【作者】 夏惠；

【导师】 张振文；

【作者基本信息】 西北农林科技大学 ， 果树学， 2010， 博士

【摘要】 干旱是影响植物分布和农作物产量的一个重要环境因子。植物在逆境下产生一系列的生理、生化反应以应对环境的变化,包括分子水平的。植物耐旱性是一个数量性状(QTL),即有多个基因控制。从植物耐旱的信号传导途径可以将这些基因分为两类:依赖于ABA(ABA-dependent)信号传导的抗旱途径和不依赖于ABA(ABA-independent)信号传导的抗旱途径。本研究是以依赖于ABA信号传导的耐旱途径的两个基因NCED和脱水素基因(DHN)为研究对象的。9-顺式-环氧类胡萝卜素双加氧酶(9-cis-epoxycarotenoid dioxygenase,NCED)是植物激素脱落酸(ABA)生物合成途径中的关键酶,因此是依赖于ABA信号传导耐旱途径的上游基因。脱水素基因(DHN)是依赖于ABA信号传导抗旱途径的下游基因,主要功能是在植物脱水状态下维持细胞结构的稳定性,可在干旱和ABA条件下诱导表达。本文首先以苹果为材料分别从叶片和果实中克隆了脱水素基因Mddehydrin和MdNCED的cDNA和DNA,并对其进行了干旱条件下的表达研究;其次,以模式植物野生番茄的两个种秘鲁番茄(Solanum peruvianum)和智利番茄(Solanum chilense)的不同群体为研究对象,分别克隆了LeNCED1和脱水素基因pLC30-15的DNA片段,并对其进行分子特性和一致性分析。最后对获得的野生番茄两个基因的序列进行遗传进化分析,试图找到群体适应性的遗传学证据。主要研究内容及结果如下:1.从苹果果实中克隆了MdNCED的cDNA和gDNA序列。序列分析表明该cDNA长1945bp,编码607个氨基酸。gDNA序列中没有内含子。在果实生长初期,MdNCED表达水平较低,随着果实成熟,表达量开始增加,当果实进入成熟期后表达水平达到最大值,以后表达量基本不变。说明本研究克隆到的MdNCED基因与果实成熟有关,负责果实成熟过程中ABA的合成。2.从苹果叶片中克隆了脱水素基因Mddehydrin的cDNA和gDNA序列。序列分析表明cDNA长848bp,编码230个氨基酸。gDNA序列由2个外显子和1个内含子组成。该基因具有脱水素的特征motif,一个S-segment,两个Y-segment和三个K-segment,是Y2SK4型脱水素基因。在0、2、4、6、8d干旱处理下,采用实时定量PCR技术分析表明:在正常水分条件下,Mddehydrin基因的表达量非常低,随着干旱程度的加重,其表达量开始逐渐增加,第6天时显著增加,并达到最大值,几乎是正常条件下表达量的200倍。随后表达量开始下降至最大值的25%左右。结果充分证实了干旱可以强烈诱导Mddehydrin基因的表达。3.以模式植物野生番茄的两个种秘鲁番茄和智利番茄为材料,克隆了6个群体的30个个体的LeNCED1基因DNA片段。获得的基因片段长1827bp,无内含子区。其推导氨基酸序列与马铃薯StNCED1基因一致性极高,为96.8%。4.以模式植物野生番茄的两个种秘鲁番茄和智利番茄为材料,克隆了6个群体的30个个体的脱水素基因pLC30-15的DNA片段,共获得60条等位基因序列。获得的基因片段长918bp包含5’端非编码区,一个内含子区和二个外显子区。在第一个外显子区的尾部有一个富含丝氨酸残基的保守序列称为S-segment;在第二个外显子区有3个富含甘氨酸和赖氨酸残基的保守序列K-segment,是SK3型脱水素基因。对SK3型脱水素基因推导氨基酸的多序列比对发现该基因与马铃薯(AY292655)的一致性很高,达到91%。5.对从两个野生番茄种克隆的60条LeNCED1基因序列的核苷酸多态性分析表明:(1)该基因所有核苷酸位点(all sites)的多态性水平在两个野生番茄种上都非常低,仅是中性位点的平均核苷酸多态性水平的二分之一;而沉默位点(silent sites)的核苷酸多态性水平与中性位点的相当。(2)相对地,智利番茄的核苷酸多态性水平高于秘鲁番茄,主要是因为智利番茄在非同义位点的核苷酸多态性水平比较高。综合以上两点可以推测,LeNCED1基因在两个番茄种上都经历过淘汰选择的作用,但作用在秘鲁番茄的选择压力要强于智利番茄。(3)无论是秘鲁番茄还是智利番茄在LeNCED1位点的dN/dS比值都非常小(<0.10),这也说明了淘汰选择曾作用于该位点。6.pLC30-15基因在两个野生番茄种所有核苷酸位点的多态性水平明显高于LeNCED1基因和中性位点的平均核苷酸多态性水平,但在保守序列区(S-和K-segments)的核苷酸多态性水平比较低。7.pLC30-15的dN/dS比值也小于1,但却是LeNCED1基因的10倍,说明淘汰选择也曾作用于pLC30-15,但作用强度比LeNCED1小。LeNCED1和pLC30-15在dN/dS比值上的差距主要体现在前者同义位点替代(dS)是pLC30-15的3倍,而非同义位点替代(dN)却只有pLC30-15的1/2。8.用Fst对群体分化水平的检验结果表明:(1)智利番茄的两个群体Tacna和Moquegua在两个位点上的分化水平都非常低(Fst接近零),这与中性位点的结果一致,因此可以将这两个群体合并为一个集合,命名为TacMoq。TacMoq和Quicacha分化水平却较高。(2)LeNCED1的Fst值是中性位点的2倍,表明有强的淘汰选择作用在基因上。(3)智利番茄的群体间分化水平高于秘鲁番茄各群体间的分化水平。(4)除Tacna-Mocquegua外,智利番茄的各群体间的分化水平都高于中性位点。(5)在pLC30-15基因上Tacna和Moquegua的高分化水平也是群体对当地环境适应的一个证据。9.应用Tajima’s DT和Fu & Li’s检验对两基因位点各群体进行了中性检验,其它各群体没有发现偏离中性理论,除了Quicacha群体在pLC30-15上的DT和DFL正显著,也高出中性位点的值。说明平衡选择或多向选择曾作用于这个群体。10.发现Quicacha群体的10个等位基因只有3个单倍体型,Haplotype 1,Haplotype 2和Haplotype 3。Haplotype 1与Haplotype 2之间只有两个核苷酸位点不同;而Haplotype 3与前两者有较大差距。对Quicacha单倍体型检验值也极显著(P=0.006)。结合Quicacha正显著的Tajima DT值考虑,是多向选择而不是群体规模变化效应(demographic effect)造成Quicacha这种单倍体型模式的。更多还原

