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小麦苗期抗旱性QTL分析和冰草BADH基因克隆

QTL Analysis of Drought Resistance in Wheat Seedlings and BADH Gene Cloning in Wheatgrass

作者: 专业:遗传学 导师:李斯深 年度:2010 学位:硕士  院校: 山东农业大学

Keywords

Wheat, drought resistance, QTL location, wild wheatgrass, BADH

        小麦是我国主要粮食作物之一。干旱是限制其产量及品质的重要非生物胁迫因素,开展小麦抗旱遗传机理研究,对我国粮食安全和可持续发展具有重要意义。本研究旨在通过小麦苗期抗旱相关性状的QTL定位及抗旱基因克隆,为研究小麦苗期抗旱遗传机理及抗旱育种提供依据,主要获得以下结果。1、以小麦RIL群体(川35050×山农483)水培幼苗为材料,在PEG-6000模拟的干旱胁迫及对照条件下,测定了根数、最长根长、苗高、地上部鲜重、地下部鲜重、总鲜重、地上部干重、地下部干重、总干重、地上部失水率、地下部失水率、叶片相对含水量、脯氨酸含量、丙二醛含量、可溶性糖含量和过氧化物酶活性共19个抗旱相关性状。在渗透胁迫及正常水分两种条件下,RIL群体各性状均有较大的变异范围,除正常水分条件下的叶片失水率、叶片相对含水量及胁迫条件下苗高、叶片相对含水量外,其余性状变异系数均大于10%。多数性状表现超亲分离,表明RIL群体具有比较丰富的遗传变异,双亲对这些性状有贡献的等位基因在其群体中得到了广泛分离,具有较大的选择潜力。RIL群体中多数性状表现正态分布,表明这些性状为数量性状,可能由多个基因控制,其抗旱机制较为复杂。2、对根数等19个性状进行QTL定位,结果在1A,1B,1D,2A,2B,2D,3B, 3D,4A,4B,5A,5D,6D和7A共14条染色体上定位了54个QTL,可解释表型变异的5.63-37.30%。其中正常条件下检测到21个QTL位点,在胁迫条件下检测到33个QTL位点。22个QTL的加性效应来自母本川35050,32个QTL的加性效应来自父本山农483。在1D、5D染色体上有3个QTL簇,在2A、3B和6D上有4个共定位QTL。获得最长根长、苗高、鲜重根冠比、地上部干重、总干重、过氧化物酶活性、脯氨酸含量共7个性状的主效QTL,分别为QMrl.sdau-4A.OS、QSh.sdau-5D.NW、QRsfw. sdau-6D.2.OS、QSdw. sdau-5D.NW、QTdw.sdau -4A.OS、QPod.sdau-2B.OS和QPro.sdau-1D.OS,贡献率分别为37.30、21.51、30.84、21.61、29.09、22.72和36.75%。3、以新疆草原区强抗旱植物野生冰草为材料,采用同源克隆方法分离甜菜碱脱氢酶基因(BADH),获得cDNA全长为1422bp,包括1185bp的开放阅读框,编码394个氨基酸,将推导氨基酸序列于NCBI网站进行Blast搜索,发现该序列包含BADH基因保守结构域,表明分离到的序列为冰草BADH基因序列,开放阅读框正确,提交至GenBank,获得注册号为GU181396。冰草BADH基因归于单子叶植物一类,与大麦、羊草同源关系最近,分别为98%和97%。4、于Expasy网站通过在线工具对BADH推导蛋白进行分析,二级结构预测结果表明该蛋白富含α-螺旋、延伸链、β-转角和无规卷曲等二级结构。亲水性预测结果表明该蛋白形成几个亲水性高峰,有利于形成兼性α-螺旋结构,该结构可达到结合水分子以保护细胞免受水分亏缺伤害的作用。
    Wheat is one of the major food crops in China. Drought is one of the most important abiotic stress factors that limit the yield and quality of wheat. Researches on genetic mechanism of drought resistance are important to the food security and sustainable development of China. The main aim of this study was to provide the basis for the study of drought resistance genetic mechanism and wheat breeding via QTL location of wheat seedlings drought related traits and drought gene cloning. The main result was as follow.1. Root number, maximum root length, seedling height, shoot fresh weight, root fresh weight, total fresh weight, shoot dry weight, root dry weight, total dry weight, shoot water loss rate, root water loss rate, relative water content, proline content, MDA content, soluble sugar content and POD activity was determined using a RIL population (Chuan 35050×Shannong 483) of wheat under the osmotic stress condition induced by PEG-6000 and normal water condition in nutrient solution. In both conditions, marked variability of characters was observed in the RIL population. The coefficient of variation of most traits were higher than 10% except for the leaf water loss rate and relative water content in normal water condition and plant height and leaf relative water content in stress condition. Most traits performance transgressive segregation, implying that RIL population has a relatively rich genetic variation, alleles for these traits contributed by parents were separated widely, the population had great potential for selection. Normal distribution was observed in most traits, indicating that these traits were quantitative traits that controlled by multiple genes, with complex mechanism.2. A total of 54 QTLs were detected on 14 chromosomes, 1A, 1B, 1D, 2A, 2B, 2D, 3B, 3D, 4A, 4B, 5A, 5D, 6D and 7A. Of which, 33 QTLs in OS and 21 QTLs in NW were determined, explaining 5.63-37.30% of the phenotypic variations. The additive effects for 22 QTLs were positive with Chuan 35050 increasing the effects, while 32 QTLs were negative with Shannong 483 increasing the effects. Three QTL clusters were located on chromosomes 1D and 5D, four co-located QTLs were detected on chromosomes 2A, 3B and 6D. QMrl.sdau-4A.OS, QRsfw.sdau-6D.2.OS, QTdw.sdau-4A.OS, and QPro.sdau -1D.OS were major QTLs, contributing 37.30, 30.84, 29.09, and 36.75% of the phenotypic variations, respectively.3. Betaine dehydrogenase gene (BADH) was isolated by homology cloning technology using the strong drought-resistant plants Xinjiang wild wheatgrass as material. The full length cDNA obtained was 1422bp, including a 1185bp open reading frame (ORF) that encoding 394 amino acids. Blast research was carried out using the deduced amino acid sequence in NCBI web site, conserved domains of the BADH gene were found, indicating that the sequence isolated was BADH gene sequence, open reading frame is correct. GenBank registrated number GU181396 was get after submit. Wheatgrass BADH gene attributable to the class of monocots, homology with barley and wildrye were 98% and 97%, respectively.4. BADH deduced protein was analyzed in Expasy website using online tools. Secondary structure prediction results showed that the protein is rich inα-helix, extended chain,β-turn and random coil. Hydrophilic predicted results showed that the protein formed a number of hydrophilic peak, and is conducive to the formation ofα-helix structure that can achieve the bound water molecules to protect cells from injury of water stress.
        

