广西抗旱耐瘠型割手密抗旱基因筛选与鉴定研究

More than 80 percent sugarcane was planted in dry upland and drought was the main factor that restricts the sugarcane productivity in china. In order to improve the cane productivity under seasonal drought in China, transgenic sugarcane with high drought resistance and water use efficiency gene is a new methodology to improve the sugarcane drought resistance. Saccharum spontaneum is one of the wild relatives of sugarcane, has many advantageous characteristics, such as drought resistant, cold resistant, barren tolerance, disease resistance, better tillering and so on, so which is the most important breeding of sugarcane wild germplasm resources. Based on saccharum spontaneum with drought and barren resistance collecting, comprehensive evaluating drought resistance of saccharum spontaneum and gene cloning of drought resistance from sugarcane, the differential genes induced under drought stress will be firstly isolated and screened using cDNA-SCoT differential display expression from the drought and barren resistance type of saccharum spontaneum, and the specific gene fragment by drought induced expression will be focus on find out and obtain a number of drought related important gene fragment. And then positive TDFs were obtained using reverse Northern blotting excluding the false positives and TDFs isolated with cDNA-SCoT were selected and verlidated by qRT-PCR analysis. Finally, those genes function were identified and analyzed. The completion of this study, will obtain some independent intellectual property rights of the important drought resistance gene of saccharum spontaneum, which would provide the premise and theoretical basis for genetic improvement of drought resistance in sugarcane, also provide candidate genes for improving other crops.

我国甘蔗80%以上种植在旱坡地上，干旱已成为我国甘蔗生产的最主要限制因子。为解决干旱给甘蔗生产带来不利影响，发掘抗旱力强、水分高效利用基因资源并应用到甘蔗育种提高甘蔗抗旱能力是当前研究新方向。割手密具有耐旱、耐寒、耐瘠、抗病、分蘖旺盛等优良特性，是甘蔗育种最重要的野生种质资源。本项目拟在广西抗旱耐瘠型割手密收集、抗旱性综合评价及甘蔗抗旱基因克隆的研究工作基础上，首先使用cDNA-SCoT差异显示构建割手密干旱胁迫诱导的基因表达谱，筛选干旱诱导表达基因片段，获得一批与抗旱相关的重要基因片段；其次对基因进行Northern印迹杂交验证和qRT-PCR表达验证分析；最后对基因进行功能鉴定研究。本项目的完成，将获得若干割手密重要抗旱基因，为甘蔗抗旱性的遗传改良研究提供前提基础和理论依据，也为其它作物的改良提供相应的候选基因。

我国甘蔗80%以上种植在旱坡地，干旱已成为我国甘蔗生产的最主要限制因子。为解决干旱给甘蔗生产带来的不利影响，发掘抗旱力强、水分高效利用基因资源并应用到甘蔗育种提高甘蔗抗旱能力是当前研究新方向。割手密具有耐旱、耐寒、耐瘠、抗病、分蘖旺盛等优良特性，是甘蔗育种最重要的野生种质资源。本项目在广西抗旱耐瘠型割手密收集、抗旱性综合评价及甘蔗抗旱基因克隆的研究工作基础上，通过使用cDNA-SCoT差异显示及转录组测序的方法筛选割手密GX83-10干旱诱导表达相关基因，并对感兴趣的基因进行了初步的鉴定。目前已收集石山地区野生割手密种质52份，初步筛选出耐旱种质12份；已获得27条SCoT引物的割手密GX83-10干旱胁迫叶片差异基因表达谱；cDNA-SCoT筛选120条割手密GX83-10叶片干旱胁迫上调基因片段序列；转录组测序筛选割手密GX83-10耐旱相关代谢通路47条，叶片干旱胁迫相关基因1325个，根系干旱胁迫相关基因5630个，揭示了割手密应答干旱胁迫基因表达信息，丰富甘蔗遗传改良研究的候选基因库；qRT-PCR表达验证分析感兴趣的基因24个，这些基因在割手密应答干旱胁迫中可能起着重要的调节作用，尚需进一步的基因功能鉴定研究；已初步构建割手密SsAPX基因pBI221-GFP植物表达载体pBI221-GFP-SsAPX，建立农杆菌转导体系，优化割手密组培技术，为割手密基因转导研究奠定基础。综合上述研究结果，本研究为探讨割手密耐旱机制及割手密基因的功能鉴定研究奠定了坚实的工作基础。项目资助发表研究论文9篇（SCI 期刊论文3 篇，北大中文核心期刊论文4篇，科技核心期刊论文2篇），已投稿论文1篇（SCI 期刊论文），实用新型发明专利授权2项。