【作者】 梁美霞；

【导师】 戴洪义； 邓子牛；

【作者基本信息】 湖南农业大学 ， 果树学， 2010， 博士

【摘要】 树型改良是苹果分子育种的重要方向。苹果生长习性直接影响树型发育及开花结果能力。柱型苹果因其节间短,分枝少,萌芽率高的生长习性而成为超高密植栽培的重要资源。研究与柱型苹果生长习性有关的细胞学和分子生物学对于柱型苹果资源的开发和利用具有重要意义。本研究旨在揭示柱型苹果与普通型苹果在细胞学、对激素的敏感性及相关生理指标的差异,并通过RACE方法克隆赤霉素信号转导途径的DELLA蛋白基因,利用实时荧光定量PCR分析DELLA蛋白基因在柱型与普通型苹果生长发育中的表达模式。研究结果如下：1.对叶片结构和叶绿体超微结构观察发现,柱型苹果叶片栅栏组织每一层内的细胞大小不等,细胞排列相对杂乱,各层细胞之间相互交错,层与层之间分界线不明显；而普通型苹果叶片栅栏组织每一层内的细胞大小相对一致,细胞排列整齐紧密,各层之间分界线明显。柱型苹果的叶片总厚度、栅栏组织厚度和栅海比均显著高于普通型苹果。柱型苹果上表皮细胞表面的褶皱明显多于普通型苹果,表皮细胞隆起的高度较高。柱型苹果的气孔密度显著高于普通型。柱型苹果叶绿体内基粒或基质片层与叶绿体长轴方向不平行,基粒片层排列不规则,片层数相对较少；而普通型苹果叶绿体内的基粒或基质片层与叶绿体长轴方向平行排列,基粒片层排列整齐致密,片层数量相对较多。2.柱型苹果对GA3、BR和IAA三种激素的敏感性既不属于激素缺陷型也不属于激素不敏感型。柱型苹果在喷施GA3150mg／L时,新梢的敏感性最强。喷施不同浓度的BR在一定程度上能够促进柱型苹果新梢的增长,喷施不同浓度的IAA在一定程度上抑制了新梢的增长。在同一生长季节内,柱型苹果和普通型苹果之间的GA3、ZT、IAA和ABA四种激素的绝对含量差异不显著,两者之间的GA3/ABA、ZT/ABA、IAA/ABA比值差异也不显著。3.外源GA3能明显促进柱型苹果节间α淀粉酶和POD活性的提高,但抑制SOD活性。外源GA3处理初期,柱型苹果节间GA3、ZT和IAA含量呈现先降后升再降的趋势,而ABA含量变化规律为先升后降再升的趋势。外源GA3浓度为150mg/L或200mg／L时,GA3/ABA、ZT/ABA、IAA/ABA的比值相对较高,对新梢的生长促进作用较大。4.用RACE方法克隆得到柱型苹果“鲁加5号”的两个编码DELLA蛋白的基因MdGAI1和MdGAI2的cDNA全长。MdGAI1基因的cDNA全长序列为2489bp,编码635个氨基酸,蛋白质等电点为5.30,分子量为69734.6；MdGAI2基因的cDNA全长序列为2274bp,编码639个氨基酸,蛋白质等电点为5.26,分子量为70235.28。MdGAI1和MdGAI2基因的核酸序列和氨基酸序列均与其它植物具有较高的同源性,在N端具有保守氨基酸结构域DELLA和VHYNP,在C端存在保守的氨基酸结构域VHVID和SAW。5.实时荧光定量PCR分析结果表明,在春季、夏季和秋季三个新梢生长时期,无论是柱型还是普通型苹果,新梢顶端的MdGAI1基因均有一定量的表达。同一生长时期内,3个柱型苹果的MdGA1I基因相对表达量均高于3个普通型苹果。春季新梢生长初期,3个柱型苹果节间MdGAI1基因的相对表达量均高于3个普通型苹果。在“鲁加5号”苹果的种子、叶芽、花芽、花、茎尖、节间和幼果中,MdGAII基因均能表达,但不同器官中的表达量存在差异。MdGAI1基因在苹果果实发育初期表达量最高,相对表达量为1.55,是种子发芽后表达量0.61的两倍多。种子萌发后,MdGAI1基因的表达量明显下降；花朵开放后,MdGAI1基因的表达量也随之下降；在整个生长季节中,春梢、夏梢和秋梢中MdGAI1基因的表达量比较稳定。更多还原