小麦苗期抗旱性QTL分析和冰草BADH基因克隆

摘要8-10
ABSTRACT10-11
1 前言12-26
    1.1 作物抗旱机理12-18
        1.1.1 形态结构与抗旱性13
        1.1.2 生理生化性状与抗旱性13-18
        1.1.3 作物抗旱性综合评价指标18
    1.2 作物抗旱性状的QTL 定位18-21
        1.2.1 形态性状19
        1.2.2 生理性状19-21
    1.3 植物基因分离的方法和策略21-23
        1.3.1 利用图位克隆技术分离基因21-22
        1.3.2 利用比较基因组学分离基因22
        1.3.3 利用突变体分离基因22-23
        1.3.4 利用基因表达技术分离基因23
    1.4 甜菜碱脱氢酶基因(BADH)及其克隆23-25
    1.5 本研究的目的和意义25-26
2 材料与方法26-42
    2.1 试验材料26-27
        2.1.1 植物材料26
        2.1.2 实验药品及菌株26
        2.1.3 主要仪器26-27
    2.2 试验方法27-42
        2.2.1 试验处理27
        2.2.2 指标测定27-30
        2.2.3 数据处理30-31
        2.2.4 QTL 定位分析31
        2.2.5 PCR 引物设计31-32
        2.2.6 总RNA 提取及检测32-34
        2.2.7 第一链cDNA 合成34
        2.2.8 中间目的片段获取34-38
        2.2.9 3’RACE 获取3’端片段38-39
        2.2.10 5’RACE 获取5’端片段39-40
        2.2.11 cDNA 全长序列的获得40-41
        2.2.12 BADH 全长基因组序列的获得41
        2.2.13 生物信息学分析41-42
3 结果与分析42-60
    3.1 RIL 群体及其亲本抗旱相关性状的表型分析42-45
    3.2 苗期小麦抗旱相关性状QTL 定位45-50
    3.3 RIL 群体抗旱性综合评价50-52
    3.4 冰草BADH 基因克隆及分析52-55
        3.4.1 总RNA 提取及检测52
        3.4.2 反转录cDNA 检测52-53
        3.4.3 中间片段的克隆及测序53-54
        3.4.4 BADH 基因3’端分离54
        3.4.5 BADH 基因5’端分离54-55
        3.4.6 BADH 基因全长 cDNA 的分离55
        3.4.7 基因组全长基因获取55
    3.5 BADH 基因生物信息学分析55-60
        3.5.1 BADH 基因cDNA 序列分析55-57
        3.5.2 BADH 基因编码蛋白序列分析57-59
        3.5.3 BADH 基因组序列分析59-60
4 讨论60-64
    4.1 QTL 定位60
    4.2 QTL 簇及QTL 间的关系60-61
    4.3 QTL 在不同条件下的分布61-62
    4.4 甜菜碱脱氢酶基因克隆62-64
5 结论64-66
    5.1 小麦抗旱相关性状QTL 定位64
    5.2 野生冰草BADH 基因克隆及分析64-66
参考文献66-76
致谢76-77
攻读学位期间发表论文表情况77
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