【Abstract】 Drought is a major factor that limit the distribution of plants and the production of crops. Drought stress induces a range of physiological and bio-chemical responses in plants, also in molecular level. Drought tolerent is a quatative trait (QTL), an assortment of genes involved in this process. These genes can be classified into two groups according to signaling pathway: ABA-independent and ABA-dependent regulatory systems. Here, we examined two genes involved in ABA-dependent pathway: 9-cis-epoxycarotenoid dioxygenase gene (NCED) and dehydrin gene (DHN). NCED is a key enzyme in ABA biosynthesis, which is on the upstream of ABA-dependent pathway; DHN, downstream gene, can be induced by drought and ABA to stabilize cellular structures and membranes against dehydration.In this research, we first cloned cDNAs and DNAs of MdNCED and dehydrin gene Mddehydrin from apple leaves and fruits respectively; and studied its quantative expression under drought treatments. Then, we cloned LeNCED1 and dehydrin gene pLC30-15 from 6 populations of two wild tomatoes: Solanum peruvianum and Solanum chilense; And made a homologus analysis. Finally, we made population genetics analysis of sequences from wild tomatoes, and try to find evidence of local adaption. Main contents and results are as below:1. We cloned a cDNA and a DNA of MdNCED from apple fruits. cDNA is 1945 bp long,encoding 607 amino acid, and there is no intron in gDNA. Expression pattern of MdNCED during fruit development were detected by real-time RT-PCR. MdNCED mRNA level was very low in young fruits, increased markedly with fruit growth, reached maximum in mature fruit, and then maintained stable. Taken together, the results indicate that MdNCED might play a key role in the regulation of fruit ripeness.2. We cloned a cDNA and a DNA of dehydrin gene of Mddehydrin from apple leaves. This gene is 1170bp long, including 2 exones and one intron. Sequence analysis shows there are several characterized motifs in the sequence: a S-segment, two Y-segments and three K-segments, so it is a Y2SK4 type dehydrin gene. Under drought treatment of 0、2、4、6、8d, experiment by real-time PCR shows Mddehydrin expression is very low under no drought treatment; while Mddehydrin mRNA level increased markedly with extent of dehydration, and reached maximum at the sixth day, which is 200-fold higher than under no treatment. Then expression of Mddehydrin mRNA decreased to 25% of maximum。It is fully proved that drought can induced expression of Mddehydrin mRNA strongly.3. We also sequenced LeNCED1 from 30 individuals of 6 populations, and totally obtained 60 sequences. LeNCED1 is 1827 bp long consisting of a 5’flanking region and a single exon. The deduced amino acid sequence of LeNCED1 shared high identity with StNCED1 of S. tuberosum, 96.8%。4. Using mode plant wild tomatoes S. peruvianum and S. chilense as plant material, we sequenced pLC30-15 from 30 individuals of 6 populations. We totally obtained 60 sequences. pLC30-15 is 918 bp long consisting of two exons, one intron and a 5’flanking region. There are one S-segment and three K-segments, blonging to SK3 type dehydrin gene. The deduced amino acid sequence of SK3 dehydrins shows pLC30-15 shared 91% identity with potato (AY292655).5. We surveyed nucleotide diversity at LeNCED1 gene, observed that (1) the nucleotide diversity of LeNCED1 at all sites is low in both species, about twofold lower than at the reference loci. In contrast, levels of diversity at silent sites are comparable with those at the reference loci. (2) Most remarkably, S. chilense shows higher nucleotide diversity (at all sites) than S. peruvianum due to its higher nucleotide