Expressive Characterization of Transcription Factors in Cotton Fibre Initiation and Cloning and Characterization of Three Transcription Factor Genes in Gossypium Hirsutum L.
|School||Nanjing Agricultural College|
|Keywords||cotton fiber development transcription factor qPCR abiotic stress subcellular localization|
Cotton is one of the world’s most important economic crops. It is an important raw material source in textile industry. It is important to elucidate fiber development process and major factors related to fiber quality by molecular biology. A variety of factors play an important role in plant growth and development, one of the main factors is transcription factor (TF)My laboratory has used+1DPA, ODPA,-1 DPA ovule from Xinxiangxiaojilintless-fuzzless mutants (XinWX) and Xinxiangxiaojilinted-fuzzless mutants (XinFLM) as the materials to acquire a set of dominant expression genes in fiber initial development phase based on gene chip technology and biological informatics analysis. In this study, we chose 104 transcription factor genes from the former ones which relate to fiber initial development, and validate their expression by qPCR in fiber initiation phase. We design diffierential primers according to genes’sequence information and 81 different genes have amplification products. Finally there are 56 different genes accord with the gene chip, and the accordant rate consistent with the chip is 69.14%.The results showed that there are some false positive results in gene chip. But most of the differential expression genes got from the gene chip are reliable, it establish a well-knit basis for the further study of cotton fiber developmental functional genomics.The reported gene sequences of the transcription factors are used as probes to scan the EST database,3 complete ORF contigs are pieced together. Differential primers are designed to isolate 3 transcription factor genes from upland cotton, including two zinc finger protein genes and one AP2/EREBP gene. Each gene contains a single complete ORF, they are 663 bp,744 bp and 561 bp. The proteins encoded by ORF contain 220,247 and 186 amino acid, with an isoelectric point and a calculated molecular mass 9.12/25.1 kD, 8.09/26.3 kD,6.39/21.0 kD, respectively. Four lintless-fuzzless mutants:Xinxiangxiaojilintless-fuzzless mutant (XinWX), XZ142WXWX, MD17, SL1-7-1; Two linted-fuzzless mutants: Xinxiangxiaojilinted-fuzzless mutants (XinFLM), N1N1; and a normal material (TM-1) were selected for the early expression research of transcription factors at the stage of initiation and elongation of cotton fiber development to clarify the genes’function. We found these three genes have high expression in the fiber initiation and elongation, so these three genes may have relationship with fiber initial development and fiber elongation. these three genes express differently in nomal materials and mutants, GhZFP4 and GhZFP5 expresse lower in XinFLM than in XinWX, and expresses lower in nomal material TM-1 than in mutants MD17, SL1-7-1, XZ142WXWX and N1N1, may have negative effect on fiber development. GhAP2/EREBP4 expresses higher in XinFLM than in XinWX, may have positive effect on fiber development.Real-time Fluorescence Q-PCR was used to detect the expression of the three genes under abiotic stresses (including drought, salt, ABA, cold, H2O2). Compare with control, GhZFP4 and GhAP2/EREBP4 were induced by salt, ABA, cold and H2O2 strongly, while GhZFP5 was dominantly induced by drought, salt, ABA, cold and H2O2. These results indicate that the three proteins play a role in stress response in cotton. Further function studies of these ZFPs are under way.Subcellular localization research showed:The three genes all express in cell nucleus.