【作者】 刘春；

【导师】 夏光敏；

【作者基本信息】 山东大学 ， 细胞生物学， 2011， 博士

【摘要】 物种在进化过程中一直伴随着基因组序列的变异,而基因组序列的变异又是物种进化的源动力。小麦是异源六倍体作物,在形成过程中因“杂合性”和“多倍性”引起的基因组冲击使小麦基因组发生了高频率的基因组变异,使多倍体“二倍体化”。本实验室利用非对称体细胞杂交方法获得了普通小麦(Triticum aestivum L.2n=42)济南177(JN177)与长穗偃麦草(Thinopyrum ponticum,2n=70)的渐渗系小麦耐盐、抗旱和优质新品种山融3号(SR3)。非对称体细胞杂交过程中会发生双亲基因组短暂共存于一个细胞核、供体染色质大规模消减和少量供体染色质渐渗到受体基因组等事件,因而是一种特殊的二倍体化,也能引起强烈的基因组冲击,并产生系统性的基因组变异。但至今非对称杂交介导的基因组变异特征和机制及其和异源多倍体化的差异还未见报道。本实验室分子标记和功能基因分析显示,SR3基因组发生了遗传学和表观遗传学变异,但变异程度和特征仍不清楚。本试验拟构建SR3和亲本小麦JN177的cDNA文库并进行大规模测序,通过EST功能注释、EST序列分析和cDNA芯片杂交,从组学水平系统分析非对称体细胞杂交对SR3基因组的基因结构和基因表达的影响及其与SR3耐盐能力的关系,为阐明非对称杂交介导的基因组变异机制提供了理论基础。一、SR3和JN177cDNA文库构建现在常用的文库构建技术具有各自的优缺点。根据课题研究目标,本试验利用Stratagene技术构建了两个噬菌体文库：SR3盐胁迫根叶混合文库和SR3旱胁迫根叶混合文库；利用Gateway技术构建了两个重组文库：SR3盐旱胁迫根混合文库和JN177盐旱胁迫根混合文库。四个cDNA文库容量均达到107以上,插入片段的平均长度超过1.3kb,满足大规模测序的需要,为系统分析非对称杂交介导的基因组变异和深入开展小麦功能基因学研究提供了坚实的基础。二、SR3和JN177cDNA文库的大规模测序和注释利用5’端测序方法,获得了18192条SR3和9770条JN177的高质量5’端EST序列,总长度分别达到11678320 bp和7131983 bp。经CAP3软件聚类分析,分别获得了9634和7107条不重复的单一EST序列,其中contigs分别为2097和1207条,sigletons分别为7537和5900条,全长序列分别为4825条和2975条。利用本地BLAST软件将这些单一EST序列和小麦EST数据库进行比较,发现没有比对结果的476条SR3序列和594条JN177序列是新的小麦EST,而且部分SR3序列可能是来源于长穗偃麦草的外源序列。利用Blast2GO工具获得的单一EST序列进行功能注释和分类,发现在SR3 cDNA文库中含有高丰度的抗逆和抗病相关EST。利用codonw软件对SR3和JN177的密码子使用偏爱性进行了分析,结果表明SR3和JN177 EST密码子第三位GC含量分别为67.4%和69.7%,远远高于小麦基因整体序列的GC含量,而且SR3整体序列的GC含量明显低于JN177。三、SR3和JN177的EST序列多态性分析为了系统分析小麦品种间序列多态性和非对称杂交介导的序列多态性,本试验进行了三组比对分析：JN177 EST序列比对小麦EST数据库(JN177-Ta)、SR3EST序列比对小麦EST数据库(SR3-Ta)和SR3 EST序列比对JN177 EST序列(SR3-JN177)。然后从中提取相似性大于等于96%的序列进行多态性分析,其中SR3-JN177有2581条,SR3-Ta有7284条,JN177-Ta有5072条。与Ta数据库相比,JN177 EST序列的单核苷酸多态性(single nucleotide polymorphism, SNP)和1-22核苷酸插入/缺失(Insertion and Deletion, InDel)频率分别为9.02SNPs/kb EST和3.81InDels/kb EST, SR3 EST序列的SNPs和InDels频率分别为9.09 SNPs/kb EST和4.29 InDels/kb EST;而与JN 177相比,SR3 EST序列的SNPs和InDels频率较高,分别为11.8 SNPs/kb EST和5.44 InDels/kb EST。这些结果表明非对称体细胞杂交可以引起高频率的基因组变异；SR3-Ta间的基因组多态性并非是非对称体细胞杂交过程新产生的多态性和JN177-Ta间原有多态性的简单叠加,暗示基因组变异可能存在可逆的恢复变异机制,而且SNPs的恢复能力更强。结果发现,不同类型SNP和InDel的发生频率具有不均衡的特点。与JN177-Ta和SR3-Ta相比,SR3-JN177中A→T和T→A在总SNPs中所占比例明显降低,而C→T所占比例则显著提高(16.6%：14.8%)。C→T的碱基替换主要通过甲基化C脱氨实现,显示非对称体细胞杂交显著改变了SR3基因组的甲基化水平。SR3-Ta和JN177-Ta之间不同类型SNP所占比例也不相同,其中G→A频率在SR3-Ta SNPs中占17%,而在JN177-Ta只占14%,表明不同SNP的可逆性存在差异。SR3-JN177中单核苷酸Insertion频率占单核苷酸InDel的62.0%,JN177-Ta中占74.6%,而SR3-Ta中占76.4%,表明Insertion和Deletion的不均衡性导致InDel的可逆性程度较低,而且非对称体细胞杂交介导的Insertion和Deletion频率的均衡性高于自然和异源多倍体进化。JN177-Ta和SR3-Ta中大片段(>22bp)Deletion频率高于Insertion频率,表明异源六倍体小麦中的二倍体化是一直发生着的进化事件。但SR3-JN177中大片段Insertion频率高于Deletion频率,暗示非对称体细胞杂交杂种和异源多倍体中“二倍化”的机制存在显著差异。分析显示,InDel相邻核苷酸的GC含量具有特异性。JN177-Ta和SR3-Ta中Insertion左边相邻核苷酸GC含量很高而右边相邻核苷酸GC含量很低,Deletion正好相反；在SR3-JN177中Insertion和Deletion均为左边相邻核苷酸GC含量较高,而右边相邻核苷酸GC含量较低。这说明InDel不是一种随机的生物学事件,GC含量可能是InDel位点的识别特征,而且非对称体细胞杂交杂种和异源多倍体中的识别机制存在差异。有趣的是,我们发现6-22nt InDel具有“3N”特征,即6、9、12、15、18和21nt InDel频率及其侧翼序列GC含量均高于相邻大小InDel,而且InDel频率与侧翼序列GC含量存在指数相关性,其中SR3-JN1 77中相关性最高。此外,SR3-JN177中有些InDel两端具有重复序列的拷贝或者存在滑动现象,而且有些序列存在转位和反向重复等特征,表明非对称体细胞杂交能够引起多样化的基因组变异。四、SR3和.JN177的cDNA芯片分析为了评估高频率的基因组变异对转录组的影响以及SR3的耐逆分子基础,我们利用cDNA芯片比较了对照和盐、旱胁迫下SR3和JN177幼苗根转录组差异。为了涵盖部分渐渗到SR3中的长穗偃麦草序列,本试验根据部分cDNA文库序列和功能数据库小麦EST拼接序列设计探针,点制了60-mer cDNA芯片,其包含15172个探针,代表小麦15000个unigene。