diversity at non-synonymous sites. Taken together, this indicates that LeNCED1 has been under purifying selection in both species and that selection pressure on LeNCED1 appears to be stronger in S. peruvianum than in S. chilense. This may be due to the effective population size, which is larger in S. peruvianum, or due to a difference in selection coefficients. (3) A very small dN/dS ratio (<0.10) for LeNCED1 in both S. chilense and S. peruvianum also indicates that LeNCED1 has been under purifying selection.6. pLC30-15 exhibits higher average levels of nucleotide diversity (at all sites) than LeNCED1 and the reference loci, except for the S-, and K-segments, where diversity is very low.7. The dN/dS ratio at pLC30-15 is smaller than 1 but almost 10 times larger than at LeNCED1, indicating that purifying selection is also operating on its coding region but to a lesser extent than on LeNCED1. This difference in dN/dS between LeNCED1 and pLC30-15 results from a threefold higher rate of synonymous substitution (dS) and an almost twofold lower rate of non-synonymous change (dN) at LeNCED1 than at pLC30-15.8. We estimated population differentiation using Fst between pairs of populations. (1) We observed that in S. chilense genetic differentiation between the Tacna and Moquegua samples is very low at both loci. This agrees with the results found at the reference loci. Therefore, we pooled the samples of Tacna and Mocquegua into one sample (called TacMoq) and measured differentiation between the pooled sample and Quicacha, which is high. (2) In particular, LeNCED1 shows twofold higher Fst values than the reference loci, which is consistent with the strong purifying selection on this gene. (3) Overall, genetic differentiation between populations of S. chilense is higher than between those of S. peruvianum. (4) Except for the pair Tacna-Mocquegua, both candidate genes are more differentiated than the reference loci in S. chilense. (5) The high Fst at pLC30-15 between TaqMoc and Quicacha indicates local adaptation.9. We used Tajima’s DT and Fu & Li’s DFL statistics to detect deviations from the standard neutral model. Most of the individual populations do not exhibit significant departures from neutral equilibrium expectations. Only the sample from Quicacha shows significant deviations in both the DT and DFL statistics at locus pLC30-15, which much higher than those of the reference loci. Since these statistics deviate in a positive direction from the expectation, some form of diversifying or balancing selection may have acted on this gene in the Quicacha population in the (recent) past.10. The Quicacha sample shows evidence for a haplotype structure at pLC30-15, with only three haplotypes of 10 alleles. While haplotypes 1 and 2 show differences at only two sites, haplotype 3 is quite diverged from the other two haplotypes. Application of the haplotype test shows that the observed haplotype diversity is significantly lower than expected (P=0.006). Consistent with the significantly high Tajima DT estimate and the much lower corresponding value at the reference loci, this suggests that the pattern of variation at pLC30-15 has been caused by positive (diversifying) selection rather than demographic effects.更多还原