沙田柚配子体自交不亲和性的分子机理研究

The newly developed Solexa/Illumina RNA-sequence and DGE high-throughput deepsequencing approaches have dramatically changed how functional complexity of the transcriptome can be studied. Self-pollinated styles and cross-pollinated styles of Citrus grandis var. Shatinyu Hort are used for material. Total RNA was isolated from the self-pollinated styles and cross-pollinated styles，and mRNAs are isolated from total RNA. Cleaved RNA fragments were subsequently purified and amplified through PCR to create the final cDNA libraries. Then, cDNA libraries of transcriptome will sequenced with high throughput transcriptome sequencing technology. For validation and annotation of assembled unigenes, sequence similarity search is conducted against the NCBI non-redundant protein (Nr), Uniprot, CDD database and the Swiss-Prot protein database. On the basis of Nr annotation, the unigene sequences are also aligned to the COG and GO database to predict and classify possible functions. Pathway assignments will be carried out according to the Kyoto Encyclopedia of Genes and Genomes pathway database. On the basis, to identify differentially expressed genes between self-pollinated styles and cross-pollinated styles samples. In addition, the genes correlated with self-incompatibililty of Citrus grandis var. Shatinyu Hort will be cloning by PCR amplification. And then, indentification of the self-incompatibility genes will carry out by transferred the genes into Arabidopsis thaliana or tobacco.Results can contribute to the in-depth study of molecular mechanism of Gametophytic self-incompatibility in Citrus grandis var. Shatinyu Hort.

以沙田柚自交和异交授粉后1～3天的花柱为材料，提取其总RNA，分离出mRNA，分别构建模板文库，采用第二代测序技术对沙田柚自交和异交花柱转录组进行高通量测序。通过与Nr、Uniiprot、及CDD等公共数据库序列比对来注释自交和异交花柱中转录组测序序列，并对所测序列进行GO、COG以及KEGG等功能注释和代谢途径分析。通过比较自交和异交花柱中基因表达图谱，了解自交和异交花柱中相关基因的表达差异。同时，根据测序结果，设计特异引物，通过PCR方法克隆参与自交不亲和的主要基因。在此基础上，将自交不亲和基因转入拟南芥或烟草中进行功能鉴定。最后通过分析沙田柚自交不亲和过程相关基因表达和协同关系，探讨沙田柚配子体自交不亲和性的分子机理。

沙田柚为芸香科、柑桔属配子体自交不亲和的典型植物。目前，芸香科植物配子体自交不亲和的分子机理尚不清楚。本项目采高通量测序技术对沙田柚自花授粉和异花授粉（酸柚X沙田柚）1-3天花柱分别进行了转录组和miRNA测序，对自交和异交花柱之间差异表达基因进行表达模式的聚类分析、GO功能显著性富集分析、Pathway显著性富集分析。转录组测序结果表明：异花授粉1天与自花授粉1天花柱之间的差异表达基因为3466个（上调1609,下调1857），异花授粉2天与自花授粉2天花柱之间的差异差异表达基因为3459个（上调1756，下调1703），异花授粉3天与自花授粉3天花柱之间的差异表达基因数量为3608（上调1069，下调2539）。差异表达的基因主要参与花粉管的伸长、植物激素信号转导和胁迫应答等过程。miRNA测序结果：异交1、2、3天花柱中的已知miRNA的个数分别为232、122和219个，新miRNA分别为1184、476和836个。自交1、2、3花柱中已知miRNA的个数分别为178、179和186个，新miRNA则分别为640、739和801个。各样品中新miRNA数量大于已知的miRNA数量。自交花柱特有的miRNA为40个，异交花柱特有的miRNA共32个。其靶基因的功能均涉及到RNA降解、转录因子、核糖体合成、植物与病原菌相互作用、激酶以及蛋白质合成等代谢过程。. 对异交和自交花柱中2个差异表达的核糖核酸酶基因cgSLA和cgSRA进行了克隆、构建原核表达载体，诱导回收目的蛋白。在花粉体外萌发实验中目的蛋白对沙田柚花粉的萌发和花粉管的生长均有较为明显的抑制作用。拟南芥转基因结果表明cgSLA和cgSRA基因对花药的发育有抑制作用。研究结果为阐明沙田柚配子体自交不亲和的分子机理提供了重要依据。