对照条件下,共有508个探针的转录水平在SR3和JN177间存在显著差异,其中在SR3中高表达的探针有241个,低表达的探针有267个。高表达探针中胁迫响应过程的富集程度最高,包括冷调节蛋白、热激蛋白和脱水素等。此外,戊糖磷酸和抗坏血酸盐代谢以及一些茉莉酸和赤霉素合成和响应途径基因表达显著增强,而碳和能量代谢相关基因表达降低。NaCl处理0.5h后,SR3和JN177中上调表达的探针分别有122和87个,其中48个共同上调；下调表达的探针分别为101和218个,其中75个共同下调。PEG处理0.5h后,SR3和JN177中上调表达的探针分别为215和210个,下调表达的探针分别为81和143个,其中同时上调和下调的探针分别为71和29个。NaCl处理24h后,SR3和JN177上调表达的探针分别为1451和1556个,下调表达的探针分别为1579和1507个,其中同时上调和下调的探针分别为950和978个有趣的是,除了4个探针外,其他非共同响应的探针均表现出品种特异性的胁迫响应特征,即在胁迫条件下只在一个品种中表达发生变化,而在另一个品种中表达量保持不变。生物途径聚类分析显示,PEG和NaCl胁迫0.5h后,SR3中非饱和脂肪酸和脂肪生物合成即α-亚麻酸代谢耐逆相关过程的活性显著高于JN177；NaCl处理24h后,抗坏血酸代谢、糖酵解／糖异生、不饱和脂肪酸和类黄酮生物合成等耐逆相关过程在SR3中被显著促进。这些途径增强与G蛋白—磷脂—ABA/DREB信号通路活性以及激素合成和调控网络的整体提升相关。将SR3与JN177 EST序列与芯片探针序列进行比对,具有匹配探针的EST序列分别有3471和1618条,但这些序列丰度与匹配探针的杂交信号间没有相关性。为了比较序列变异对基因表达的影响,我们从这些序列中提取出SR3和JN177同源且匹配探针表达变化的序列61条。上调表达探针匹配的序列共有17条,均具有SNPs,其中15条含有InDel,包括一个414bp的大片段InDel。下调表达探针匹配的序列有42条,其中39条具有SNPs,27条含有InDel。根据EST多态性分析和探针功能注释结果,发现SR3和JN177间基因表达差异是以下遗传学和表观遗传学因素系统改变的结果：1、序列发生了高频率的SNP和InDel变异；2、来源于供体亲本长穗偃麦草的渐渗序列：3、逆转录转座子和转座子转录活性的改变；4、一系列组蛋白变体和组蛋白修饰相关基因表达模式的转变导致染色体重塑；5、DNA甲基化模式的改变。综上,通过对SR3和JN177cDNA文库的高通量测序、序列比较和cDNA芯片分析表明,非对称体细胞杂交过程中基因组发生了有规律的高频率序列变异和表达变异,从而改变了SR3耐逆能力和发育潜力；非对称体细胞杂交与异源多倍体化和自然进化中的基因组变异存在明显的区别。本实验对这些变异规律的研究在一定程度上为理解非对称体细胞杂交和耐盐的机制提供了重要信息。更多还原

【Abstract】 Genomic variation is a fundamental driving resource for species evolution, and is always going on during the whole period of evolution. Wheat is an allopolyploid, and during its formation the hybridity and polyploidy played a strong genomic shock, which induced a high frequency of genomic variation and made a diploidizaton of the allopolyploid. Using the asymmetric somatic hybridization method, we have released the bread wheat cultivar Shanrong No.3 (SR3), a somatic hybrid introgression line obtained from hybrids of common wheat Jinan 177 (JN177) as a recipient with Thinopyrum ponticum as a donor. During asymmetric somatic hybridization, transitory co-existing of biparent genomes in one nucleolus, large-scale extinction of donor chromatins and introgression of a few donor chromatins may occur. These bio-events are of a specific diploidization, which may also play a strong genomic shock and induce genomic variation. However, the characteristics and mechanism of asymmetric somatic hybridization induced genomic variation as well as its difference from allopolyploidization induced one has not been well documented.Biomarker and functional gene analysis indicated that genetic and epigenetic variation had been taken place in the genome of SR3, but the extent and characteristics of this variation is still unclear. In this work, we proposed to construct cDNA libraries of SR3 and its parent JN177, and perform a large-scale EST sequencing, EST functional annotation and cDNA microarray hybridization, and then outline the effect of asymmetric somatic hybridization on the structure and expression of SR3’s genome as well as its relationship with the high salt tolerance capacity of SR3, with the aim to provide evidence for elucidate the mechanism of asymmetric somatic hybridization induced genomic variation.1. Construction of cDNA libraries of SR3 and JN177 Based on the object of this work, four cDNA libraries were constructed with two methodologies:SR3 salt-stressed