Gene Cloning And Characterization Of B3 And B1 Subgroup Transcription Factors Of ERF Family From Gossypium Barbadense L

Cotton is an important cash crop, however, a variety of diseases, especially Verticillium wilt have exerted serious impact on cotton production. The development of genetic engineering technique provides a new way for cotton disease resistance breeding. Transcription factors of ERF (Ethylene responsive factor) family play an important role in resistance and/or tolerance to biotic and/or abiotic stresses. All members of ERF family contain a highly conservative DNA binding domain, which endow the transcription factors with the capacity to recognize and combine with the GCC-box and nonGCC-box. Via these interactions, they are able to regulate the expression of downstream genes such as PRs (pathogenesis-related protein genes) which contain GCC-box in their promotors, thereby changing the plant traits of disease resistance and stress tolerance. ERF family transcription factors can be divided furhter into many different groups or subgroups. The transcription factors of B3 subgroup are able to serve as activators and up-regulate the transcription activities of GCC-box-promoted genes, and in turn enhance the plant resistance to diseases. On the contrary, B1 subgroup members can inhibit the expression of GCC-box-promoted genes, for preventing overactivation of defense genes. To clone genes of the two subgroups and research their function have significance in genetic improvement of the disease resistance of crops.For the purpose to isolate the B3 and B1 subgroup genes related to resistance to verticillium wilt, in this study, we firstly cloned in silico 29 genes including 20 B3 and 9 B1 members utilizing the EST (Expression sequence tag) data of Gossypium hirsutum. Then, according to the homology between Gossypium barbadense and Gossypium hirsutum, and the conservative characteristics of ERF family transcription factors, we cloned 29 genes, containing complete coding region homologous to upland cotton, by PCR using cDNA extracted from Sea Island cotton seedlings infected with verticillium dahliae as templates, and 27 of the 29 genes are novel. RT-qPCR(Real-time Fluorescence Quantitative Polymerase Chain Reaction)analysis showed that, compared with the control without inoculation, after treated with Verticillium dahliae, the expression of GbB3002,GbB3003,GbB3009,GbB3016-20,GbB1006 and GbB1007 genes in roots tissue were up-regulated obiviously, more than 7 times; however, the GbB3012 and GbB3015 genes were down-regulated at 2-3 fold, and the GbB3010 didn't basically change, and the transcripts accumulation of other genes had a slight increase less than 5 fold. Then, we cloned the full length cDNAs of the four genes of GbEREB3(GbB1007),GbEREB4(GbB1006),GbEREB5(GbB3009) and GbEREB6(GbB3015) by RACE (Rapid amplification of cDNA end) technique, and submmitted them to NCBI GenBank data and received the accession numbers : JN003806, JN003807, JN003808 and JN003809.On the basis of bioinformatics analysis, constrution of over-expression vectors inserted respectively with the four genes fragaments and transformation into onion epidermis cell were performed for transient expression analysis, and the results showed that they were all nucleus-localized proteins, and meanwhile, the results of EMSA (Electrophoretic mobility shift assay) proved that they were all able to combined with GCC-box. Semi-quantitative PCR and RT-qPCR analyses in transgenic tobacco showed that GbEREB3/4/5/6 had the capacity to activate or inhibit the expression of PRs. Furthermore, the results of RT-qPCR analysis also showed that the expression levels of both chitinase (CHI)andβ-1,3-glucanase (BGL)genes decreased in transgenic cotton plants, in which GbEREB2 (GbB1004) over-expressed. In addition, experiments demonstrated that the leaf water loss rate of transgenic cotton plants with GbEREB2 increased compared with non-transgenic cotton lines. These data implied that (Gb)EREB2, a member of B1subgroup genes, involved in the resistance to diseases and water metabolism.This study shows that these genes are involved in the precesses of cotton defense responses to attacks from Verticillium dahliae, and may regulate the resistance of cotton to verticillum wilt. The identification and analysis of cotton resistance against Verticillium dahliae are in progress. And this study provides the basis for screening of genes relative to cotton resistance to verticillium wilt.