Studies On Auxin Transportation And Distribution And Expression Of Related Genes In Pegging Stage Of Peanut

Peanut (Arachis hypogaea L.) is the unique plant which blooms overground, fructifies underground in the plant kingdom. It is generally acknowledged that pegging and podding are regulated by phytohormones. Peanut was taken as materials, and the contents and distribution of plant hormones, expression of ABP1 and PIN1 were studied in the pegging stage after IAA and its inhibitors treatments, while the regeneration system of peanut was also studied for further investigation. These will provide some theoretics to support the development regulation and physiological phenomenon of pegging stage in peanut.The main results are as the follows:1. A method was established to detect both the IAA and IAA-Ile based on solid phase extraction by LC-MS. This method can be used to determine the concentration of IAA and IAA-Ile in different tissues of peanut.The contents of IAA and IAA-Ile were highest in all the tissues of full-bloom stage, while decrease to the lowest in fruit maturation stage. In full-bloom stage, the IAA and IAA-Ile contents are both highest in peg. Being treated with exogenous IAA, IAA and IAA-Ile had a remarkable increase in all tissues of peanut, but the content of IAA was obviously increased in leaf. After CHPAA treatment, the content of IAA was increased and IAA-Ile was reduced in leaf and stem, while IAA reduced and IAA-Ile increased in the root and peg. The content of IAA and IAA-Ile was not changed obviously in the blossom. TIBA was used to result in the increase of IAA and decrease of IAA-Ile in stem and leaf, decrease of IAA and IAA-Ile in root and peg, not obvious variations in blossom. The analysis showed that there was a significant correlation between free IAA content and conjugated IAA content in stem and leaf. After exogenous IAA or its transport inhibitor treatments, the IAA and IAA-Ile contents of root and peg was highly correlated in full-bloom stage.2. A method was established to extract and purify JA based on petroleum ether extraction and SPE. The contents and distribution of JA, Z, ZR and ABA were studied by LC-MS and HPLC.The contents of JA and Z were higher at full-bloom stage and early-bloom stage, and the pegs get a highest JA contents peak at podding stage. The content of ABA was higher in fruit maturation stage than other stages, while the content of ZR was not changed obviously in all the stages. After IAA treatment, JA and Z increased in root, stem and leaf, ABA reduced in stem and peg, and increased in leaf. ZR was first reduced and then increased in stem, while increased firstly and then reduced in leaf and peg. JA was increased in root by CHPAA treatment, while the JA contents of stem and leaf were increased first, and then decreased. After TIBA treatment, JA contents became lower in stem and leaf, and higher in root, which was the highest with the TIBA concentration of 10μM. After NPA treatment, ABA content was decreased first and then increased in root, leaf and peg, and increased in stem. Z content decreased firstly and then increased in all the tissues. The contents of ZR was increased and then decreased in root, stem and leaf.3. ABP1 (auxin binding protein 1) in peanut was cloned which was a 635 bp molecule and could be translated into putative protein with 189 amino acids. It was designated as AhABP1 according to the auxin binding protein gene nomination habit and was submitted to GenBank with an accession number FJ360751. The homology of nucleotide sequence was more than 70% of the other plants ABP1 while the amino acid sequence was more than 75%. The expression of ABP1 could be found in root, stem, leaf of the full-bloom stage, which was highest in leaf and lowest in root. The expression level of ABPl was highest in full-bloom stage, and lowest in fruit maturation stage. ABP1 was expressed highest after exogenous IAA treated 8h, which was lower than the control after TIBA treatment. The level of ABPl expression was lower than the control after the CHPAA treated 4h and 8h, but was higher than the control after treated 12h.4. PIN1 gene was detected in root, stem, leaf and peg, which was expressed highest in root, and least in leaf. The highest expression of root was in full-bloom stage, and the lowest was in early-bloom stage. PIN1 was expressed highest in the roots when the plant was treated by IAA for 8h while the inhibitors both reduced the expression of PIN1.After treated by IAA and its inhibitors, the PIN1 expression was distinct in peg and leaf5. Using stem tip as explants, more callus and cespitose buds were obtained on the MS culture with 1.0 mg/L BA and 1.0 mg/L NAA. Then the cespitose buds were induced to grow roots in the MS culture with 2.0 mg/L NAA and 0.2 mg/L BA.