Cloning Of Stress-Related Homolog Genes And Their Responses To Abiotic Stresses In Upland Cotton

Drought, salinity, cold and freeze are the key environment factors affecting plant growth and development, and seriously limit the increase of yield and improvement of quality of crops. Under abiotic stress, plants could sense and transduce the signals caused by stress to transcription factors through a series of phosphorization responses. The functional genes related to stress tolerance are then initiated; a series of physio-chemical reactions are activated to alleviate the damage caused by abiotic stress, finally the stress tolerance of plants is enhanced.NAC transcription factor family genes are plant specific transcriptional regulators, and this gene family is one of the biggest gene families in plant. The NAC domain was originally characterized from consensus sequences from petunia NAM and from Arabidopsis ATAF1, ATAF2, and CUC2. NAC transcription factor genes play very important roles in stress response. The basic helix-loop-helix (bHLH) proteins are a group of functionally diverse transcription factors found in both plants and animals. Recently, hundreds of bHLH genes were identified in organisms whose genome sequences were available. Several plant bHLH-type proteins have been characterized, which act as development and growth and stress response. However, the biological roles of most members of this gene family in plant remain to be studied.Cotton (Gossypium spp.) is a major cash crop for both textiles and food. Upland cotton (Gossypium hirsutum L.) accounts for>95%of world cotton production. However, cotton-growing areas are subject to extreme drought, salinity, and temperature, each of which can impede cotton growth and production. Our research focused on identifying the NAC type and bHLH type ranscription factor genes involved in abiotic from Gossypium hirsutum L cv Jinmian 19(a widely used stresses-resistant cultivar in china for breeding) and providing new targets for producing tolerance-enhanced transgenics. It is of important significance to cotton stress-tolerant breeding and clarification of stress-tolerant molecular mechanism.Progresses of this research are following: We identified six abiotic stress related NAC-type transcription factors (GhNAC1-GhNAC6) for the first time in cotton. In A. thaliana, NAC-domain proteins contribute to abiotic stress signal transduction pathways. We extend these results to cotton. NAC domain protein sequences from A. thaliana were used as queries to screen the G. hirsutum L. EST database, and contigging the candidate ESTs. We obtain six putative full-length cDNA sequences encoding NAC-like proteins in cotton. We designed Nested-PCR prime pairs and PCR prime pairs of full-length ORF based on this six putative cDNA sequences, then six abiotic stress related NAC-type transcription factors (GhNAC1-GhNAC6) were isolated from leaves cDNA of upland cotton cv Jinmian 19. For six cDNA sequences of GhNAC1 to GhNAC6, each contained a single complete ORF encoding a predicted 276,299,298,346, 356 and 327-amino acids protein with a calculated molecular mass of 31.9,33.8,33.9,38.4, 40.2 and 37.0 KDa, respectively, as well as an isoelectric point of 5.83,5.74,6.54,8.87, 8.17 and 5.39, respectively. All six have conserved intron-exon structure, although they differ in intron length and chromosomal location. The predicted proteins, GhNAC1-GhNAC6, are similar in sequence, especially in the NAC domain. Semi-quantitative RT-PCR reveals that all GhN AC genes were highly expressed in leaves while they had little to no expression in stems, roots and 7-day-post anthesis fibers. Based on real-time quantitative RT-PCR, the genes were differentially regulated under drought, high salt, cold and/or ABA conditions. Based on a phylogenetic analysis, six GhNAC belong to four NAC protein subfamilies, ATAF, AtNAC3, NAP and NAM. Three of these subfamilies, ATAF, AtNAC3 and NAP, include a large number of stress-regulated NAC genes in other plants.We identified an abiotic stress related bHLH-type transcription factor (GhbHLHl) for the first time in cotton. The phytohormone ABA was known to play a vital role in modulating plant responses to drought stress, and AtMYC2 (a GhbHLH1 homolog) had been proved to act as transcriptional activator involved in the dehydration and ABA response pathway in Arabidopsis. Here, using AtMYC2 amino acid sequence as a querying probe to screen Gossypium hirsutum L. EST database and contigging the candidate ESTs, the putative cDNA sequence of cotton bHLH transcription factor was assembled. Further a pair of specific primers flanking the sequence of open reading frame of cotton bHLH transcription factor was designed based on this assembled cDNA sequence, then a key gene GhbHLHl, positively regulating ABA response in cotton (Gossypium spp.) was isolated from leaves cDNA of drought-treated upland cotton cv Jinmian 19 by RT-PCR approach. The full-length cDNA of GhbHLHl has an open reading frame of 2,025 bp, encoding a protein of 674 amino acids with a calculated molecular mass of 73.6 kDa and an isoelectric point of 5.46. Sequence alignment shows that GhbHLH1 contains a 60 amino acid long bHLH domain. We found that the deduced amino acid sequence of GhbHLHl showed high homology with bHLH domain proteins in Arabidopsis, especially with AtMYC2, which plays an important role in response to stress stimuli. Semi-quantitative RT-PCR reveals that GhbHLH1 is strongly expressed in 7-day-post anthesis fibers but weak in roots, stems and leaves. Based on real-time quantitative RT-PCR, the expression of GhbHLHl in leaves was transitorily induced by ABA and PEG treatments, although its transcripts were accumulated in various organs. However, its expression was not affected by salt and cold treatments. In addition, the further functional analysis of GhbHLH1 gene is in progress.