ERF Transcription Factors From Gossypium Barbadense L And Their Relationship With Cotton Drought Resistance And Salt Tolerance

Cotton is one of the important fiber cash crops, its planting area is0.53million hm²in China. Butgrowth of cotton has been affected by Verticillium wilt or other stresses such as drought and highsalinity. Transgenic technology has been an effective way to cultivate different cotton that is moreresistant. Ethylene Responsive Factor family （ERF） can bind specifically with GCC-box or nonGCC-box of downstream genes promoter such as pathogenesis related genes, to regulate theirexpression and make responses to diseases and varied environments. Studies have shownover-expression of these genes in wild type can ensure plants more adapt to different stresses.In this research, GbEREB4/5/6were three ERF genes amplified from cDNA of disease resistantvariety of Gossypium barbadense7124, they are induced by Verticillium wilt. Based on its earlyresearch, We have cloned their promoters, made some bioinformatics analysis and some experimentanalysis, and researched their expression after drought and salt. Meanwhile We constructedover-expression vector and transformated Arabidopsis, results are as follows:1. Analysis of GbEREB4expressionDisease-free seedling of Gb7124and CCRI24were obtained by water planting that was treatedwith PEG （17%）. Results show that after drought treatment, GbEREB4gene can response to the abioticstress and present expression pattern in short time; after3h s same treatment, expression of this gene inGossypium barbdense were higher than that in CCRI24.2. Promoter cloning of GbEREB5/6and analysis of cis-elementsWe obtained promoter of each gene from Gb7124and their homologous genes from CCRI24byTail-PCR, named Pro7124GbEREB6and ProCCRI24EREB6. Besides, We analysed cis-elements ofeach promoter sequence; found stress resistant cis-elements and different elements between each gene intwo varieties.3. Transient expression activity of promoters EREB6Plant expression vectors were constructed based on pCBI vector and then activity of GUS drivenby Pro7124EREB6and ProCCRI24EREB6were detected. Results by particle bombardment show theyhave some activity in common MS medium, while they are cultivated by MS medium containing10%PEG or NaCl, promoters activity will be higher, ProCCRI24EREB6tend to be more obvious.4. Over-expression of GbEREB6in Arabidopsis and resistance function analysisOver-expression vector were constructed, and floral-dip method were used to Arabidopsistransformation. After Kanamycin screening, GUS staining and PCR, We obtained7positive plants,5ofthem were homozygous lines, name of which are1/2a/2b/3/4. Abiotic stresses such as350mMNaCl,17%PEG and natural drought were used in this research. Results are as follows: after48h NaCl andPEG treatment, contents of small molecules like proline in transgenic plants are higher than that in wildtype Arabidopsis; after PEG48h, contents of MDA （malondialdehyde） and soluble sugar in transgenicplants are higher than in wild type; through DAB staining after salt stress of4h, comparison with wildtype, root of seedling has a lower accumulation; on MS medium that containing100mmol L^-1NaCl,elongation of taproot and growth of lateral root were suppressed, while suppression degree of transgenic plants is lighter than that of wild type; After natural drought of3weeks, contrast to wild type,transgenic lines have a higher chlorophyll. In all, a preliminary conclusion was over-expression ofGbEREB6gene can make transgenic gene better responsive to drought and salt.