【作者】 李菲；

【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】 本实验室以普通小麦济南177(JN177)原生质体为受体,经380gw/cm2紫外线照射30s的长穗偃麦草原生质体为供体进行非对称的体细胞杂交,获得了可育杂种植株,从中筛选出一系列高产、耐盐、耐旱、优质的小麦渐渗系新品种和新种质。本实验室前期的SSR分析表明小麦体细胞杂种渐渗系不仅含有长穗偃麦草的遗传物质,而且亲本JN177的DNA序列也发生了较大变异。对亲本济南177/长穗偃麦草和体细胞杂种渐渗系的高/低分子量麦谷蛋白亚基基因家族的克隆和序列比较分析表明,体细胞杂交过程产生了许多亲本小麦济南177所不具有的新的高、低分子量麦谷蛋白亚基基因。体细胞杂种渐渗系中的这些新基因,一部分是长穗偃麦草的基因渐渗进入济南177基因组,或者是济南177原有基因的变异,包括点突变和复制滑动,还有些来自双亲基因的重组。4个优质杂种渐渗系的所有22个麦谷蛋白亚基基因中,两个来源于长穗偃麦草,其它20个均在体细胞杂交过程中发生等位变异。进一步在全基因组水平研究体细胞杂交过程引起的遗传和表观遗传变异及其机制是将体细胞杂交技术应用于小麦作物改良的基础。本研究选择分别具有高产和耐盐新性状的两种渐渗系新品种和新品系山融1号(SR1)及山融3号(SR3)为材料,利用各种分子标记在全基因组水平上对体细胞杂交过程引起的小麦重复序列和基因编码区的DNA序列变异进行分析,探讨这种变异的可能机制。此外还对体细胞杂种渐渗系的基因表达变异进行分析,进而了解渐渗系表型和性状变异的原因,为更好地将体细胞杂交技术及杂种新种质用于育种实践奠定理论基础。另外,探究体细胞杂交引起的基因启动子区和编码区DNA甲基化修饰的变化可以帮助了解体细胞杂种渐渗系基因表达变化的机制,有助于解释杂种渐渗系和亲本济南177间的表型差异,也有助于我们对小麦耐盐机制的理解。本研究的主要结果如下：1.非对称体细胞杂交引起的遗传变异分析利用SSR/AFLP/EST-SSR/IRAP/REMAP技术系统分析体细胞杂种渐渗系SR1、SR3和JN177在重复序列、基因编码区、反转录转座子等的差异,并与多倍体化过程相比较,探讨体细胞杂交引起DNA序列变异的机制。利用基因组SSR位点分析可以了解杂种基因组简单重复序列的多态性及其产生的原因。通过对两个体细胞杂种渐渗系、JN177及长穗偃麦草基因组116个微卫星位点的研究,我们发现杂种渐渗系含有少量长穗偃麦草的遗传物质。与JN177相比,体细胞杂种渐渗系SR3和SR1在40(34.48%)个位点上发生了变异。两个体细胞杂种渐渗系间在检测的基因组SSR位点上没有出现差异。另外对体细胞杂种渐渗系SR3\SR1 R2和R10代间在SSR位点上的变异进行检测,我们发现体细胞杂种渐渗系早代和晚代间遗传序列保持稳定。为了研究体细胞杂交引起的基因编码区的简单重复序列(SSR)的变异,并与基因组SSR序列变异相比较,本实验利用根据小麦EST序列设计的SSR引物对体细胞杂种渐渗系的R2代与两个亲本JN177、长穗偃麦草的基因组进行分析。在所有检测的108对引物中,22(20.37%)对引物在SR3与JN177间表现出多态性的带型,比SR3与济南177间基因组SSR序列的差异(34.48%)小得多。同样,R2和R10代间的EST-SSR序列亦保持稳定。AFLP分析是在全基因组水平上对体细胞杂交引起的变异进行全面分析的技术。通过对杂种后代以及亲本基因组904个位点进行AFLP分析发现在杂种渐渗系SR3中消减的条带总数为28条,新出现的条带总数为20条。非对称体细胞杂交过程中亲本JN177约有3.54%的位点发生变异。本实验利用IRAP和REMAP技术检测体细胞杂种渐渗系反转录转座子的激活情况。在JN177和杂种渐渗系SR1/SR3中共检测了116个位点,其中10(8.26%)个位点在JN177和山融3号中表现出多态性。通过对部分发生变异的条带进行DNA序列分析,并在Genbank搜索其同源序列以了解变异序列的信息,我们发现反转录转座子的激活可能影响到其位点附近的功能基因。本研究利用SSCP技术检测体细胞杂交引起的JN177基因序列的变异。结果显示非对称体细胞杂交引起JN177基因组基因序列大片段的插入和删除,并影响对应基因的表达。另外非对称体细胞杂交引起JN177基因组基因序列的点突变也是一个广泛存在的现象。大量新的基因片段的出现是体细胞杂交不同于多倍体化过程的一个显著特点。以上结果表明：体细胞杂交过程同样引起亲本基因组序列的大规模变异,各种不同的序列组分在体细胞杂交过程中变异的比率有显著的差异。基因组中的易变位点,如SSR序列和反转录转座子序列变异比率分别高达34.4%和8.26%,而基因序列的变异比率(包括基因序列丢失/插入和SNP)为4.17%。各种不同序列所处染色质位置的不同结构可能是各种序列变异比率不同的原因。调控染色质结构的表观修饰机制在这一过程中的作用是个值得进一步的研究的问题。体细胞杂交引起的重复序列、基因编码序列等的变异与常规远缘杂交多倍体化过程都有明显的不同。同样经历了异源基因组造成“冲击"(shock),体细胞杂交与多倍体化存在显著的差异。此外,体细胞杂交还同时反映了远缘杂交和渐渗过程中的基因组变异的特点,是研究植物远缘杂交中植物基因组应对外源“冲击"(shock)和进化的一个不同于多倍体化的新的体系。2.非对称体细胞杂交引起的表观修饰变异包括DNA甲基化修饰在内的表观遗传修饰机制对基因表达调控具有重要的作用。本实验利用MSAP技术检测体细胞杂交引起的基因组水平的胞嘧啶甲基化修饰变异。在对照培养条件下,我们发现渐渗系SR3和亲本济南177相比在84(23.33%)个位点上出现甲基化修饰差异。体细胞杂交中DNA甲基化修饰状态发生变异的位点绝大多数在两个杂种渐渗系中保持一致。这说明体细胞杂交过程中DNA甲基化修饰状态的变异不是随机发生的。对甲基化修饰状态发生变异的位点进行测序比对发现变异序列主要来自反转录转座子。本实验还对盐处理条件下亲本济南177和渐渗系基因组DNA甲基化修饰状态进行分析,发现盐处理仅对基因组DNA甲基化修饰状态有很小的影响。体细胞杂交引起的DNA甲基化修饰变异是渐渗系与亲本济南177间DNA胞嘧啶甲基化修饰状态差异的主要原因。为了具体了解体细胞杂交引起的DNA胞嘧啶甲基化修饰状态的变化对基因表达的影响,本实验还选择了5个山融3号中已经鉴定功能的耐盐相关基因和启动子序列进行重亚硫酸盐测序,在碱基水平上精细分析基因序列中胞嘧啶甲基化修饰状态的变异,以及这种变异对基因表达的影响。本实验发现小麦基因启动子区域被甲基化修饰的比例较高,基因表达与启动子区域胞嘧啶甲基化修饰水平呈负相关。本实验检测的3个小麦基因编码区中,仅有1个基因编码区被甲基化修饰。体细胞杂交引起了SR3与JN77基因启动子区域广泛的胞嘧啶甲基化修饰变异,变异的比率根据基因的不同有所不同,从最低0.00%(TaWRKY1-7)到最高25.00%(TaCHP),平均变异比率为12.26%。我们发现非对称体细胞杂交引起的基因启动子序列胞嘧啶甲基化修饰程度变异对杂种渐渗系中对应基因的表达有重要的影响。但是除TaFLS2基因外,启动子区域胞嘧啶甲基化修饰变异趋势与基因芯片检测对应基因的表达变异情况并不完全对应。DNA甲基化修饰与激活或抑制基因表达的各种组蛋白修饰协调作用,共同形成调控基因表达的严密而灵敏的机制。DNA甲基化修饰作为表观修饰机制的一种,与基因表达量不能完全对应是正常的。我们还需要进一步研究体细胞杂交引起的其它表观遗传变异,综合理解体细胞杂种渐渗系中基因表达变异的机制。