Cloning And Expression Analysis Of A Mitogen-activated Protein Kinase Gene In Peanut

With the development and progress of agriculture, peanut breeding have attracted more and more attention. Plants often encountered in the growth of external damage,although not by moving to avoid damage, but it can quickly sensory signals and take the initiative to adapt to the environment Existing research shows that the MAPK in plant resistance to adversity stress response pathway plays an important role. This study Huayu 19 as experimental materials and arabidopsis thaliana AtMAPKl（GenBank: NP₁81907.1） protein sequences as the reference sequence,and we use the homologous comparison method and RT-PCR and RACE-PCR from the peanut clone to a new MAPK genes. We are on the sequence alignment,expression characteristics analysis.Specific results are as follows:（1） Using peanut research center of peanut cDNA library building and using the AtMAPK 1 as The reference sequence,and via a large number of sequences of expressed sequence tag （EST） and RACE clone, so a new MAPK gene is isolated from peanut. The sequence of MAPK cDNA is 1492 bp, and ORF is 1191 bp.The number of deduced amino acid is 397.（2） Alignment results of the amino acid sequences of MAPK gene in different species indicated that the gene contains typical Ser/Thr conserved motifs,and its homology with arabidopsis,soybean,and nicotiana benthamiana is 86%,94%和90%, respectively.We found that its homologous and evolutionary relationship with arabidopsis MAPK6 recently, so it belongs to the group A classification of MAPK family. It is named AhMAPK6a and put it on the GenBank registration, registration number for KP329549.（3） Fluorescence quantitative PCR （qPCR） results for tissue expression analysis indicated that the expression of AhMAPK6a is the highest in peanut leaves, and next in the roots. The expression level is very low in the stem, flower, cotyledon and hypocotyls.（4） Fluorescence quantitative PCR （qPCR） results for AhMAPK6a under ABA,PEG,JA,SA induced expression has a specific analysis. We find that AhMAPK6a volume obvious changes in gene expression. In ABA treated leaves and roots, the expression of AhMAPK6a decreases and then increases,and there is no significant change in the expression of the gene after several hours. In 15%PEG treated leaves, the expression of gene obviously decreases,but the expression in root decreases and then increases, and there is no change in gene expression. The expression of this gene significantly increases in JA treated leaves. In the SA treated leaves, the expression of the gene is negative.（5） We will construct the justice gene expression vector pCAMBIA1301-AhMAPK6a,transformed by Agrobacterium EHA105,with the transfer device to absorb the agrobacterium,which contains the target gene, and drip to the arabidopsis thaliana is not open buds. We will after the infection of arabidopsis thaliana seed sterilization is arranged in the containing 20mg/L card kanamycin of 1/2MS medium plate, placed in the dark at 4 DEG C for 3-4 days, training room dark cultured for a week or so, long and healthy seedling of arabidopsis thaliana,and 5 transgenic plants are obtained by PCR.This article studies the molecular mechanism and peanut resilience resilience genetic engineering to provide the scientific basis for obtaining peanut stress-resistant varieties provide candidate genes.