棉花种子质量突变体ims-15及其近等基因系种子发育期转录组分析

Transcriptome Analysis of Cotton Seed Weight Mutant ims-15 and Its Near Isogenic Line During Seed Development

为了解析棉花种子质量突变体ims-15千粒质量降低的相关机制,以该突变体和其近等基因系Ji737系为试验材料,利用Illumina平台测定了开花后30 d种胚的转录组,并利用实时荧光定量PCR技术对测序结果进行验证。共筛选获得4239个差异表达基因,其中,在ims-15中上调表达基因2229个(52.6%),下调表达基因2010个(47.4%)。GO功能富集分析表明,差异表达基因显著富集在生物过程中的细胞壁高分子代谢、光合作用-光能捕获、细胞壁高分子代谢、光合作用等7个条目,分子功能中的叶绿素结合、四吡咯结合、铁离子结合等4个条目和细胞组分中的光系统、类囊体、光合膜等9个条目,其中光合作用相关条目注释到的差异基因中下调基因占比达85.11%,且光能捕获、叶绿素结合和光系统Ⅰ等3个条目注释到的差异基因均为下调基因。KEGG富集分析表明,光合作用-天线蛋白、类黄酮生物合成、植物激素信号转导、苯丙素的生物合成、MAPK信号途径、糖酵解/糖异生、淀粉和蔗糖代谢等20个代谢通路显著富集,其中光合作用-天线蛋白通路的富集程度最高且该通路注释到的18个差异表达基因均在ims-15中下调表达,植物激素信号转导通路中富集到的差异基因最多。此外,发现有大量转录因子如WRKY、Dof和AP2/EREBP等家族成员影响种子粒质量的形成。qRT-PCR和RNA-seq数据相关系数R2=0.9559,验证了RNA-seq结果的可靠性。基于各项分析结果,明确了一些与种子质量发育密切相关的代谢途径,尤其胚性光合作用途径在种子质量形成中起重要作用;发现了植物激素信号转导途径和转录因子在种子质量形成中起着重要调控作用。

In order to elucidate the mechanism of thousand kernel weight(TKW)reduction in ims-15,the transcriptomes of 30 days post anthesis(DPA)embryos of ims-15 and its near isogenic line Ji737 were compared,and the sequencing results were verified by Real-time quantitative PCR.A total of 4239 differentially expressed genes(DEGs)were screened,including 2229(52.6%)up-regulated genes and 2010 down-regulated genes(47.4%)in ims-15.GO functional enrichment analysis showed that the DEGs were significantly enriched in seven items of biological processes such as cell wall macromolecular catalysis,photosynthesis-light harvesting,cell wall macromolecular metabolism and photosynthesis,and in four items of molecular functions such as chlorophyll binding,tetrapyrrole binding and iron ion binding,and in the nine items of cellular components such as photosystem I,thylakoid part,photosynthetic membrane and thylakoid.three categories of biological processes,molecular functions,and cellular components.In the category of biological processes,seven items such as cell wall macromolecular catalysis,photosynthesis-light harvesting,cell wall macromolecular metabolism and photosynthesis were enriched.And in the category of molecular functions,four items such aschlorophyll binding,tetrapyrrole binding,and iron ion binding were enriched.And in the category of cellular components,nine items such as photosystem I,thylakoid part,photosynthetic membrane and thylakoid were enriched.Down-regulated genes annotated in photosynthesis-related items accounted for 85.11%of all DEGs in these items.And all of the DEGs annotated in photosynthesis-light harvesting,chlorophyll binding and photosystem were down regulated in ims-15.KEGG enrichment analysis showed that 20 metabolic pathways,including photosynthesis antenna protein,flavonoid biosynthesis,phytohormone signal transduction,phenylpropanoid biosynthesis,MAPK signaling pathway,glycolysis/gluconeogenesis,starch and sucrose metabolism,were significantly enriched.The enrichment of photosynthesis-antenna protein pathway was the highest and the 18 DEGs enriched in this pathway were down regulated in ims-15.The number of DEGs enriched in the plant hormone signal transduction pathways was the most.Transcription factor analysis obtained a large number of transcription factors that affect kernel weight formation,such as WRKY,Dof,AP2/EREBP et al.The correlation coefficient between qRT-PCR and RNA-seq data was 0.9559,which verified that the results of RNA-seq were accurate and reliable.Based on the results of GO functional enrichment analysis,KEGG enrichment analysis and transcription factor analysis,some metabolic pathways closely related to the development of grain weight had been clarified.Especially,the embryonic photosynthetic pathway played an important role in the formation of kernel weight.It was discovered that plant hormone signal transduction pathways and transcription factors played an important regulatory role in kernel weight formation.