【Abstract】 The tree form improvement of apple is one of important targets of molecular breeding. The growing habit can directly affect the tree form, flowering and fruit-setting ability. Columnar apple is believed to be an ideal variety resource for high density planting due to its particular growing habit such as short internodes, few branching and high bud burst rate. In this paper, the anatomical structure and chloroplast ultra structure of columnar and standard apples were studied using paraffin section method, scanning electronic microscope and transmission electronic microscope. The sensitivity to different hormones and physiological differences between columnar and standard apples were also evaluated. The DELLA protein genes related to gibberellins signaling transduction pathway were cloned using RACE and the expression patterns were showed by real time PCR. The results were as follows:1. The size of palisade tissue cells within the same layer of columnar apple was different and its array was irregular. The cells of different layers were intervened each other, and the border of different layers was not clear. The size of palisade tissue cells within the same layer of standard apple was equal and its array was quite regular and close together. The border of different layers was obvious to distinguish. The leaf total thickness, palisade tissue thickness and the ratio of palisade tissue thickness and spongy tissue thickness of columnar apple were obviously higher than those of standard apple. There were more wrinkles on the surface of the upper epidermal cells of columnar apple and the bulges of epidermal cells were higher compared with standard apple. There were no obvious difference for the length of stomata between columnar apple and standard apple, but the stomata density of columnar apple was obviously higher and its stomata width was shorter than those of standard apple. The grana or stroma lamellae of chloroplast of columnar apple were not parallel to the long axis of the chloroplast, grana lamellae were arrayed irregularly and the number of chloroplast lamellae was small. Whereas grana or stroma lamellae of standard apple were parallel to the long axis of the chloroplast, grana lamellae were arrayed regular and closely and the number of chloroplast lamellae were relatively more than those of columnar apple.2. According to the sensitivity to GA3, BR and IA A, the columnar apple was neither hormone defective nor hormone insensitive type. The new shoots were mostly sensitive to the spray of GA3 at 150 mg/L. Spray of BR at different concentrations could promote shoot growth of columnar apple, whereas IAA could inhabit the growth of the new shoots. During the period of same growing season, there were no obviously differences of the absolute contents of GA3, ZT, IAA and ABA and the ratios of GA3/ABA, ZT/ABA, IAA/ABA between columnar and standard apples.3. Exogenous GA3 could promote the activities of a amylase and POD, and inhibit the activity of SOD in the internodes of columnar apple. After spraying GA3 at the early stage the contents of GA3, ZT and IAA in the internodes of columnar apple showed a changing tendency as being decreased firstly, being increased then and being decreased finally. But the changing tendency of ABA contents was different, that is, being increased firstly, being decreased then and being increased finally. Higher concentration of GA3 (150mg/L or 200mg/L) could obviously promote the lengthening of the new shoots and was associated with the higher ratios of GA3/ABA, ZT/ABA and IAA/ABA.4. Full-length sequences of two genes encoding DELLA proteins named MdGAI1 and MdGAI2 were obtained from columnar apple "Lujia No.5" by means of RACE. The full length sequence of MdGAI1 was 2489bp, encoding 635 amino acids, and the full length sequence of MdGAI2 was 2274bp, encoding 639 amino acids. Their nucleic acid sequences and amino acid sequences were highly homologous with those of other plants. Sequence analysis of the products of the MdGAI1 and MdGAI2 indicated that they shared a highly conserved N terminus that contained two recognizable motifs DELLA and VHYNP, and a highly conserved C terminus that contained two recognizable motifs VHVID and SAW.5. Real-time quantitative PCR analysis showed that MdGAIl was moderately expressed in shoot apices of columnar and standard apples during the spring, summer and autumn growing season. In the same season, the MdGAI1 of three cultivars of columnar apple were expressed at higher level than that of three cultivars of standard apple. In the early spring, the MdGAI1 are expressed at higher levels in internodes of three cultivars of columnar apple compared with that of standard apple. MdGAI1 was moderately expressed in different tissues and organs at various developmental stages of columnar apple "Lujia No.5", with highest level in early stage of fruit development, the relative expression was 1.55, twice more than that of germinating seeds (0.61). After seeds germination and flower blooming, the relative levels of MdGAI1 gene expression were significantly decreased. During the whole growing season, the MdGAI1 gene of shoot apices were expressed stably.更多还原