我们认为非对称体细胞杂交过程中,在细胞核中共存的长穗偃麦草和济南177的基因组包括同源序列和非同源序列间存在着复杂的相互作用,引起各种遗传和表观遗传变异。遗传和表观遗传变异之间并不是相互独立的。DNA胞嘧啶去甲基化引起反转录转座子的激活,同源序列间的相互作用可能引起基因沉默等。各种遗传和表观遗传变异结合起来,共同促进了体细胞杂种渐渗系优良表型的出现。3.非对称体细胞杂交引起的基因表达变异本实验利用SSCP技术分析了JN177和体细胞杂种渐渗系SR1/SR3在盐处理和对照培养条件下根和叶的基因表达情况。在检测的80对引物中,我们发现对照培养条件下有6(7.5%)对引物在渐渗系中发生了一个同源基因表达被抑制的情况。此外我们还发现了三例(3.75%)某一同源基因在渐渗系中被激活的现象。在SSCP分析中没有发现体细胞杂种渐渗系SR1/SR3间基因组DNA序列的差异。但是在叶的mRNA分析中我们发现两例(2.5%)SR1/SR3间基因表达差异的情况,而且SR1/SR3间基因表达差异具有器官特异性。这很可能是由于两个渐渗系间基因表达调控的差异引起的。渐渗系中基因表达的激活或沉默部分是由DNA序列的变异、基因表达调控网络和表观修饰的变化引起的。我们发现在渐渗系中酪蛋白激酶CK2和葡萄糖-6-磷酸脱氢酶基因被激活。这两个基因的激活对渐渗系的表型差异可能有重要的影响。本实验利用SSR/EST-SSR/AFLP/IRAP/REMAP/SSCP等多种分子标记技术对亲本济南177和两个具有优良表型的体细胞杂种渐渗系新材料SR1和SR3间的遗传、表观遗传和基因表达变异进行系统的研究。通过本论文的研究发现,非对称体细胞杂交过程引起了济南177基因组遗传、表观遗传水平的广泛变异。SSR序列和反转录转座子的变异对济南177的基因序列产生了影响。同源重组和非同源末端连接(NHEJ)等DNA修复方式也造成了基因编码序列的变异。表观修饰的变异对渐渗系的基因表达水平产生影响。遗传和表观遗传水平的各种变异综合起来促进了渐渗系表型的改善。非对称体细胞杂交过程引起的遗传和表观遗传变异与远缘杂交和多倍体化过程有相似的地方,也有自己独特的特点。因此体细胞杂种渐渗系是研究植物基因组进化的一个全新的体系。更多还原

【Abstract】 We obtained various fertile introgression lines through asymmetric somatic hybridization between protoplast of common wheat (Triticum aestivum) cv. JN177 and UV-irradiated protoplast of tall wheatgrass (Thinopyrum ponticum) and these lines have inherited to F10 generation. Some of these introgression lines showed stable superior agronomic traits compared to JN177, including salt and/or drought tolerance, dwarf habit, disease resistance, high yield and good processing quality etc. SSR analysis conducted by Chen et al.(2004) indicated that genetic materials of Tall wheatgrass have been introgressed into the genome of JN177, and the SSR sequences of JN177 was altered by asymmetric somatic hybridization. Cloning and sequence analysis of HMW-GS/LMW-GS genes of various introgression lines and the two parents JN177 and tall wheatgrass showed that novel glutenin genes not discovered in JN177 appeared in somatic hybrid lines.These novel glutenin genes appeared in the introgression lines come directly from tall wheatgrass or from the recombination between HMW-GS/LMW-GS gene sequences of the two parents or gene sequence alterations of JN177 glutenin genes.14 of 37 cloned JN177 HMW-GS/LMW-GS genes were altered by asymmetric somatic hybridization. Further studies on the manner and mechanisms of genetic variation induced by asymmetric somatic hybridization are the basis for understanding these variations and will be helpful for wheat improvement utilizing these hybrid lines. To explore genomic variation derived from "genomic shock" via parental genome merging and donor genome introgression, a genetic analysis was performed with two asymmetric somatic introgression lines (released cultivars) involving bread wheat(Triticum aestivum), and its relative tall wheatgrass(Thinopyrum ponticum Podp). The two cultivars have proven to be phenotypically stable through a number of selfing generations. A spectrum of cytogenetic assays and DNA profiling techniques was applied to understand the nature of the genetic and epigenetic changes which had been induced by somatic hybridization. At the chromosome level the cultivars appeared very similar to their wheat parent but containing introgressed chromatin. The molecular profiling revealed many