Identification Of The Chilling Resistance Of Cotton And The Expression Of Cold Resistance Related Genes

1. Identification of the chilling resistance of upland cotton from germination stage to seedling stageSpecific identification ways of cold resistance suitable for different growth stages should be established correspondingly. The relative cotyledons unfolding rate under normal condition growth for7 d after 0℃ treatment for 4d can serve as a cold resistance identification index for germination stage.The relative cotyledons unfolding rate under normal condition growth for 7d after 4℃ treatment for 5d can be used as chilling resistance identification index of the budding stage. Chilling resistance index under normal condition growth for 7d after 0℃ treatment for 24 h can evaluate the chilling resistance of cotton at cotyledon stage. Cotton has different cold resistance ability during different growth periods. At the same time, we screened 7 cotton materials, 4 cotton materials, 7 cotton materials with high chilling tolerance respectively at germination period, budding stage and the cotyledon stage. Based on the variation trends of enzyme activity of three enzymes, we speculated the cold-resistance of cotton at budding stage has relation with antioxidant enzymes activity.2. The effect of low temperature on the growth of upland cotton seedling and the expression of chilling resistance related genesThe treatment of 4℃ 24 h low temperature produces greater growth inhibition on chilling sensitive cotton materials than chilling tolerant materials and root growth inhibition is greater than the part on the ground. The measurement of the cell membrane permeability indicated that leaf cell membrane was more sensitive to low temperature stress than stem and root. Leaf cell membrane of chilling resistant cotton materials is more stable than chilling sensitive ones. The root cell membrane of 4 different kinds of cotton materials can stay relatively stable under low temperature stress. The activities of POD, SOD,and CAT decreased in the roots of two cotton materials after low temperature stress. The activities of 3kinds of antioxidant enzymes and soluble protein content in the roots of Yu 2067 decreased and the 3kinds of antioxidant enzyme activities of cold resistance materials Henan 2067 declined less than cold sensitive ones. There were more up-regulated genes than down-regulated genes of 5 chilling tolerance related genes in the leaves of Yu2067. On the contrary, the amounts of down-regulated genes were higher than up-regulated ones in roots. The result of gene expression is consistent with root growth inhibition.3. Whole genome transcription analysis of Upland Cotton under low temperature stressThe transcripts were sequenced using the Illumina HiSeqTM 2000 sequencing platforms using Yu2067 plants at three leaf stage subjected to 4℃ low temperature stress about 24 h and their corresponding controls(28℃). A total of 2487 unigenes were found to be differentially expressed under low temperature stress(4℃) versus their corresponding controls(28℃). About 54.36%(1352) of these genes were down-regulated, at the same time, about 45.64%(1135)were up-regulated under chillingstress. The extremely up-regulated unigenes were identified as candidate genes including ethylene-responsive transcription factor ERF109-like, momilactone A synthase-like, 15 kD oleosin-like protein, delta-8 sphingolipid desaturase, probable disease resistance protein At1g12280-like, nbs-lrr resistance protein, multidrug resistance pump, auxin-responsive protein and some predicted proteins.Then we mapped these cold-induced genes to the KEGG database indicated that pathways significantly enriched involved in “plant hormone signal transduce”, “circadian rhthm-plant", “plant-pathogen interaction”, “fatty acid elongation”et al.. Together, a total of 31 cold-inducible transcription factors were identified, including subsets of AP2/ERF, Zinc figure, DREB1, NAC, and MYB family members.4. Whole genome analysis of CIPK gene family in cotton and theirs response to low temperature stressIn the present research, identification of the CIPK genes were performed in the whole genomes of diploid cottons(Gossypium raimondii and Gossypium arboreum L.) and specific bioinformatics analysis of these members was conducted to investigate the phylogenetic relationship. The initial flowering stage transcriptome and abiotic expression analysis of the CIPK genes were also analyzed in different tissues within different periods. The results demonstrated that the CIPK gene family is unevenly distributed whether in two diploid cotton genomes(D genome and A genome).Fortty-one and thirty-nine CIPK genes were identified in the D genome in the A genome respectively which can be classified into 2 types- rich introns(>3) and few introns(<3) according to the structures of these genes. The abiotic stress expression analysis of CIPK genes in different tissues at trefoil stage demonstrated that stresses(drought, salt and low temperature) can induce the expression of CIPK genes, but the response to different stresses is different. Differential gene expression analysis of CIPK genes under stresses shows that the number of CIPK genes up-regulated in the A genome was higher than that of the D genome indicating that the CIPK genes in the A genome play an important role in response to different stresses.5. Isolation and characterization of GhDHN1 gene from cotton and its expresstion in response to low temperature stress.We cloned a gene named Gh DHN1 from the upland cotton variety. The cDNA of the dehydrin gene is 726 bp, and encodes 211 amino acids Gh DHN1 protein was sorted into K2 S subfamily of dehydrins. The analysis of the transient expression in onion epidermal cells showed that GhDHN1 was mainly localized near the cell plasma membrane. Real time fluorescence quantitative analysis showed that Gh DHN1 was up-regulated after low temperature stress. There had significant different expression of GhDHN1 between the cold resistance materials and cold sensitive materials, and the expression of GhDHN1 was positively correlated with cotton cold resistance. The expression of GhDHN1 can be thusemployed as a marker of cotton chilling resistance. At the same time Gh DHN1 gene can serve as an important candidate gene in cotton cultivation of resistant materials especially cold resistance materials.