端粒相关基因对侧柏衰老的调控作用

The longevity of animals and plants has the close relationship with the telomere, which is regulated not only by telomerase, but also by many other regulated genes. At present,it is one of the interesting problems about the relationship between the age of old trees and telomere length in the field of plant aging, however,the results are a bit of differences. In this study, the trees of Platycladus orientalis with 20, 100, 1000 and 2000 years old were selected as materials, and the profiles of gene expressions at the different ages of leaves were established, based the sequence of the transcriptomics, by RNA-seq combined with the digital gene expression (DGE) technique; And then, the different expression genes were analyzed among ages, and telomere regulated genes were further mined and analyzed by qRT-PCR under the different stress factors,and the genes' functions were identified by overexpression and loss of function in Arabidopsis. Combination with telomere length, telomerase activity and anatomical observation,the relationship of telomere regulated genes, ages and stress factors were discussed,which will benefit to reveal that telomere regulated genes whether or not regulate and maintain telomere length for the life extensions of the old trees. Finally, the mechanism of aging resistance in Platycladus orientalis was deciphered, which will benefit the genetic improvement and molecular breeding of trees, crops and flowers.

端粒与动植物的寿命存在着密切关系,不仅受端粒酶影响,还受许多端粒调控相关基因的调控。古树寿命与端粒长度及基因调控间的关系是近年来植物衰老领域感兴趣的研究之一，但他们之间的关系目前仍存在分歧。本项目拟以20、100、1000、2000年古侧柏为研究材料，通过对不同树龄侧柏针叶转录组测序及建立年龄相关的数字基因表达谱，比较分析不同树龄侧柏的差异基因的表达情况，挖掘与端粒长度调控相关的基因，并在不同胁迫因子处理的1年生幼苗中进行qRT-PCR表达分析；利用过量表达和功能丧失技术，在拟南芥中进一步研究这些相关基因的功能；结合端粒长度、端粒酶活性、生理生化、解剖观察等手段，综合分析端粒调控基因与端粒长度、树龄和胁迫因子三者间的关系；进而，探讨端粒调控相关基因是否能通过调控或维持端粒长度从而延长古树寿命，最终揭示侧柏抗衰老的分子机制。这将为农林作物或花卉抗性遗传改良和分子育种奠定基础。

古树寿命长达几千年仍不见衰老，其生理与分子调控机制如何？成为近年来植物抗衰老研究领域的热点之一；端粒与动植物的寿命存在着密切关系，不仅受端粒酶影响，还受许多端粒调控相关基因的调控。研究结果如下：（1）提取高质量的侧柏RNA是在分子水平研究侧柏的基础，采用两步除多糖和多酚的方法改良试剂盒，改良方法提取侧柏RNA的28S rRNA：18S rRNA约2:1，A260/A280为2.03，成功提取了高质量的侧柏和其他5种柏科植物的RNA，这有利于在分子水平上研究柏科植物。（2）通过不同侧柏树龄针叶的高通量差异表达谱，我们对418个表达差异基因分成8 clusters，分析发现氧化还原和光合作用相关的基因随树龄升高下调表达，胁迫响应和转录相关的基因随树龄升高表达上调，结果说明侧柏衰老过程中光合作用降低，活性氧也起到了重要的作用。（3）以侧柏幼苗为试验材料研究外施ABA对H2O2胁迫下侧柏幼苗活性氧代谢的影响。侧柏幼苗叶片活性氧代谢相关基因Cu/Zn-SOD、CAT、GR、APX、MDAR和GST表达水平较对照CK均有显著性提高；正常和H2O2胁迫下侧柏幼苗外施0.5 μmol·L-1 ABA相较于200 μmol·L-1更有利于提高侧柏叶片活性氧代谢相关基因Cu/Zn-SOD、CAT、GR、APX、MDAR和GST的表达量。低浓度0.5 μmol·L-1 ABA有效地增强抗氧化系统的防御能力，减弱幼苗的氧化胁迫和膜脂过氧化水平，从而降低活性氧对侧柏的伤害。（4）克隆了500年生古侧柏的端粒相关基因PoWHY1、PoKub3和PoTRF1的全长。随着侧柏年龄的增长，PoWHY1和PoTRF1基因呈先升后降的趋势；PoKub3基因表达水平逐渐增加。（5）PoKub3基因正调控拟南芥对NaCl和干旱胁迫的抗性。PoKub3过表达提高拟南芥种子对ABA的敏感性。相较于WT和VC，PoKub3过表达提高了拟南芥中RAB18和DREB2A基因表达水平且降低了JAZ1和ABI2基因表达水平，证明了PoKub3过表达提高拟南芥抗盐性。PoKub3提高对干旱的抗性是通过调控一些胁迫相关基因如JAZ1、LOX1、DREB1A、DREB2A、GSTF6和APX1的表达来实现的。降低MDA含量、电解质外渗率和气孔导度，以及提高相对含水量和可溶性糖，有助于提高植物抗旱性。