root and leaf mixed library and SR3 drought-stressed root and leaf mixed library were constructed with the Stratagene method; SR3 salt- and drought-stressed root mixed library and JN177 salt- and drought-stressed root mixed library were constructed by the Gateway method. The result showed that the capacities of these four libraries were all more than 10’, and the average lengths of inserted sequences were all larger than 1.3kb, showing that these high-quality libraries can be used for large-scale sequencing.2. Large-scale sequencing and annotation of cDNA libraryWith the 5’-sequecing method, we obtained 18192 SR3 and 9770 JN177 5’-ESTs with high-qualities, and their total lengths were 11678320 bp and 7131983 bp, respectively. These sequences were assembled into 9634 SR3 and 7107 JN177 unigenes using the CAP3 software, of which contigs were 2097 and 1207, and sigletons were 7537 and 5900, respectively. These unigenes were subjected to local-BLAST against the public wheat EST database, and 476 SR3 and 594 JN177 unigenes had no identified alignments, which were new wheat ESTs or exogenous sequence of Thinopyrum ponticum. GO annotation and classification were performed with the Blast2GO software, and high abundance of stress and pathogen resistance associated ESTs were found in SR3. The codon bias of SR3 and JN177 ESTs were analyzed using the codonw software, and the result showed that the GC contents of third nucleotide of codon of SR3 and JN177 ESTs were 67.4% and 69.7%, respectively, significantly higher than those of whole sequences, and that the GC content of SR3 ESTs were significantly lower than that of JN 177.3. Polymorphism analysis of SR3 and JN177 ESTsIn order to comprehensive analysis the sequence polymorphism among wheat cultivars and that induced by asymmetric somatic hybridization, three alignments were carried out:JN177 ESTs vs public wheat EST database (JN177-Ta), SR3 ESTs vs public wheat EST database (SR3-Ta), SR3 ESTs vs JN177 ESTs (SR3-JN177). Then the aligned sequences with identity more than 96% were selected for polymorphism analysis, of which SR3-JN177 had 2581, SR3-Ta had 7284, JN177-Ta had 5072.In comparison with Ta, the frequencies of single nucleotide polymorphisms (SNPs) and 1-22nt insertions/deletions (InDels) were 9.02 SNPs/kb EST and 3.81 InDels/kb EST in JN177 ESTs, and 9.09 SNPs/kb EST and 4.29 InDels/kb EST in SR3 ESTs. In comparison with JN177, the frequencies of SNPs and InDels were up to 11.8 SNPs/kb EST and 5.44 InDels/kb EST in SR3 ESTs. These results indicated that asymmetric somatic hybridization can induce high frequency of genomic variation, and the polymorphism of SR3-Ta were not the plus of polymorphism of JN177-Ta to new-occurred one during asymmetric somatic hybridization, which suggested that there may have a reversible variation mechanism, and InDels has a lower reversible potential.The results indicated that the frequencies of different types of SNPs and InDels were unequal. In comparison with JN177-Ta and SR3-Ta, the proportion of A→T and T→A in total SNPs were lower in SR3-JN177, whereas the proportion of C→T was remarkably higher (16.6%:14.8%). The substitution of C→T is majorly achieved