【作者】 陈全家；

【导师】 陈章良； 康定明；

【作者基本信息】 中国农业大学 ， 作物遗传育种， 2014， 博士

【摘要】 陆地棉和海岛棉是目前世界上最具有商业价值的栽培棉种。陆地棉由于产量高和较强的环境适应能力而被广泛种植,占棉花产量的90%。相反,海岛棉仅占棉花产量的5-8%,由于其在长度、强度等方面具有优良纤维品质而价格昂贵。海岛棉(G. barbadense)和陆地棉(G. hirsutum)都是异源四倍体棉花栽培品种,但是他们的纤维品质和发育进程显著不同。虽然科研工作者在揭示棉纤维发育机制上,已经开展了大量的研究,但是目前对于海岛棉优异的纤维品质机理还不清楚,因此,本文对两个棉种胚珠进行转录本测序并进行分析,并对海、陆棉开花后5、10、15、25DPA的棉纤维进行了数字基因表达谱分析,筛选纤维发育各时期表达有差异的基因,以期揭示两个棉种在纤维发育时期的基因组表达差异。获得结果如下：1.对两个棉种胚珠进行转录本测序并进行分析,从海岛棉和陆地棉中分别获得79,686和67,450个unigenes。将获得的unigenes基因与最近公布的二倍体棉的参考基因组进行比较表明,大约有3/4的胚珠基因与二倍体棉蛋白编码的基因具有高度的同源性,同源性高达90.7%；其次,两个栽培品种胚珠的差异基因主要富集于胚后发育,这与他们纤维发育差异是一致的。最后通过SNPs (single nucleotide polymorphism,单核苷酸多态性)分析表明,两个转录组各有超过200,000个SNPs映射到二倍体基因组。两个四倍体品种间SNP差异可以解释他们间遗传差异。大多数核苷酸变异的基因参与了胚后发育和纤维发育,包括调控细胞大小和表皮毛分化。因此,本研究提供了海岛棉基因的完整列表,揭示数百个新的基因涉及两个棉种之间的不同纤维发育机制。2.根据棉花同一时期不同棉种之间,以及两种棉种同一时期之间的差异基因分析,两个棉种不同时期差异基因表达数目相似,在5DPA和10DPA,陆地棉和海岛棉共同差异表达基因有1,924个,其中上调的有1,419,下调的505个；在10-15DPA时期,共有936个差异基因,上调的基因只有292个,下调的基因有644个；在15-25DPA时期,共有2,169个差异基因,上调基因1,268个,下调基因901个。3.找出海岛棉和陆地棉之间的高可信度的差异表达基因,并对差异表达基因进行功能聚类分析(GO),发现海岛棉胚珠发育初期生毛细胞分化基因上调促进其棉纤维发育,海岛棉中纤维细胞的伸长相关基因上调促进其纤维发育,海岛棉纤维伸长期长于陆地棉,这也促进了棉纤维的发育。通过海岛棉和陆地棉纤维相关基因的共性和差异性,发现了海岛棉棉纤维品质优越的决定基因和分子机制。4.从海岛棉中扩增得到MYB转录因子基因GbMYB25与GbDET2基因,经测序验证、Blast比对与同源性分析,并实时荧光定量PCR分析表明,GbMYB25与GbDET2基因可能参与调控棉花纤维起始发育。更多还原

【Abstract】 Gossypium hirsutum and Gossypium barbadense are the most commercially cultivated cotton in the world G. hirsutum occupies about90%of the cotton production in the world due to its high yield and strong adaptation to diverse environmental conditions. On the contrary, G. barbadense accounting for only5-8%of the production is much more expensive due to its unprecedented quality of the fiber length, fitness and strength. Gossypium hirsutum and Gossypium barbadense are allotetraploid cotton cultivars differing greatly in their fiber quality and developmental programs. Although a huge number of studies and approaches have been carried out to uncover the cotton fiber developmental mechanisms, it remains unclear how G. barbadense achieves its superior fiber characteristics. Because the transcriptome of the fertilized ovule at the day of anthesis reflects the cotton genome expression at the fiber initiation stage, we sequenced their ovule transcriptomes and analyed the fiber digital gene expression profiling of Gb21and Gh36from5DPA to25DPA (including5,10,15and25DPA). The results were as follows:1.Here we sequenced and analyzed their ovule transcriptomes, and obtained79,686ovule unigenes for G. hirsutum and67,450for G. barbadense.The confidence of these unigenes was confirmed by three independent analyses. First, comparison with the recently published diploid cotton reference genome showed that90.7%of diploid protein-coding genes are homologous to over three quarters of these ovule unigenes. Second, the ovule unigenes differentially assembled between the two cultivars were strongly enriched in post-embryonic development, consistent with their fiber developmental difference. The two transcriptomes each harbored over0.2million SNPs when mapped onto the diploid genome, consistent with the reported phylogenetic distance. SNPs between the two tetraploid cultivars were recovered to address their genetic difference. Strikingly, the unigenes with most nucleotide variation were those involved in post-embryonic development, and in fiber development including regulation of cell size and trichome differentiation. Therefore, this study provides a comprehensive list of unigenes for G. barbadense, and revealed hundreds of novel unigenes involving in the different fiber developmental mechanisms between the two cultivars.2.Based on the analysis of differential gene expression between different strains of cotton during the same period and the same period of two strains,the number of differentially expressed genes were similar in different periods of two cotton varieties. At5DPA and10DPA, the differentially expressed genes of Gossypium hirsutum and G. barbadense were1,924, of which1,419were up-regulated,505were down-regulated, During the period of10-15DPA, there were936differential expressed genes, only292up-regulated genes,644down-regulated genes. During the period of15-25DAP, there were2169differential expressed genes, only1,268up-regulated genes,901down-regulated genes.3. We performed the Gene Ontology (GO) analysis for the highly confidence differentially expressed genes between Gossypium hirsutum and Gossypium barbadense, discoved that the up-regulated hairy cell differentiation genes in the initial stage of ovule development and fiber elongation-related genes promote sea-island cotton fiber development. The fiber elongation stage of sea-island cotton was longer than the upland cotton also facilitates the sea-island cotton fiber development. According to the anlysis of similarities and differences of the sea island fiber related genes, found the genes and molecular mechanisms of sea-island cotton fiber quality superior to upland cotton.4. Furthermore,one MYB transcription factor GBMYB25and GbDET2were amplified from the sea-island cotton, and verified by sequencing, Blast alignment and homology analysis. By the analysis of real-time fluorescent quantitative PCR showed that the two genes GbMYB25and GbDET2were involved in regulating the fiber developmen.更多还原