicken and five genotypes viz. DI, JK, KK, KL, and KM with six different alleles viz. D, I, J, K, L, and M in Tellicherry breed of chicken. The homozygous genotypes viz. GG and HH in Naked Neck and DD, II, JJ, LL, and MM in Tellicherry chicken was not identified. The nucleotide substitution rate estimated to be in the range of 0.004–0.011. The identified genetic variation can be helpful for better insight to disease resistance property of the Mx1 gene.     

Identification and cloning of a submergence-induced gene OsGGT (glycogenin glucosyltransferase) from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) by suppression subtractive hybridization

A submergence-induced gene, OsGGT, was cloned from 7-day submerged rice (Oryza sativa L. plants, FR13A (a submergence-tolerant cultivar, Indica), using suppression subtractive hybridization and both 5- and 3-rapid amplification of cDNA ends (RACE). The full-length OsGGT cDNA contains 1,273 bp with an open reading frame of 1,140 bp (17–1,156) that encodes 379 amino acids. Its deduced amino acid sequence is homologous with glycogenin glucosyltransferase. We found that the OsGGT gene is located in the 17,970–20,077 bp region of genome fragment AAAA01002475.1 of the Indica cultivar and in the 53,293–51,186 bp region of genome fragment AC037426.12 of chromosome 10 of the Japanica cultivar. A time-course study showed that OsGGT-gene expression increased in FR13A during submergence but decreased in IR42 (submergence-intolerant cultivar, Indica). The expression of the OsGGT gene in FR13A was induced by salicylic acid and benzyladenine. The accumulation of OsGGT mRNA in FR13A also increased in response to ethylene, gibberellin, abscisic acid, drought and salt treatment, but methyl jasmonate treatment and cold stress had no effect on expression. These results suggest that the OsGGT gene could be related to submergence stress and associated with a general defensive response to various environmental stresses.The nucleotide sequences of OsGGT cDNA has been submitted to GenBank DDBJ under accession numbers AB164463.     

A melanin polyketide synthase (PKS) gene from Nodulisporium sp. that shows homology to the pks1 gene of Colletotrichum lagenarium

The melanin polyketide synthase (pks) gene of Nodulisporium sp. MF5954 (ATCC74245) was cloned by exploiting its homology to the Colletotrichum lagenarium pks1 gene. Sequence analysis demonstrated that this gene is 70% identical to the C. lagenarium pks1 gene. A gene disruption construct, designed to replace both the ketoacyl synthase and acyl transferase domains with a hygromycin resistance (Hy^r) gene, was synthesized, and used to disrupt the Nodulisporium melanin pks1 gene via homologous recombination, resulting in a mel(−) phenotype. Sequence analyses of the gene and of cDNA segments generated by RT-PCR indicate that there are three introns in the 5′ half of the gene. The proposed 2159-amino acid product is 72% identical and 78% similar to the 2187-amino acid sequence deduced from the C. lagenarium pks1 gene. This similarity is notable, considering that C. lagenarium is a member of the order Phyllachoales or Sordariales, whereas Nodulisporium is generally believed to be member of the order Xylariales. However, despite the strong resemblance between the amino acid sequences in the acyl transferase domains of the two proteins, only one in five codons are conserved in the DNA sequences that encode this motif. The Nodulisporium sp. pks1 gene sequence and the amino acid sequence deduced from its coding region have been deposited in Genbank under Accession No. AF151533. Received: 15 May 1999 / Accepted: 26 July 1999