through deamination of methylated cytimidine, demonstrating the shift of methylation in genome during asymmetric somatic hybridization. The proportions of different types of SNPs between JN177-Ta and SR3-Ta were also inequable, of which G→A shared 17% in SR3-Ta and 14% in JN177-Ta, suggesting the difference of reversion among SNPs. One-nucleotide insertions contributed to 62% of InDels in SR3-JN177,74.6% in JN177-Ta, and 76.4% in SR3-Ta, showing the role of inequilibrium between insertion and deletion in low reversible potential of InDels, and the higher equilibrium between insertion and deletion during somatic hybridization than natural and allopolyploidized evolution. The higher frequencies of large (>22bp) deletions than those of insertions in JN177-Ta and SR3-Ta indicated that diploidization is an on-going evolutionary event in allopolyploidy wheat. However, the higher frequency of larger insertions than that of deletions implied the obvious distinction between diploidization mechanisms in asymmetric somatic hybrids and allopolyploids.The analysis showed that the GC content of InDel flanking sequences had specificity. In JN177-Ta and SR3-Ta, the GC contents of the left flanking nucleotide of insertions were very high whereas those of the right one were very low, but the result of deletion was quite opposite. In SR3-JN177, the GC contents of the left flanking nucleotide of insertion and deletion were both high whereas those of the right ones were both low. This revealed that InDels are not random bio-event and GC content may be the identification label of InDel sites, and that the identification mechanisms may be somewhat distinct from each other in asymmetric somatic hybrids and allopolyploids. Interestingly, we found the "3N" rule of 6-22nt InDels, that the frequencies of 6,9,12, 15,18 and 21nt InDels as well as the GC contents of their flanking sequences were all higher than those of InDel with adjacent sizes. Also a correlationship between InDel frequency and GC content of flanking sequences was found, and the correlationship in SR3-JN177 was the highest.Besides, repeated, slippage and translocated sequences as well as inverted repeats were found in SR3-JN177, showing diverse patterns of genomic variation were taken place during asymmetric somatic hybridization.3. cDNA microarry analysisIn order to reveal the effect of high frequency of genomic variation on transcripteome and the molecular basis of SR3’s stress tolerance, we performed a cDNA microarry analysis to compare the difference of transcriptome between roots of SR3 and JN177 seelings as well as the difference of their response to salt and drought stress. In this work, a customized 60-mer cDNA microarray with 15172 probes designed from public wheat EST assembled sequences and part of our cDNA library sequences was used to cover the exogenous sequences from Thinopyrum ponticum.Under the control, there had 508 probes with differential expressions between SR3 and JN177, including 241 with high and 267 with low transcription levels in SR3. GO term ’response to stress’ was significantly enriched in highly transcribed probes, including cold-regulated protein, heat-shock protein and