【作者】 郝岗平；

【导师】 杨清； 黄丛林；

【作者基本信息】 南京农业大学 ， 生物化学与分子生物学， 2004， 博士

【摘要】 本研究以生长于不同生态气候条件下的32个拟南芥核心生态型为材料，分析了它们的抗旱指标和综合抗旱性，同时通过直接测序法分析了抗旱转录因子CBF4，组氨酸蛋白激酶ATHK1和ABA合成关键酶基因NCED3的单核苷酸多态性(SNP)。 结果表明，不同的拟南芥生态型具有差异明显的抗旱性，抗旱综合聚类分析将32个生态型聚合为4类：强抗旱、抗旱、中等抗旱和干旱敏感型。 CBF4基因位点核苷酸多态性的频率为每35.8bp一个SNP，每143 bp一个插入／缺失(Indel)。编码区SNP的频率为每96.4bp一个SNP，其中1034位(以GenBank No.AB015478序列第19696位的核苷酸为1)碱基变化：GT，引起第205位氨基酸变化：glyval。基因非编码区的多态性是编码区的4倍，表明编码区承受着更大的选择压力。Tajima’S D统计学检验结果是各区域都不显著，说明该区域核苷酸变化符合中性突变假说，较高的核苷酸多样性是净化选择的结果。相关性分析表明CBF4基因5′端非编码区的核苷酸变化与拟南芥综合抗旱性相关，氨基酸变化与综合抗旱性不相关。 ATHK1基因位点核苷酸多态性的频率为每142 bp一个SNP，每1705bp一个Indel。编码区SNP的频率为每190.6 bp一个SNP，非编码区SNP的频率为每87.9bp一个SNP，非编码区的多态性是编码区的2.17倍。Tajima’S D，Fu Li’D°test和Fu Li’F°test统计学检验结果表明ATHK1基因位点的整个区域和编码区显著，这说明编码区域核苷酸变化不符合中性突变假说，是自然正选择的结果。Ka／Ks的比率为0.727，说明ATHK1基因属于中等速度进化基因。相关性分析表明ATHK1基因5′非编码区的序列变化与抗旱综合能力可能没有相关性。257 av和266 av第1198位氨基酸Ser变为终止密码子可能与它们较低的综合抗旱性相关。 NCED3基因位点核苷酸多态性的频率为每87.4bp一个SNP，每502bp一个Indel。编码区SNP的频率为每94.6 bp一个SNP，非编码区SNP的频率为每51 bp一个SNP，基因非编码区的多态性是编码区的1.85倍。Tajima’S D，Fu Li’D°test和Fu Li’F°test统计学检验结果表明NCED3基因位点的整个区域和编码区不显著，5′端非编码区显著，这表明编码区域核苷酸变化符合中性突变假说，5′端非编码区的核苷酸变化不符合中性假说，可能有平衡性选择作用。整个编码区的Ka／Ks的比率为0.270，表明NCED3基因正经受较强的净化选择，属于相对保守基因。相关性分析表明157 av拟南芥抗早候选荃因的SN甲及其抗早性状的相关性分析以及才7了IKI基因的功能研究5‘非编码区第”位的A.C与170位的A.C可能与其较高的脱落酸(^BA冷t有关;2 57 avs‘非编码区的87位的C.T可能与其较低的杭早综合能力相关.163a，NCBD3蛋白质第46位氛基故cys“^rg的互换可能与^B人含童相关性较高.NcED3蛋白的氛基酸变化与各生态型的综合杭早性没有关系. 以拟南芥ws仪“5 1 lewskija)生态型及其才刀济2基因T一DN人插入所产生的缺失突变体为材料，分析了它们在生理和基因表达方面的差异.结果表明突变体的离体叶片失水率明显大于歼生型;在3。%PEG胁迫后，歼生型和才刀深了突变体的细胞膜离子外渗率比胁迫前分别增加了50%和80%.PEG一6000胁迫48h时突变体的英蔫程度明显大于歼生型ws.以上结果说明A刀澄I突变体的抗渗透胁迫能力低于野生型，即才劲盯基因参与了拟南芥适应逆境的调节反应.利用DDRT一PcR技术研究二者在PBG一6 000胁迫36h后的基因表达差异，分离到9个在野生型中被PEG诱导表达而在突变体中未被诱导的参与逆境应答的基因片段，其中包括蒯乃以了了矛和丝氛酸/苏氮酸蛋白激醉基因，即才刀澄2基因失活引起下游基因响应渗透胁迫的能力减弱，进一步说明A几澄1基因参与拟南芥适应逆境的调节反应，且ATHKI可能在逆境信号转导组分MAPK的上游起作用，很可能是植物中的渗透感受器.更多还原

