Study Of Allelic Variation And Functional Analysis Of Transcription Factor AREB1 Responding To Drought Stress In Arachis Species

Our country is the largest country in the world in production and consumption of peanuts. Arid, semi-arid hilly region is the main growing area of peanut. Now, the drought has affected peanut yield and quality, and has been the primary limiting factor of peanut production. AREB1 is one of most widely studied transcription factors, which is a drought-related transcription factors. In this study, we chose 16 cultivars and 28 copies of germplasm contained 21 kinds of peanut as materials to clone AREB1 genes and analysis allelic variation. 11 peanut varieties with different drought res istance were used to research the relative expression of AhAREB1 and drought coefficient under PEG osmotic stress. Meanwhile, We constructed an expression vector of AhAREB1 which has obtained transgenic seedlings. The major results are abstracted as follows:1. Allelic variation analysis of AREB1 in peanut cultivarsTwo sequences of AREB1-ORF were cloned in the same cultivar, which were named AhAREB1a-OFR, AhAREB1b-OFR.The length of the two sequences were 1767 bp, 1775 bp, containing 23 SNP. Compared with AhAREB1a-OFR, AhAREB1b-OFR have nucleotide deletion mutations in intron regions. Both of them encode 445 amino acids, which have 7 differences. We tested 16 species, 15 varieties have no difference between alleles. There are differences in 1761 bp of AhAREB1a-OFR and 1769 bp of AhAREB1b-OFR in Yuanza 9102 and other varieties, respectively.Two sequences of AREB1-5,UTR were cloned in Yuhua 15, which were named AhAREB1a-5,UTR, AhAREB1b-5,UTR. There is a SNP in these sequences, causing differences in a cis-acting elements. AhAREB1a-5,UTR was cloned in Fuhua 12、Yuanza 9102 and 02P175, AhAREB1b-5,UTR was cloned in other 12 cultivars.Two sequences of promoter of AREB1 were cloned in Yuhua 15, which were named AhAREB1a-P, AhAREB1b-P. The length of the two sequences were 919bp、900bp, containing 16 SNP. Compared with AhAREB1a-OFR, AhAREB1b-OFR have 3 deletion variants, 2 insertion mutation, response to the drought in AhAREB1a-P is more than AhAREB1b-P. AhAREB1a-5,UTR was cloned in Fuhua 12 、Yuanza 9102 and 02P175, There are some sequences difference in allele; AhAREB1b-P was cloned in other 12 cultivars, No difference in allele.2. Genetic diversity of AREB1 in wild speciesThere are 11 sequences of AREB1-ORF in 28 wild species, Aw1AREB1-ORF and AhAREB1a-ORF have same sequence, also Aw9AREB1-ORF and AhAREB1b-ORF. Compared wild species with cultivars, there are 68 differences site, where 63 is the SNP. There have SNP in exon region, insertion or deletion fragments in intron. AREB1 in wilds encodes 6 type protein, containing 14 different, mainly in the non-conserved region of bZIP conserved N-terminal region. 5 sequences of AREB1-5,UTR were cloned, Aw5AREB1-P have no TCA elements. There are 18 AREB1-P in wild species, Aw1AREB1-P and AhAREB1a-P have same sequence. There are 63 differences site,where 47 is the SNP. The same sequence of AhAREB1b-P had not cloned in wild species.3. Analysis of the expression of AhAREB1 of different varieties in PEG osmotic stress11 peanut varieties with different drought resistance were used for PEG osmotic stress test. In this test, the relative expression of AhAREB1 shows that high-resistance cultivars presented a higher and quickly relative expression of AhAREB1. AhAREB1 expression level of transcription had a significantly positively correlated with drought resistance. It indicated that the levels of AhAREB1 expression is a key factor to drought resistance.4. Construction of AREB1 expression vector and genetic transformationWe built a plant overexpression vector p GBAREB1 carrying AhAREB1. The pGBAREB1 plasmids were transformed into Agrobacterium tumefaciens LBA4404. Then We obtained transgenic plants by genetic transformation.