Jiong Fei

CDNA cloning and characterization of the Ve homologue gene StVe from Solanum torvum Swartz.

Verticillium wilt is a disastrous disease causing significant yield losses of many crops. Isolation of verticillium wilt resistance gene is a fundamental work for controlling this disease through genetic engineering. In this report, we describe the cloning and characterization of a Ve like gene (StVe) from Solanum torvum Swartz. The nucleotide sequence of StVe is 3640 bp long with an open reading frame of 3414 bp encoding a protein precursor of 1138 aa. Sharing high homologies to tomato verticillium wilt disease resistance genes Ve1 and Ve2, the leucine rich (15.89%) protein StVe has a calculated molecular weight of 126.48 kDa with an isoelectric point of 5.62. It possesses a hydrophobic N-terminal signal peptide of 20 aa and 38 predicted leucine-rich repeats containing 32 potential N-glycosylation sites (28 being significant). Fifty-seven predicted phosphorylation sites (36 for S, 8 for T and 13 for Y) distribute in StVe protein. A PEST-like sequence and a mammalian endocytosis signals YCVF are found within the C-terminal region. The C terminus of StVe concludes with the residues KKF similar to the KKX motif that confers endoplasmic reticulum localization in plants as well as mammals and yeast. The sequence analysis of the StVe gene implies that the StVe is a potential verticillium wilt disease resistance gene encoding a cell surface-like receptor protein.

Molecular cloning and characterization of a novel mannose-binding lectin gene from Amorphophallus konjac

A new lectin gene was cloned from Amorphophallus konjac. The full-length cDNA of Amorphophallus konjac agglutinin (aka) was 736 bp and contained a 474 bp open reading frame encoding a 158 amino acid protein. Homology analysis revealed that the lectin from this Araceae species belonged to the superfamily of monocot mannose-binding proteins. Molecular modeling of AKA indicated that the three-dimensional structure of AKA strongly resembles that of the snowdrop lectin. Southern blot analysis of the genomic DNA revealed that aka belonged to a low-copy gene family. Northern blot analysis demonstrated that aka expression was tissue-specific with the strongest expression being found in root.

Molecular cloning and characterization of a novel lectin gene from Zephyranthes candida

A new lectin gene was cloned from Zephyranthes candida by using RACE-PCR. The full-length cDNA of Zephyranthes candida agglutinin (ZCA) was 647 bp and contained a 477 bp open reading frame encoding a 159 amino acid protein. Zephyranthes candida lectin gene was found to encode a precursor lectin with signal peptide and had extensive homology with those of other plant lectins. Molecular modeling of ZCA indicated that the three-dimensional structure of ZCA strongly resembles that of the snowdrop lectin, implying ZCA may have the similar insecticidal functions with GNA.

An Efficient Method for Rapid Amplification of Arisaema heterophyllum Agglutinin Gene Using a Genomic Walking Technique

Abstract The genomic sequence of Arisaema heterophyllum agglutinin (AHA), a mannose‐binding lectin (MBL), was cloned through a novel genomic walking technique. Adaptor ligation reactions and subsequent amplifications with adaptor primer and multiple specific primers were used to generate specificity in this method. The method allowed for the amplification of over 1 kb of genomic DNA sequence immediately upstream and downstream from the 5′ and 3′ ends of full‐length cDNAs. For aha gene, the upstream regions contained a putative transcription initiation start site and other sequences commonly found in eukaryotic promoters. The downstream regions of aha contained two polyadenylation signals. Our study demonstrated that aha had no intron like mannose‐binding lectin genes cloned from other plant species so far. This efficient method, based on a genomic walking technique, was useful for the cloning of promoters, insertion sites, and other sequences of interest without constructing and screening genomic libraries.

[Isolation and expression profiling of the Pto-like gene SsPto from Solanum surattense].

A novel Pto-like gene (designated as SsPto) is cloned from yellow-fruit nightshade (Solanum surattense). The full-length cDNA of SsPto is 1331 bp long with an open reading frame of 960 bp encoding a polypeptide of 320 amino acid residues. The deduced SsPto protein has a calculated molecular weight of 36.21 kDa with an isoelectric point of 6.18. Multiple sequence alignment shows that the SsPto protein shares 71.4% and 71.6% identities with Pto proteins from Lycopersicon pimpinellifolium and L. hirsutum, respectively. Genomic Southern blot analysis indicates the presence of a small family of SsPto in the S. surattense genome. SsPto is found to be constitutively expressed in the S. surattense plant with the highest expression in stems. However, under induction by TMV for 6 days, SsPto is expressed the highest in roots. Further expression analysis reveals that the signaling components of defense/stress pathways, such as methyl jasmonate (MeJA), salicylic acid (SA), gibberellic acid (GA3), and hydrogen peroxide (H2O2), up-regulate the SsPto transcript levels over the control. Nevertheless, cold treatment has no significant effect on SsPto expression, whereas SsPto expression is down-regulated by dark treatment. Our findings suggest that this novel stress- and pathogen-inducible SsPto from S. surattense may participate not only in the defense/stress responsive pathways, but also in diverse processes of plant growth and development.

Isolation and Characterization of an IAA-responsive Gene from Gossypium barbadense L.

The full-length cDNA of an IAA-responsive gene was cloned from Gossypium barbadense L. (designated as Gbiaa-Re) by rapid amplification of cDNA ends (RACE). Gbiaa-Re gene was 1043-bp long and contained a 573-bp open reading frame encoding a polypeptide of 190 amino acid residues. Homology analysis revealed that Gbiaa-Re strongly resembled known plant IAA-responsive genes. The conserved integrated domain “AUX_IAA, AUX/IAA family” resided within the region from L11 to V190 of GbIAA-RE, and the 4 typically conserved domains of IAA-responsive gene family were all found in GbIAA-RE. The secondary structure of GbIAA-RE consisted of 20.53% alpha helix, 13.68% extended strand and 65.79% random coil. In total, 12 phosphorylation sites, 1 N-glycosylation site and 4 O-beta-GlcNAc attachment sites were predicted. Southern blot analysis indicated that Gbiaa-Re belonged to a low-copy gene family. Semi-quantitative PCR analysis indicated that the expression of Gbiaa-Re gene was inducible by IAA. Our studies suggested that Gbiaa-Re was a new member of plant AUX/IAA gene family.

Genomic cloning and characterization of a Pto-like gene SsPto-2 from Solanum surattense

A Pto-like gene (designated as SsPto-2) was isolated from Solanum surattense by using genomic walker technology which encoded a cytoplasmically localized serine-threonine protein kinase. Analysis of the 2365 bp segment revealed a gene including a 905 bp 5′ flanking region, a 924 bp open reading frame (ORF) and a 536 bp 3′ flanking region. The deduced amino acid sequence of the SsPto-2 gene shared high homology with other known Ptos. The deduced SsPto-2 protein contained no signal peptide with a calculated molecular weight of 34.61 kDa. The analysis of SsPto-2 promoter region and terminator region was also presented. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that SsPto-2 transcripts were up-regulated by defense-related factors such as gibberellic acid (GA3), salicylic acid (SA) and down-regulated by darkness. The cloning of the SsPto-2 gene will allow us to further study its potential role in disease resistance.