The Study On AhbHLH1 Involved In Regulating The FAD2 Gene Expression In Peanut (Arachis Hypogaea L.) Seed

Basic helix-loop-helix proteins (bHLHs) constitute one of the largest families of plant transcription factors. bHLH transcription factors play crucial roles in the plant transcriptional regulation of growth and developmental processes, as well as in the biological processes of hormone signaling and stress responses (drought, salt, cold).A homologous gene of SebHLH from peanut, designated as AhbHLHl, was isolated. By yeast one-hybrid system, we found that the AhbHLH 1 may interact with the AhFAD2 promoter region. Through the analysis of the regulatory elements in AHFAD2 promoter, four E-BOX elements existed in the -200bp region upstream from start codon ATG, and which located at-155bp site,-90bp-78bp site (including two consecutive E-BOX), and -42bp site, respectively. The recombinant AhbHLH1 protein containing His-tag was purified through Ni-chelating affinity chromatography from soluble E. Coli extracts. Three double-stranded DNA probes with biotin-label at 5’ terminal, which correspond to different position of the E-BOX elements, were synthesized. The binding reaction between AhbHLH1 and probes was determined by electrophoretic mobility shift assay (EMSA). In the meantime, in order to identify whether AhbHLH1 directly interacts with E-BOX in AhFAD2 promoter in vivo, The reporter vectors and their relative effector vectors for transient expression assay were constructed. The plant expression vectors of overexpressing the AhbHLH1 gene and inhibiting its expression by RNAi strategy were constructed for further studying of AhbHLH1 functions. Both of the resultant plasmids were introduced into peanut by the Agrobacterium-mediated transformation method respectively.The main results include the following three aspects:1. The binding reaction between AhbHLH1 protein and the AHFAD2 promoter by EMSA assay. The procaryotic expression vectorpLM1-AhbHLH1 was constructed and the AhbHLH1 protein tagged with a 6xHis residuses at N-terminal, was purified by Ni- chelating affinity chromatography. The DNA probes P1, P2 and P3 with 5’biotin-labelled, which respectively are about 28 bp containing two continuous E-BOX (located at-90bp--78bp) and its flanking sequence, about 19bp with one E-BOX (located at-155bp) and its flanking sequence, and approximately 19bp including an E-BOX (located at -42bp) and its flanking sequence, were synthetized by Sangon Biotech (Shanghai) Co., Ltd. The combination status between AhbHLH1 and above three probes in vitro was determined by EMSA. The results showed that AhbHLH1 interacts with P1 specifically and there are no interaction with P2 and P3. It is suggested that AhbHLH1 protein mediates transactivation of the AhFAD2 gene through binding to the E-box elements located at -90bp--78bp from ATG.2. The construction of the reporter vectors and the effector vectors for GUS transient expression assay. The original pBI121 vector carries a complete GUS expression framework driven by CaMV 35S promoter. The CaMV 35S promoter was replaced by a 100bp minimal 35S promoter in the basic reporter vector RO. Based on the position of E-BOX elements in the AhFAD2 promoter, three fragments I1(-167bp-24bp), I2(-148bp-35bp), and I3(-167bp-55bp), was cloned and respectively inserted into the front of minimal 35S promoter using the in-fusion PCR cloning system of Clontech Laboratories, Inc., resulting that the corresponding reporter vectors R1,R2, and R3 were constructed.3. The construction of RNAi vector for AhbHLH1 and peanut genetic transformation. A 400bp region downstream from start codon was selected as target interference fragment after analyzing the siRNA sites of AhbHLH1. Both of the overexpression vector and RNAi vector were introduced into peanut successfully. The transgenic plants were confirmed by kanamycin selection and PCR verification. Three overexpressing transgenic plants and three RNAi transgenic plants have been obtained.In our following research, the interaction between AhbHLH1 and AhFAD2 promoter will be further confirmed by transient expression assay and ChIP-Seq. The function of AhbHLH1 in regulation of AhFAD2 involved in storage lipid synthesis will be investigated by analyzing AhFAD2 expression and the composition of storage lipid in transgenic plants.