Cloning and characterization of a FLO/LFY ortholog in Gossypium hirsutum L.

KEY MESSAGE: GhLFY was cloned from G. hirsutum L. Its expression, subcellular localization, and function were analyzed, as well as the in vivo regulation of GhLFY by the MADS-box protein SOC1 (GhSOC1). ABSTRACT: Flowering is a very important phase during which plants produce the organs for sexual reproduction. The FLORICAULA/LEAFY (FLO/LFY) homologs play a major role in the initiation of flowering. To understand the mechanism of the transition from the vegetative to reproductive phases in Upland cotton (Gossypium hirsutum L.), we isolated a candidate LFY gene from G. hirsutum L. (GhLFY) that showed a high degree of similarity to other plant homologs of FLO/LFY. qPCR analysis showed that GhLFY was highly expressed in the shoot apex, with substantial upregulation at the third true leaf expansion stage during floral bud differentiation. Subcellular localization studies revealed GhLFY localization in the nucleus. Ectopic expression of the GhLFY coding region in Arabidopsis resulted in early flowering. The expression of the GhLFY coding region under the control of the 35S promoter complemented the lfy-5 mutation in transgenic Arabidopsis lfy-5 mutant plants. Furthermore, a chromatin immunoprecipitation assay revealed that GhLFY may function downstream of GhSOC1 during the initiation of flowering in G. hirsutum L. GhLFY was likely to be regulated by GhSOC1, which binds to the LFY promoter in Arabidopsis. These results suggest that GhLFY is a FLO/LFY ortholog that may be involved in controlling flowering time and floral development.

[Development and appraisement of functional molecular marker: intron sequence amplified polymorphism (ISAP)].

Molecular markers are playing an increasingly important role in map construction, QTL analysis, gene mapping and marker-assisted selection. Researchers hope the target gene and locus are as close as possible, one locus can present one gene, or linked with some important trait, then, individuals with useful trait can be selected through molecular markers selecting, and it's the functional molecular marker. PCR-based molecular markers such as RAPD, SSR, AFLP amplified non-coding regions, or the whole genome randomly, the locus is far away from the gene of targeted trait, this limit the ap-plication of these molecular markers. This study established a kind of functional molecular markers based on intron of gene sequence, trying to link loci with gene sequence to achieve the purpose of its function. It used the conservative consistent sequence of intron splicing sites as its core sequence of amplification. ISAP is a PCR-based marker system, it has two kinds of primers: forward primer and reverse primer, both primers are 18 bases. Any of the primers can be used to construct a primer combination with the other kind of primers. Seventeen primers, 9 forward and 8 reverse, were used to construct 72 primer combinations, 67 of them showed polymorphism in a G. hirsutum cv. CCRI36 x G. barbadense cv. H7124 F2 population and a total of 212 loci were obtained. Together with 164 SRAP loci, these 212 loci were used to construct a genetic linkage map. ISAP markers distributed evenly in the entire linkage group, part of the region had a high saturation, might be the coding sequence-rich region. Sequencing results of 20 fragments showed that 85% of the sequences announced homology with published EST sequence stored in the NCBI which indicated that they were amplified adjacent to expressed sequences. These results showed that ISAP marker system was simple, efficient, reliable, and had a relatively high polymorphism, furthermore, it directly targeted gene sequence, was a functional molecular marker system. ISAP was also used to amplify other plants and good results were achieved.

Cloning and expression of the chloroplast copper/zinc-superoxide dismutase gene in upland cotton (Gossypium hirsutum L.).

A full-length 1,043-base-pair cDNA clone encoding a chloroplast copper/zinc superoxide dismutase (Cu/Zn-SOD) of upland cotton was first isolated by rapid amplification of cDNA ends (RACE) from the leaves of the cotton (Gossypium hirsutum L.) variety 'CRI36'. Nucleotide sequence analysis of the clone revealed that it contained the complete coding sequence of the mature SOD isozyme subunit, along with a 60-amino acid transit peptide at N-terminal. The amino acid sequence predicted from the full-length clone showed 66%-74% homology with the amino acid sequences of Cu/Zn-SOD from several other plants. This gene was found to be expressed in the leaves and stems, but not in roots, flowers, and hypocotyls, indicating that the gene was expressed only in green tissues. Also, its expression was found to be most active at seedling stage and declined gradually in later development stages. Expression of this cotton Cu/Zn-SOD gene by using the pET-21a (+) expression vector in E. coli BL21 (DE3) led to the production of a novel 29 kD polypeptide with SOD enzyme activity, confirming that the cloned cotton Cu/Zn-SOD cDNA was indeed encoding a functioning SOD enzyme.

In vitro cytotoxicity and in vivo acute and chronic toxicity of Xanthii Fructus and its processed product.

Xanthii Fructus (XF), the fruit of Xanthium sibiricum Patr., was used in the treatment of rhinitis and related nasal disease. Adverse effects of Xanthii Fructus are frequently reported these years. In the paper, in vitro renal cytotoxicity and in vivo acute and chronic toxicity researches of Xanthii Fructus (XF) and its processed product (processed Xanthii Fructus (PXF)) were carried out. Water extraction of XF displayed no cell membrane damage effects even in the highest concentration (100 µg/mL); however, it might affect the function of renal cell mitochondria. Acute toxicities were observed only in high and middle dosage groups. Fortunately, the single dose administration of XF or PXF was safe even at the highest daily dosage. Twelve-week chronic toxicity assays were performed in SD rats with low, middle, and high dosage. Notable changes in body weight and blood cell and BUN and Scr changes sporadically occurred in middle and high groups after the 9th week. Serum HA and HPCIII values were sustained increasing from the 4th week to the 8th week in Group V male rats, which indicated that the renal fibrosis risks still existed although no fibrosis was found in the pathological examination of the liver and kidney.