【Abstract】 The levels of drought tolerance and nucleotide polymorphism at transcription factor CBF4, ATHK1 and key enzyme regulating abscisic acid (ABA) synthesis NCED3 locus in a worldwide sample of 32 core accessions of Arabidopsis thaliana were examined. The results showed that different accessions had quite difference in adaptation to drought stress. Thirty-two core accessions of Arabidopsis thaliana were classified in four clusters by synthetical analysis of drought tolerance.The frequency of nucleotide polymorphism at the CBF4 locus, including single nucleotide polymorphism ( SNP ) and insertion/deletion( Indel ), was high, on average 1 SNP per 35.8bp and 1 Indel per 143bp. In coding region, SNP frequency was 1 SNP per 96.4bp, one nonsynonymous mutation was detected, which was the 205th site amino acid variation: gly val caused by the 1034th site (corresponding to 19696 site nucleotide of GenBank No.ABO 15478 as 1) nucleotide variation: G T. Nucleotide polymorphism in non-coding region was three times higher than that in coding region. No significance (NS) Tajima’D test in all region regions indicated that the neutral mutation hypothesis could explain the nucleotide polymorphism in CBF4 gene region. The higher polymorphism was the result of purification selection. This result might also indicate a recent relaxation of the selection pressures on non-coding region of CBF4 gene. Association study showed that nucleotide variation of 5’flanking sequence of CBF4 was associated with the synthetical drought tolerance, but the amino acid variation of CBF4 was not associated with the synthetical drought tolerance.The frequency of nucleotide polymorphism at the ATHKl locus was on average 1 SNP per 142 bp and 1 Indel per 1705 bp. SNP frequency was 1 SNP per 190.6 bp in coding region and 1 SNP per 87.9 bp in non-coding region, SNP in non-coding region was 1.17 times higher than that in coding region. Significant Tajima’D test hi whole region and coding region indicated that the neutral mutation hypothesis could not explain the nucleotide polymorphism in ATHKl gene region, the higher polymorphism was the result of natural selection. The ratio Ka/Ks, being 0.727, indicated that ATHKl was the middle evolution-speed gene. Association study showed that nucleotide variation of 5’flankingsequence of ATHK1 was not associated with the synthetical drought tolerance, but the the amino acid variation of ATHKl of 257av and 266av, which was 1198 site Ser stop code, was associated with their lower synthetical drought tolerance.The frequency of nucleotide polymorphism at the NCED3 locus was on average 1 SNP per 87.4 bp and 1 Indel per 502 bp. SNP frequency was 1 SNP per 94.6 bp in coding region and 1 SNP per 51 bp in non-coding region. SNP in non-coding region was 0.85 times higher than that in coding region. No significance Tajima’D test in whole region and coding region indicated that the neutral mutation hypothesis could explain the nucleotide polymorphism in NCED3 gene region, The higher polymorphism was the result of purification selection. But significant TajimaT) test in 5 ’flanking sequence indicated that nucleotide variation of 5 ’flanking sequence was not explained by the neutral mutation hypothesis but by balancing selection. The ratio Ka/Ks, being 0.2727, indicated that NCED3 was the highly conservative gene. Association study showed that the 33th site A C and 170th site A C nucleotide variation of 5’flanking sequence of NCED3 of 157av was associated with its higher ABA content; 87th site C T of NCED3 of 257av was associated with its the lower synthetical drought tolerance. The amino acid variation of NCED3 of 163av, which was 46th site Cys Arg, was associated with its higher ABA content. The amino acid variation of NCED3 was not associated with their synthetical drought tolerance.Differences in physiology and gene expression between ATHK1 knock-out mutantcaused by T-DNA insertion and wild type (WT) of Wassilewskija (WS) accession of Arabidopsis thaliana were analysed. Water loss ratio of detached leaf of ATHKl -mutant was更多还原