基于精细取样和多组学整合分析发掘柑橘珠心胚起始的关键基因和调控路径

During citrus apomixis, some nucellar cells deviate from somatic cell fate, and develop directly into nucellar embryos. Seedlings developed from nucellar embryos are identical to the maternal parent in genetic composition, which hinders cross breeding of citrus, but is applied widely in citrus rootstock propagation, seedling rejuvenation and scion improvement via somatic variation of nucellar embryos. However, the mechanisms that regulate the initiation and formation of nucellar embryo remain to be elucidated. In our previous study, comparative analysis of transcriptome and small RNAs were conducted in ovules between the mono- and poly-embryonic citrus varieties. In the present proposal, we plan to collect the nucellar tissue before nucellar embryo initiation and the nucellar embryo initial (NEI) cells with enlarged nucleus and condensed cytoplasm through laser microdissection (LMD). Through analysis of transcriptome, DNA methylation and small RNA profile of the above mentioned specific cell lineages, we intend to identify the genes and epigenetic regulations involved in nucellar embryo initiation. The function of potential regulatory genes will be validated first through the time-saving genetic transformation of citrus in vitro callus tissue, and then by transformation into the wild kumquat (Fortunella hindsii), the close relative of Citrus genera with short juvenility. The implementation of present proposal is expected to provide candidate genes and regulatory information for elucidating nucellar embryo initiation and formation, which also holds potential application for citrus variety improvement and rootstock propagation.

柑橘无融合生殖过程中，珠心细胞偏离体细胞分化途径，经特化直接发育形成珠心胚；珠心胚苗与母体遗传组成一致，是柑橘常规杂交育种的障碍，但被广泛用于柑橘砧木繁育、苗木复壮和接穗品种的珠心胚苗变异改良；然而，柑橘珠心胚如何起始和形成？其机理尚不清楚。基于申请人所在课题组以成对的柑橘单/多胚性材料而发掘的胚珠间的差异转录组和小RNA等前期基础，本申请项目拟以单、多胚柑橘的杂交分离群体和珠心多胚程度高的椪柑为材料，采用激光显微切割术精细取样珠心胚起始前的珠心组织和核大质浓的珠心胚起始细胞，通过转录组、DNA甲基化和小RNA组学整合分析，发掘珠心胚起始相关基因和表观遗传调控路径，筛选关键基因，并以柑橘愈伤组织体细胞胚发生这一离体快速转化体系和短童期的山金柑遗传转化体系验证候选基因的功能。本项目的实施将为解析柑橘珠心胚起始和形成机理提供候选基因和调控信息，对柑橘品种改良和砧木繁育也具有潜在的应用价值。

柑橘独特而稳定的珠心多胚性是典型的孢子体无融合生殖类型，被广泛用于繁育整齐一致的砧木和保持接穗种性，但也是柑橘杂交育种的主要障碍。然而，珠心胚起始发生的调控路径和生物过程尚不明晰。为发掘柑橘珠心胚起始的关键基因和调控路径，本项目采用激光显微切割技术精细取样结合转录组（RNA-Seq）和重亚硫酸盐测序（BS-Seq），分析无融合生殖细胞的基因表达和DNA甲基化特征。研究结果发现胚发育和氧化逆境响应过程在无融合生殖细胞显著上调富集，鉴定了无融合生殖细胞特异表达基因CitRWP和CitZFP，发现CitRWP基因编码区的DNA甲基化水平在无融合生殖细胞中较高，多胚型胚珠相比单胚型胚珠积累更多活性氧物质（ROS）。由此提出柑橘珠心胚起始工作模型，即CitRWP基因区DNA超甲基化增强了其在无融合生殖细胞表达，CitRWP促进锌指蛋白转录因子基因CitZFP表达，进而激活下游胚发生途径，诱导柑橘珠心组织向胚性细胞转变，完成珠心胚起始发生。此外，在多胚的甜橙和单胚的柚基因组鉴定B3转录因子超家族基因，发现CsFUS3在生胚过程高表达，证实CsFUS3通过调节愈伤组织中ABA/GA含量比促进胚发生。优化柑橘愈伤原生质体瞬时转化体系，使转化率高达50%-70%，蛋白亚细胞定位成功率高于60%，编辑效率10.7%，为再生无外源基因插入的基因编辑植株提供了技术体系。本研究的实施提出柑橘珠心胚起始发生的工作模型，筛选了珠心胚起始关键基因并解析了胚发生调控通路，完成了项目目标，为高效利用无性胚发生提高柑橘育种和繁育效率提供了理论参考，丰富了植物胚发生的理论认知。已在Journal of Experimental Botany、DNA Research等国际学术期刊发表论文5篇，在园艺学报发表摘要1篇，授权发明专利1项，培养博士生4人、硕士生1人。

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