温水处理中国完全甜柿脱涩过程中LTR逆转座子激活转座及参与脱涩机制研究

A Chinese pollination-constant non-astringency (C-PCNA), namely 'E Shi 1', which is at the stage of non-astringency development, will be taken as the examined plant material in this project. Firstly, warm water is used to remove astringency, and then, partial fragments of LTR retrotransposon which are actived and of different expression are revealed via transcriptome sequencing. Afterwards, the complete sequences of these LTR retrotransposons are isolated by using RACE or cDNA library selection method to get the information of LTR section. Next, basing on the LTR sequences of retrotransposons, cDNA gene pools flanking both 5' LTR and 3' LTR are constructed in order to obtain the flanking genome sequences of actived retrotransposon insertion sites. Finally, gene probes are designed basing on the sequences of candidate gene fragments and northern blots are conducted in the CK and treated samples, basing on the difference in the size of resolved fragments, to screen the candidate genes destroyed due to insertion of retrotransposon. Simultaneously, using both gel mobility shift assay and yeast one-hybrid system, potential transcription regulators correlated with the trigger of read-through transcription of retrotransposons are expected to study. Altogether, taking advantage of the characteristic of activation of retrotransposon by stress factors, this project will investigate both the relationship between LTR retrotransposon and the trait of astringence and the mechanism of removing astringence under the treatment of artificial deastringency. These attempts would provide guidance for future research on obtaining the genes or regulators relevant to the deastrigence of C-PCNA.

本项目拟以非脱涩期的中国完全甜柿'鄂柿1号'为试材，先施以温水处理使柿果脱涩；再通过转录组测序获得差异激活表达的逆转座子片段；接着，通过RACE或cDNA 文库筛选获得这些LTR逆转座子的全长；然后，根据逆转座子全长中的LTR序列信息，分别构建LTR 5'和3'端侧翼cDNA基因池，获得逆转座子侧翼基因序列；最后，以这些基因序列设计探针，脱涩前后的试材为模板进行Norther杂交，通过检测同一基因转录本片段大小间差别与否，筛选可能发生结构变异的候选基因。与此同时，还采取凝胶迁移率分析以及酵母单杂交手段，对可能与逆转座子LTR区特异结合的转录因子蛋白进行分析，以获得启动LTR逆转座子发生通读转录调控因子。本项目利用逆转座子逆境激活转座特性，针对逆转座子与中国原产完全甜柿脱涩性状间的关系和作用机制展开研究，将为今后获得与中国原产完全甜柿自然脱涩相关的基因或调控因子，探究其自然脱涩机理提供参考。

本研究借助基因组逆转座子逆境激活特点及特殊转座特性，通过温水脱涩可控试验，对中国完全甜柿自然脱涩相关基因进行筛选。通过同源基因克隆、cDNA-IRAP、荧光定量PCR确定了1个Ty1-copia类LTR逆转座子DKRE1在柿温水脱涩过程中可被激活并发生转座，为开展cDNA-SSAP转录差异带的实施提供了必要条件；基于DKRE1-cDNA-SSAP构建中国甜柿温水脱涩前后差异转录的cDNA文库，转录水平上检出远超过DNA水平的多态条带，对差异条带GO注释和KEGG分析，结合转录组测序和对候选片段及部分已知基因的表达分析，认为醛类及果胶积累是柿果温水脱涩主要原因；基于cDNA-SSAP差异文库，选取候选片段开展与中国完全甜柿自然脱涩相关基因的分离和功能验证，首次在柿上获得14条DkPK基因cDNA全长，初步明确其中3个DkPK基因参与中国甜柿的自然脱涩，联合前人进展，提出了中国甜柿自然脱涩分子机制模型图；利用酵母单杂交，进而获得与DkPK1互作的上游调控因子。本项目中国完全甜柿自然脱涩相关基因及转录因子的获取，为诠释甜柿自然脱涩调控机理研究和未来新种质创建增添新的内容。发表SCI论文4篇。