摘要
林木多倍体形态上的巨大性受到林木育种工作者的广泛重视,研究林木染色体加倍后基因转录调控的变化,对于深入理解林木多倍体的变异具有重要意义。本研究以前期获得的白桦四倍体为试材,对其表型特征、光合特性、生理指标、花粉特性等进行观察,同时开展了基于RNA-Seq技术的转录组学研究,为深入理解白桦四倍体的变异、白桦重要性状关键调控基因的克隆以及白桦基因工程育种提供了参考。研究结果如下：
白桦四倍体的叶部形态具有明显的巨大性,其叶面积和气孔长度分别高于二倍体的10.54%和95.73%。四倍体的光合作用明显强于二倍体,其净光合速率、PSⅡ实际光化学效率和光化学淬灭分别比二倍体高13.33%、12.38%和10.68%。四倍体在生理指标和激素含量方面与二倍体的差异达到显著水平,四倍体的可溶性糖含量、可溶性蛋白含量、叶绿素含量、生长素含量、脱落酸含量和赤霉素含量分别比二倍体高8.24%、12.52%、6.21%、32.93%、13.15%和39.43%。白桦四倍体的花序也表现出巨大性,雄花序长度和雄花主苞片长度分别比二倍体高44.02%和9.30%,果序长度和果序直径分别高于二倍体16.40%和33.69%,花粉直径较二倍体高27.75%。白桦四倍体的不育性主要体现在雄配子体上,白桦四倍体的花粉萌发率比二倍体低64.66%。此外,不同白桦四倍体的生长和生殖特性也具有明显的差异。
采用Solexa技术测定了白桦四倍体和白桦二倍体的转录组,通过转录组的测序和组装,共得到132427个unigene,其中47.61%的unigene得到注释。根据Nr数据库的注释结果,55.78%的unigene与已知序列具有较高的同源性；17.99%的unigene与已知序列具有较高的相似度；来源于葡萄的unigene最多,占注释unigene的34.08%。白桦四倍体与二倍体相比,共有1166个unigene上调表达,890个unigene下调表达。GO分析表明白桦四倍体与二倍体在其他器官细胞凋亡、细菌起源分子的细胞响应、细胞凋亡铁离子结合、蛋白丝氨酸-苏氨酸磷酸酶活性、血红素结合、单加氧酶活性、高亲和性铜离子跨膜转运蛋白活性和四吡咯结合等9个方面差异显著。Pathway分析表明白桦四倍体与二倍体在RNA聚合酶、嘧啶代谢、植物与病原体相互作用、嘌呤代谢、二苯乙烯类化合物、二芳基庚酸类化合物和姜辣素的生物合成、柠檬烯和蒎烯的降解和次生代谢产物生物合成等7个途径差异显著。白桦四倍体与二倍体以及不同白桦四倍体之间在RNA聚合酶、基础转录因子、核糖体、植物激素合成及信号转导、糖代谢以及昼夜节律方面发生了明显变化,初步认为白桦四倍体与二倍体以及不同白桦四倍体之间在生长和生殖上的差异与其在RNA聚合酶、基础转录因子、核糖体、植物激素合成及信号转导、糖代谢以及昼夜节律方面的变化有关。
Tree breeders have focused much attention on polyploid trees because of their importance to forestry. To evaluate the impact of intraspecies genome duplication on phenotype and transcriptome, morphology, photosynthesis, physiology, pollen characterist and transcriptome were measured between diploid and autotetraploid. These results shed light on variations in birch autotetraploidization and help identify important genes for the genetic engineering of birch trees. These results were as follows:
There were significant variations in leaf morphology between diploid and autotetraploid. The leaf area and stoma length of autotetraploid were increased10.54%and95.73%respectively compared to those of diploid. The photosynthsis of autotetraploid was superior to that of diploid. The net photosynthetic rate, actual photochemical efficiency of PS Ⅱ and photochemical quenching of autotetraploid were increased13.33%,12.38%and10.68%respectively compared to those of diploid. There were significant variations in physiology and plant hormones between diploid and autotetraploid. The content of soluble sugar, soluble protein, chlorophyll, auxin, abscisic acid, gibberellin of autotetraploid were increased8.24%,12.52%,6.21%,32.93%,13.15%and34.93%respectively compared to those of diploid. The size of flower was larger in autotetraploid compared with diploid. The length of catkin and primary bract from staminate flowers, pollen diameter, the length and diameter of fruit of autotetraploid were increased44.02%,9.30%,27.75%.16.40%and33.69%respectively compared to those of diploid. The sterility of autotetraploid was reflected in male gamete. The germination rate of pollen in autotetraploid was decreased64.66%compared to that of diploid. In addition, there were significant variations in growth and reproduction in different autotetraploids.
In the present study, we compared differences between transcriptomes of diploid and autotetraploid using Solexa sequencing technology. Short reads were assembled into unigenes, taking the distance of pair-end reads into account. All of the sequences were assembled, yielding132427non-redundant unigenes. Approximately47.61%of the unigenes were annotated by Blastx and Blastn against six public databases (Nr, Nt, Swiss-Prot, KEGG, COG and GO), with a threshold of10-5. The E-value distribution of the top hits in the Nr database revealed that55.78%of the mapped sequences showed strong homology (E-value≤10-30), and17.99%of the sequences shared greater than80%similarity. Nearly34.08%of the unigenes could be annotated using sequences from Vitis vinifera which was the most top-hit specie. Transcriptome data revealed numerous changes in gene expression attributable to autotetraploidization, which resulted in the upregulation of1166unigenes and the downregulation of890unigenes. GO enrichment analysis revealed that significant differences were seen between autotetraploid and diploid in the GO categories killing of cells of other organism, cellular response to molecule of bacterial origin, cell killing, iron ion binding, protein serine/threonine phosphatase activity, heme binding, monooxygenase activity, high affinity copper ion transmembrane transporter activity and tetrapyrrole binding. Differences in the dynamics and absolute levels of unigene expression between autotetraploid and diploid were seen in the following categories:RNA polymerase, pyrimidine metabolism, plant-pathogen interaction, purine metabolism, stilbenoid, diarylheptanoid and gingerol biosynthesis, limonene and pinene degradation and biosynthesis of secondary metabolites. Transcriptome analysis revealed that143genes involved in RNA polymerase, basal transcription factors, ribosome, plant hormone biosynthesis and signal transduction, sugar metabolism, circadian rhythm were altered after genome duplication, which may have contributed to phenotypic changes.
引文
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