dehydrins. Besides, the expression of phosphate pathway, ascorbate and aldarate metabolism and some JA and GA biosynthesis and response associated probes was enhanced, but carbon and energy metabolism associated probes were restricted.Under 0.5h NaCl treatment, up-regulated probes in SR3 and JN177 were 122 and 87, and down-regulated were 101 and 218, of which 48 and 75 were shared by two cultivars. Under 0.5h PEG treatment, up-regulated probes were 215 and 210, down-regulated were 81 and 143, and 71 and 29 were overlapped, respectively. Under 24h NaCl treatment, up- and down-regulated probes were increased to 1451/1556 and 1579/1507 in SR3 and JN177, and 950 and 978 were found in both of cultivars. Interestingly, except for four probes, all other un-co-responsive probes showed cultivar-specific responsive pattern, that is those responded to stress in one cultivar and kept constant in the other. Bio-process enrichment showed that unsaturated fatty acids and lipids synthesis and alpha-linolenic acid metabolism were obviously promoted in SR3 under 0.5h PEG and NaCl treatment; Ascorbate and aldarate, glycolysis/gluconeogenesis, unsaturated fatty acids and flavoniods synthesis were markedly accelerated in SR3 under 24h NaCl treatment. The enhancement of these stress-responsive bio-processes partially contributed to the systematic improvement of G protein-phospholipid-ABA/DREB signaling pathways and hormone synthesis and regulatory network.The alignment of ESTs with cDNA microarry probes extracted 3471 and 1618 probe-matched ESTs in SR3 and JN177, respectively, and correlationship between EST abundances and matched probes’signals was not found. In order to address the effect of variation on expression, we selected 61 SR3 ESTs that were homologous between SR3 and JN177 and had matched probes. Of them,17 had matched probes with higher abundance in SR3, all of which had SNPs, and 15 of which had InDels; 42 had matched probes with lower abundance in SR3,39 of which had SNPs and 27 of which had InDels.According to EST polymorphisms and probe annotations, we found the difference in gene expression between SR3 and JN177 was partially resulted from following genetic and epigenetic factors:1. high frequency of genomic variation such as SNPs and InDels; 2. the introgression of exogenous sequences from Thinopyrum ponticum; 3. change in transcriptional activity of retrotranspons and transpons; 4. chromatin remodeling resulted from the change in expression of histone variants and histone modification genes; 5. the alternation of DNA methylation patterns.Taken together, the results of large-scale sequencing of SR3 and JN177 cDNA libraries, comparison of sequence and cDNA microarry analysis indicated that high frequency of genomic variation and gene expression were taken place during asymmetric somatic hybridization, which changed the stress tolerance capacity and development potential of SR3; the mechanisms of genomic variation during asymmetric somatic hybridization, allopolyploidization and natural evolution were largely distinct. This work provided important information for understanding the mechanisms of asymmetric somatic hybridization and salt tolerance in plants.更多还原