Changes Of Hormone And Cloning Of Senescence-related Gene In Short Season Cotton

The seeding of cotton cultivars Liaomian No 9 was derooted. Then in darkness they were cultured in vitro by addition of 6-BA, ABA, GSH, H2O2, CaCL2, A23187 and A23187 and CaCL2 respectively. The endogenous hormones, SOD activity and malondialdehye (MDA) contents in leave of derooted cotton seeding incubated with different test solutions, were investigated during dark -induced senescence. Treatment such as 6-BA, GSH and calcium ions, which retarded dark-induced leaf senescence, decreased MDA level in leaf of derooted cotton seeding. ABA and H2O2, which accelerated dark-induced leaf senescence, increased MDA level in leaf of derooted cotton seeding. The current results suggested that endogenous hormone and free radical scavengers, such as GSH and SOD enzyme, might be involved in regulating dark-induced leaf senescence. Evidence was presented to show that the dark-induced leaf senescence of derooted cotton seeding may first attributed to decreasing cytokine contents, then blocking the entrance of calcium ions into the cytosol, and then reducing SOD activity and increasing the level lipid peroxidation, consequently promoting protein loss and chlorophyll degradation.Changes of endogenous hormone were investigated in the stem leaves of 3 short season cotton varieties with different senescent type on development processes. The results suggested that early mature short season cotton was extensively coincident with the peak content of IAA and Z+ZR in stem leaves on early development processes. The earlier the peak of IAA and Z+ZR content appeared the earlier the yield apparatus developed. The premature senescent short season cotton varieties were associated with the content peak of ABA, ethylene and IAA, counteracted with the peak of iP+LPA, in stem leaves on late development processes. After boll opening, the premature senescent variety showed low contents of iP+iPA and high contents of ABA and IAA in stem leaves. On the contrary, the premature senescent resistance varieties showed high contents of iP+iPA and low contents of ABA and IAA in stem leaves.A cysteine proteinase gene from cotton (Gossypium hirsutuni) was isolated by rapid amplification of cDNA ends using polymerase chain reaction (RACE-PCR), a set of consensus oligonucleotide primers was designed to anneal to the conserved coding region of cysteine proteinase. The PCR products were cloned and sequenced. The cDNA, which designated Ghcysp gene, contained 1368 bp terminating in a poly (A)+ trail, and included a putative 5 (98 bp) and a 3 (235 bp) non-coding region. A signal sequence AATAAA was located 76 bp upstream from poly (A) tail. The opening reading frame (ORF) could encode polypeptide 344 amino acids with the predicted molecular mass of 37.88 KD and theoretical pI of 4.80. DNA gel blot analysis using Ghcysp gene as a probe detected two bands in the tetraploid AD genome of cotton, This finding suggested that the two bands seen in the G. hirsutum cotton are homeologous genes, representing one Ghcysp gene originating from the A and D genome progenitors. The sequence of Ghcysp was submitted to GenBank, with an accession number ofAY604196. A comparison of the deduced amino acid sequence with the sequence in the Swiss-PROT database has shown considerable sequence similarity to a novel family of cysteine proteinase. This putative cotton Ghcysp protein showed from 67% to 82% identity to the other plants. All of them shared catalytic triad of residues, which were highly conserved in three regions. Hydropaths analysis of the amino acid sequence showed that the Ghcysp was a potential membrane protein, which had a transmemberane helix between resides 6-21.The expression of Ghcysp gene was determined using northern blot analysis. The Ghcysp mRNA levels were high in development senescent leaf but below the limit of detection in root, hypocotyle, flower, 6 days post anthesis (DPA ) ovule, and young leaf. The transcript level of Ghcysp gene in the senescent leaf decline rapidly and continuously with the cytokinin and sucrose treatment. On the contrary, the transcr...