盐胁迫下石竹幼苗中清除H2O2相关基因的克隆及功能分析

In preliminary studies, it had been determined that H2O2 moved from root tip to cotyledons through stem under long-term salt-stress in Dianthus chinesis L. seedlings. There were significant differences in H2O2 contents among the organs of Dianthus chinesis L. seedlings, which the highest were in leaves, following by stems and roots. The total activities of CAT and POD decreased with the increasing of NaCl, and SOD, POD and APX showed no significant changes in comparison with an untreated control. Meanwhile, the highest activities of SOD, POD and APX were in roots, following by stems and roots, and GR was in the opposite situation. Based on the relationship between H2O2, Na+ and antioxidant enzymes we deduced that H2O2 and Na+ triggered different detoxification systems under salt-stress in Dianthus chinese L. seedlings. Therefore, in the present work seedling leaves harvested under NaCl and H2O2 are used to construct two substracitve cDNA libraries, and then the up-regulated genes expressed in both treatments will be isolated and identified in Dianthus Chinese L. Our aim is to analyze their actions on H2O2 and to further elucidate mechanism of detoxification system associated with H2O2 induced by salt-stress on molecular level. Consequently, it will beneficial to develop new salt-tolerant varieties intentionally according to salt-tolerant mechanism on gene expression level, which will meet with the aim of economic decoration in urban by efficient utilization of saline soil.

通过对中国石竹幼苗耐盐机理的研究，发现持续NaCl胁迫下，H2O2在种子萌发过程中从根尖沿木质部移动到茎，再经叶脉移动到子叶；不同器官间H2O2含量差异显著，叶〉茎〉根；总CAT和POD活性随NaCl浓度升高而下降，SOD、APX、GR活性与NaCl浓度无明显相关性；根中SOD、POD、APX活性最高，其次为茎、叶，GR则相反；依据H2O2、NaCl与抗氧化酶的关系，推测盐胁迫下H2O2与Na+在石竹幼苗体内诱导的清除系统不同。因此本项目以石竹幼苗为研究材料，通过筛选差减cDNA文库，对在NaCl及H2O2诱导下均表达上调的基因进行分析和功能研究，探究它们在盐胁迫下对H2O2的清除作用，为进一步探明石竹盐胁迫诱导的H2O2清除系统的分子机理奠定理论基础。从基因表达水平深入了解石竹的耐盐机理，进而有目的的培育抗盐新品种，可以达到有效利用盐碱土、减少资金投入的节约型绿化目标。

盐害是植物生长面临的重要逆境危害之一。植物处于胁迫状态会引起 H2O2和O2•-的过量产生。中国石竹耐旱、耐寒、耐盐碱，因此在绿化中广泛应用。目前，对中国石竹的基因功能组学研究的报道较少且耐盐机理尚不清楚。本研究首先以中国石竹幼苗和离体花蕾为材料，以石竹中八氢番茄红素脱氢酶（Phyteone Desaturase,PDS）基因和查尔酮合成酶(chalcone synthase,CHS)基因为报告基因,对构建的VIGS体系进行了评价，其次以中国石竹幼苗为材料，研究了H2O2改变对盐胁迫下石竹的影响，并对盐胁迫及去除盐胁迫后的转录组进行了测序、组装、分析，主要结果如下：.1）克隆得到了1651bp的DcPDS （KU318404）和898bp的DcCHS （KX893854）。.2） 构建了两套VIGS诱导沉默体系，一是采用pTRV2/DcPDS509浸染2-3对真叶石竹幼苗，15℃下培养7d再在正常条件下培养，呈现叶漂白现象，二是采用pTRV2/DcCHS407浸染石竹离体花蕾，15℃下培养4d，呈现花瓣漂白现象。qRT-PCR表明这两个基因的转录水平显著降低。.3）DMTU和DPI对中国石竹体内的H2O2有清除作用；盐胁迫增加了石竹幼苗体内的H2O2含量，胁迫前人为改变H2O2含量会改变石竹的抗性。.4）组装后获得62,371 unigenes ，31863 unigenes (51.08%) 可在7个数据库Nr, Nt, Swiss-Prot, KEGG, GO, KOG 和 Pfam 中的至少1个数据库上成功注释。盐胁迫下每个时间点根中的DEGs分别是叶中的19.90、1.25和2.6倍。通过筛选这些DEGs的表达模式，叶和根中分别获得上调的盐诱导基因155和1539个，根中的盐诱导上调基因数量是下调的1.97倍。.5）qRT-PCR验证了盐诱导基因中涉及氧化还原过程或细胞氧化还原平衡的基因的表达模式。.本研究表明VIGS可对石竹的基因功能进行快速、有效验证。H2O2在盐胁迫中扮演重要的作用。盐胁迫首先大量激发根中与激素类信号物质有关的基因，引起糖代谢、氧化还原等一系列反应，随后叶中发生响应，根中大量盐诱导基因的上调表达对维持根部的基础代谢起了重要作用，这可能是石竹能在盐胁迫下存活的原因。获得的盐诱导基因对今后石竹属植物遗传、育种研究具有重要意义。