genetic and epigenetic differences, including the elimination of genomic DNA, the altered regulation of gene expression, changed patterns of cytosine methylation, and the reactivation of retrotransposons. Repetitive sequences combined with the epigenetic regulation of gene expression and/or retrotransposon transposition appear to be largely responsible for the phenotypic differences between the introgression cultivars and their wheat parent. These results confirmed that asymmetric somatic hybrid intogression lines provide useful materials to explore the nature of the genetic and epigenetic variation of the derivatives induced by genomic shock.The results of my research are listed as follows:1. Genetic alterations induced by asymmetric somatic hybridizationWe utilizd various molecular markers, including SSR/AFLP/EST-SSR/IRAP/ REMAP to analyse the genomic sequences of the introgression lines and the two parents, so as to obtain the data about the alterations of repetitive sequences/ restrotransposons and gene sequences of JN177 genome induced by asymmetric somatic hybridization, and then analyse underlying mechanisms.An examination of the genomic SSR profiles of cv. JN177, SR1 and SR3 revealed no polymorphism between SR1 and SR3, but at 40 of the 116 genomic SSR loci, the cv. JN177 profile differed from that of R2 SR1 and SR3 individuals. At two loci, SR1, SR3 and tall wheatgrass shared the same allele. No variation in SSR profile between the R2 and R10 generations of SR1 or SR3 was observed (data not shown). Given the genotypic stability of SR1 and SR3 over many generations, the remaining fingerprinting analysis was restricted to the R2 generation.Analysis of EST-SSR alterations between JN177 and the two introgression lines indicated that 22(20.37%) of 108 loci were altered in SR3 when compared with JN177, a percentage much lower than that of genomic SSR alterations(34.48%).AFLP profiling revealed 904 cv. JN177 and 777 tall wheatgrass fragments, of which 388 were common to both templates. The comparisons of cv. JN177 with SRI and SR3 revealed 872 monomorphic fragments. Eighteen cv. JN177 fragments were absent from both SRI and SR3, while another 4 and 10 fragments were eliminated from SRI and SR3 respectively; except for the eliminated fragments, SRI and SR3 showed respectively 16 and 20 novel bands.IRAP and REMAP were used to analyze retrotransposon movement in response to somatic hybridization. Of 116 fragments detected,10 were polymorphic between cv. JN177 and SR3. Five of the 10 fragments were absent, and other 5 fragments were present in SR3 but not in cv. JN177. Eleven bands were polymorphic between cv. JN177 and SRI, with 6 bands unique to JN177 and 5 unique to SRI. There were 3 polymorphisms between SRI and SR3. In an attempt to identify the genes associated with retrotransposon activation, some of the unexpected fragments were sequenced, but the analysis of these sequences only recognized homology related to one (present in cv. JN177, absent in SR3), which identified a Ty3-gypsy subclass retrotransposon (ABA97230.1). A second fragment present in SR1/SR3, but absent from cv. JN177 was highly homologous to a wheat pore-forming toxin-like protein Hfr-2 (AAW48295.1), involved in plant defence.SSCP analysis was utilized to detecte the alterations of JN177 gene sequences. Gene loss and insertion were detected in the two introgression lines and gene sequence alterations effects corresponding gene expression. SNPs between JN177 and the two introgression lines were also a commonly observed phenomeon. Novel gene bands in the introgression lines were a unique feature of asymmetric somatic hybridization, different from that of polyploidization.These results indicated that asymmetric somatic hybridization induced large scale DNA sequence alterations of JN177, but different components of JN177 genome showed largely different tendency to alternation. Highly mutantable sequences, genomic SSR sequences showed an alternation percentage of 34.4%, while only 4.17% gene sequence of JN177 was altered in somatic hybrids.We supposed that different chromosome configurations between different sequence types were the main cause of different alternation tendencies. Different epigenetic modifications, including cytosine methylation/histone modifications/chromatin remodeling, around different sequences prabably contribute to this process. We also found that the genetic alterations induced by somatic hybridization showed different characters when compared with that of ployploidization. Asymmetric somatic hybrid intogression lines provide useful materials to explore the nature of the genetic and epigenetic variation of the derivatives induced by genomic shock.2. Epigenetic alterations induced by asymmetric somatic hybridizationThirteen pairs of selective primers were used to determine methylation status at 360 CCGG loci. Of these, SR3 and cv. JN177 differed for 84 fragments, with 35 appearing to be hypermethylated and 49 hypomethylated in SR3; while SRI and cv. JN177 differed for 77 fragments, with 31 appearing to be hypermethylated and 46 hypomethylated in SRI. Although rarely happened, the hypermethylation from H to M banding pattern or hypomethylation from M to H banding pattern was detected in the introgression lines. There were less MSAP polymorphisms between SRI and SR3 (25,6.9%) than between cv. JN177 and the two introgression cultivars, of which 12 were hypermethylated and 13 hypomethylated in SRI. Sequence analysis of a number of differentially methylated fragments showed that three of the five hypomethylated fragments in SR3 were highly repeated retrotransposons (TREP acession number: TREP255, TREP1418, TREP3251), and two showed no significant similarity to any known sequence. While the five fragments hypermethylated in SR3 also showed no significant similarity to any known sequence.We also analysed the cytosine methylation pattern of JN177 in gene promoter and coding sequences using bisulfite sequencing, in order to investigate the cytosine methyaltion alterations and its effects on gene expression. The results indicated that the gene promoter sequences of wheat were highly methylated, and cytosine methylation in gene promoter negatively affects corresponding gene expression. Only one of three analysed gene body sequence was methylated. The cytosine methylation level in wheat gene promoter was altered by asymmetric somatic