基于RNA-seq技术分析雄性不育宁夏枸杞花药不同发育时期基因表达特征

Analysis of Gene Expression Characteristics at Anther Development Stages of the Male Sterile in Wolfberry(Lycium barbarum)Based on RNA-seq

本研究利用半薄切片法对比分析了宁夏枸杞(Lycium barbarum)雄性不育系‘宁杞5号’和可育系‘宁杞1号’花药不同发育时期的细胞学特征,并进一步采用RNA-seq技术分别对‘宁杞5号’和‘宁杞1号’小孢子母细胞时期、四分体时期和单核时期的花药进行转录组比较分析。结果显示,不育系‘宁杞5号’的绒毡层在四分体时期开始出现异常,同时四分体外胼胝质壁并不发生降解,最终导致不能产生可育花粉粒。花药发育三个时期的转录组分析共筛选到2619个差异表达基因(differentially expressed genes,DEGs),其中1609个上调,1010个下调,另有70个DEGs在三个不同发育时期均有出现。GO及KEGG分析均表明,2619个DEGs主要参与胼胝质合成与降解、脂质代谢、类黄酮生物合成等过程。RT-qPCR验证结果与转录组测序结果基本一致。通过对转录组DEGs的分析,筛选到9个可能与花粉败育相关的胼胝质合成与降解的DEGs,以及19个与花药发育相关的差异表达转录因子。该研究为深入解析宁夏枸杞花药雄性不育的分子机制提供新的基因资源,奠定了一定的基础。

In this study,the different developmental stages of anthers cytological characteristics of Lycium barbarum male sterile line′Ningqi No.5′and fertile line′Ningqi No.1′were compared and analyzed by semi-thin section,and the transcriptomic profiles of anthers of′Ningqi No.5′and′Ningqi No.1′in microspore mother cell stage,tetrad stage and mononuclear stage were compared by RNA-seq technology.The results showed that the abnormal tapetal layer of the sterile line′Ningqi No.5′began to appear in the tetrad period,and the callose wall outside the tetrad did not degrade,resulting in the inability to produce fertile pollen grains.A total of 2619 differentially expressed genes(DEGs)were screened by transcriptome analysis at the three stages of anthers development,of which 1609 were up-regulated and 1010 were down-regulated,and 70 DEGs were found at all three different developmental stages.GO and KEGG analysis showed that these 2619 DEGs are mainly involved in callose synthesis and degradation,lipid metabolism and flavonoid biosynthesis.The results of RT-qPCR were basically consistent with those of transcriptome sequencing.By analyzing the transcriptome DEGs,we identified 9 DEGs which may be related to callose synthesis and degradation,and 19 different-expressed transcription factors related to anther development.This study provides a new genetic resource and lay a foundation for further analysis of the molecular mechanism of male sterility in anther of Lycium barbarum.