【作者】 刘霞；

【导师】 肖浪涛；

【作者基本信息】 湖南农业大学 ， 植物学， 2010， 博士

【摘要】 花生(Arachis hypogaea L.)是植物王国里独有的地上开花、地下结果的植物。一般认为,下针和结荚过程受植物激素调控。本研究以花生为材料,通过外施IAA及其运输抑制剂,对花生开花下针期植物激素的含量与分布、生长素结合蛋白基因ABPl和生长素输出蛋白基因PIN1的克隆与表达以及花生的再生体系进行了研究,有望为解析花生开花下针期的生长调控及生理现象提供一定理论基础。主要研究结果如下：1、建立了一种基于固相萃取同时提取纯化游离态IAA和部分结合态IAA的LC-MS检测方法,并对花生不同组织中IAA及IAA-Ile含量进行了测定。花生根、茎、叶、花、果针中IAA和IAA-Ile含量均在盛花期最高,饱果成熟期最低。IAA和IAA-Ile含量在果针中最高,叶片次之,花中最低。外施IAA后,各组织中IAA和IAA-Ile含量均增加。其中,叶片中IAA含量增加显著,而IAA-Ile则先降低再增加。外施生长素输入抑制剂CHPAA,茎和叶中IAA含量升高,IAA-Ile含量则下降；根和果针中IAA含量降低,IAA-Ile含量在根中先升后下降,果针中降低；花中IAA和IAA-Ile含量变化不明显。外施生长素输出抑制剂TIBA,叶和茎中IAA含量增加,IAA-Ile含量下降；根和果针中IAA和IAA-Ile含量均降低；花中IAA和IAA-Ile含量变化不明显。花生茎和叶中IAA含量与IAA-Ile含量显著正相关。外施IAA及其运输抑制剂后,盛花期根和果针的IAA含量和IAA-Ile含量高度正相关。2、建立了以石油醚萃取过柱法提取纯化茉莉酸的LC-MS方法,并对花生不同生长时期的JA、Z、ZR及ABA含量和分布进行了研究。JA含量在盛花期和初花期较高,但果针中以结荚期最高；ABA含量在饱果成熟期最高,在幼苗期、初花期及盛花期的含量都较低；Z含量在初花期和盛花期较高,在饱果成熟期较低；ZR含量在各生长时期的变化不大,但各时期均以果针中的含量最高。外施不同浓度IAA后,花生根、茎和叶中JA均升高；根中ABA变化不明显,茎和果针中ABA含量降低,叶片中ABA含量上升；Z在根、茎、叶的含量呈降低趋势,果针中增加；ZR在根中变化不明显,茎中先降低后增加,叶片和果针中先增加后再降低。CHPAA处理后,茎和叶中JA含量先增加后降低,而根部JA含量则明显增加,并在浓度10μM时最高；TIBA处理使茎和叶中JA含量降低,根中则明显增加,并在浓度10μM时最高。NPA处理使根、叶、果针中的ABA含量先降低后增加,茎中则呈上升趋势；根、茎、叶、果针中Z含量先降低后升高；茎和叶中ZR含量则先升高后降低,根中ZR含量在浓度10μM时最高,而ZR在果针中的含量则先降低后增加。3、得到全长635 bp,共编码198个氨基酸的花生ABP1基因,将其命名为AhABPl, GenBank登录号为FJ360751。同源性分析表明核苷酸序列与拟南芥等植物ABP1基因具有较高同源性,均达到70%以上；氨基酸水平上与拟南芥等植物ABP1氨基酸序列一致性均在70%以上,相似性在75%以上。ABP1在花生盛花期根、茎、叶中均有表达,叶片中表达最高,根中表达极少；外施IAA8h时ABP1相对表达量最高；TIBA处理使ABP1表达低于对照；CHPAA处理4h和8h后表达减少,处理12h表达高于对照。4、PIN1在花生根、茎、叶、果针中均有表达,根中表达最高,叶片表达最少。在盛花期根部表达最高,初花期最弱。浓度为10μM的IAA处理使PIN1在根部的表达最强,而CHPAA及TIBA处理后表达均低于对照。相关分析表明：IAA及其运输抑制剂处理使PIN1在在果针和叶中显著相关。5、以花生茎尖为外植体,在MS+1.0 mg/L BA+1.0 mg/L NAA培养基上可以获得较多的愈伤组织和丛生芽；在MS+0.5mg/LBA+0.2mg/LNAA培养基上可以获得较好的茎尖增殖培养；在MS+2.0 mg/L NAA+0.2 mg/L BA培养基上可以得到生根较好的再生植株。更多还原

【Abstract】 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.更多还原