hybridization. The percentage of cytosines with altered methyaltion pattern was different between different genes, from 0.00%(TaWRKY1-7) to 25%(TaCHP). The average alternation percentage was 12.26%. The alternation of cytosine methylation level between JN177 and SR3 was not always correlates with gene expression alteration, except for TaFLS2. We suppose that epigenetic modifications except for cytosine methylation, such as histone acetylation/methylation and chromatin remodeling also contributes to gene expression alterations in the somatic hybrids. Further study on histone modification/chromatin remodeling alterations between JN177 and introgression lines is needed to fully understand the mechanism of gene expression alterations.We suppose that homologous sequences between JN177 and tall wheatgrass may experience complex interactions and induce various genetic and epigenetic varitions in the introgression lines. And genetic and epigenetic alterations all contribute to gene expression alteration and superior agronomic traits of the somatic hybrids.3.Gene expression alterations induced by asymmetric somatic hybridizationTo survey the extent of gene sequence alterations and homoeologous gene silencing or activation induced by somatic hybridization, SSCP analysis was performed using genomic DNA from cv. JN177, SR1 and SR3 and RNA from the shoots and roots of cv. JN177, SR1 and SR3 seedlings. Of the 80 SSCP amplicons,11 were informative between cv. JN177 and SR1/SR3. In four of six missing cDNA fragments, the corresponding genomic amplification products were also missing. Other two missing cDNA was absent from one or both of SR1/SR3 compared to cv. JN177. In two activated cDNAs, products were present in SR1/SR3 but not in cv. JN177. For other three of these genes, the migration of the corresponding cDNAs was also affected. Similar to genomic SSR analysis, we did not find any difference between SR1 and SR3 in genomic sequences in SSCP analysis. However, SR1/SR3 shoot showed qualitatively different gene expression in two genes tested. It is likely that these two genes were differently regulated in SRI and SR3.In conclusion, somatic hybridization between wheat and tall wheatgrass has generated a series of introgression lines displaying a variety of novel genetic and epigenetic changes relative to their wheat parent. The genetic alterations induced by somatic hybridization showed different characters when compared with that of ployploidization. Asymmetric somatic hybrid intogression lines provide useful materials to explore the nature of the genetic and epigenetic variation of the derivatives induced by genomic shock. Moreover, we found repetitive sequences, epigenetic regulation of gene expression and/or retrotransposon transposition mainly relative to the novel phenotypic and agricultural traits. Therefore, somatic hybrid introgression lines provide a unique means to explore the nature of the genetic and epigenetic variation induced by genomic shock. An understanding of these mechanisms should improve our understanding of the genetic basis of agriculturally important phenotypic variation and may shed light on the variation generated by sexual wild hybridization.更多还原