Construction And Transformation Of The Vectors To Express Genes Which May Enhance The Resistance Of Pericarp And Seed Coat To Aspergillus In Peanut

Peanut is an important economic crops and oil crops of China.Peanut aflatoxin contamination caused by Aspergillus flavus infection affects seriously peanut production,processing and trade.A.flavus can infect peanut the whole growth periods including vegetative and fruiting stages and during drying,storage and processing of seeds.Preharvest stage is the key stage of A.flavus infection.The characteristics of pericarp and testa have been known to play important barrier to A.flavus invasion.By changing structure and composition of pericarp and testa may develop new varieties with high and stable resistance to A.flavus,and solve aflotoxin contamination problem at last.In the paper two transcription factors,MYB61 and TTG2,were isolated from an available Arabidopsis library and two genes,COMT and PTI,were cut from pMD18-T-COMT plasmid and pMD18-T-PTI plasmid(constructed in our laboratory).PTI was evaluated for its resistance to A.flavus via in vitro inhibition to growth of A.flavus with prokaryotic expression product.The results are as follows:A 240bps band was got by PCR using primers designed according to gene sequence at both ends and it showed 100%identity with the original one reported on the interact.The gene was inserted into the expression vector pGEX4T-1 which was then transferred into BL21,a prokaryotic expression host.After let it express in BL21 in certain condition,a 10 kDa PTI specific expression protein product was abtained on the PAGE gel.The coar extraction and purification product of expression protein appeared inhibiting the growth and sporulation of A.flavus after BL21 was cultured and induced to express the protein of interest for several hours.2.Constructing plant expression vector with MYB61,TTG2 and COMT:According to known gene sequences,primer pairs for MYB61,TTG2 and COMT were designed, and a specific band of 1100 bp for MYB61 gene,a 1050bps band for TTGs and a 772 band for COMT have been amplified by PCR.The bands were subjected to sequencing and were identified by BLAST on Genbank showing an identity of 95% to the original one for MYB61 gene,98%for TTG2 gene and 99%for COMT gene. Then these fragments were constructed on an vector pCS1300 with available 35S promoter and Nos terminal,inserting between the 35S and Tnos,respectively.The derived plant expression vectors were named pCS1300-MYB61,pCS1300-TTG2 and pCS1300-COMT and evaluated by sequencing.The above vectors were transferred into Agrobacterium tumefaciens strain EHA105.Transformation was done on the peanut and Arabidopsis both. Transformation of peanut mediated by Agobacterium tumefaciens is on the way.And we have got transformant seeds from Arabidopsis thaliana by the method of inflorescence dipping,and the seeds are assayed next.Based on the study,three plant expression vectors with MYB61,TTG2,COMT were got and the resistance of PTI to A.flavus was evaluated.The work laid the foundation for anti-Aspergillus contamination study and supplied with experience for peanut transformation.