基于RNA-Seq小黑麦耐旱性相关基因克隆及功能验证

Drought stress is one of the most serious ecology problems that humand must deal with. And drought stress will be more severe with the globle warming and decreasing of the freash water resource. It is very important in application to understand the response mechanism of crops under drought stress, find new genes related with drought tolerance in crops and develop new crop varities with higher drought tolerance and higher ability and water use efficiency. Triticale (× Triticosecale) is a hybrid specie of wheat (Triticum) and rye (Secale) , which was first bred in the late of 19th century. This new crop combines higher yield potential and good grain quality of wheat with the disease and environmental tolerance (including soil conditions) of rye. It is grown mostly for forage or food, and become one of important crops in Xinjiang. Based on the previous study and investigation, we make sure that triticale has a very stronger adaptation ability to environments, especially with higher drought tolerance ability. This project aims to clone genes related with dought tolerance in tritciale based on RNA-Seq technique and verify the function of the new cloned genes. After the comparison of expression profiles of drought stressed tritcale and the control, the different expression of transcriptomes will be found, and the higher expression transcriptome were selected. Based on bioinformatic anlysis, the genes with a higher expression level will be figured out, then the gene will be cloned by homology clone technique or RACE .The expression profile of cloned genes will be tested in triticale by real time PCR in leaf, root, spkie under drought stress and normal enviornment. Over expression vecotr of the cloned gene will be constructed. The constructed vector will be transformed into Arabidopsis, and the transformed Arabidopsis will be treated under drought stress and normal enviornments, and physiology and metablic profile will be assayed to infer the possible funcation of the new cloned genes. The study will facilitate enriching of the genes with dought stress tolerance, and understanding the mechanism in plant to endure dehydration. This project will also empower the improvement of triticale or wheat drought stress tolerance by genentic manipulating.

干旱是人类面临的最严重生态问题之一。随着全球变暖及可利用水资源日益减少，干旱问题呈日益严重趋势。理解作物对干旱胁迫的应答机制，发掘作物耐旱基因，对于培育耐旱节水作物新品种具有重要现实意义。 小黑麦是人工将小麦与黑麦杂交并经染色体加倍培育成的新型作物，具有粮食、饲料等多种用途和良好发展前景，已成为新疆重要的饲草或饲料作物之一。前期大量研究和观察中，发现六倍体小黑麦具广泛的适应性，尤其是强耐干旱性突出。 本项目拟以RNA-seq为主要手段，通过对干旱胁迫前后六倍体小黑麦转录本的比较，发掘六倍体小黑麦中对干旱胁迫优势表达的功能基因，继而对其进行克隆，探索其表达特点，并通过转化拟南芥来验证其功能。该研究对于丰富植物耐旱新基因、理解六倍体小黑麦耐干旱的分子机制具有重要意义，将为六倍体小黑麦乃至小麦耐旱性改良提供理论指导。

干旱是影响农业生产的主要非生物胁迫因素。小黑麦具有小麦籽粒产量高、品质优以及黑麦耐逆性、适应性强的特点，已成为重要的粮、饲作物。探索小黑麦耐旱机制，克隆其耐旱基因对于小黑麦乃至小麦改良与利用具有重要意义。. 本项目以抗旱性不同的小黑麦基因型为材料，在田间开花后进行干旱胁迫处理，借助转录组测序技术，鉴定耐旱性不同小黑麦在干旱胁迫下的转录组以及基因表达量差异。两种耐旱性不同小黑麦基因型转录组差异明显，共涉及到2335个基因，表达量差异较大的大多是调节基因（如DNA结合蛋白rav1等）、功能蛋白（主要调节因子，如转录因子，调节蛋白、转移酶类等。表达量下调差异较大的主要是转录因子（如NAC结构域蛋白77和NAC结构域转录因子)等。实时荧光定量PCR对其表达量验证表明，大多数表达量变化趋势与表达谱测序结果相似。. 在此基础上，候选并克隆到了小黑麦NAC转录因子基因全长cDNA，多胺氧化酶基因全长cDNA以及茉莉酸诱导蛋白cDNA全长。小黑麦NAC基因在根和幼粒中表达量明显高于茎和叶中，在受到干旱胁迫时表达量上调。. 基于该测序数据分析，共开发了SSR分子标记5204条。对部分标记验证表明，大部分在六倍体小黑麦、黑麦、硬粒小麦、普通小麦中都能够扩增，可见在六倍体小黑麦、黑麦、硬粒小麦、普通小麦中SSR标记具有通用性。. 该耐旱基因型在籽粒灌浆早期热胁迫下千粒重、容重下降、灌浆期缩短，总淀粉、直链以及支链含量均较对照降低，直链/支链淀粉的比例升高，蛋白质含量提高。这与籽粒中AGPPase、SSS、DBE酶活性降低, SBE酶活性提高，α-淀粉酶活性升高，GS、GOGAT、GOT、GPT活性升高，9个与淀粉合成相关酶基因的表达量下调，7个与临时淀粉降解相关基因表达量上调，6个与淀粉合成相关基因表达量下降相关。. 本研究为理解小黑麦耐旱与耐热机制，小黑麦耐旱转基因育种，以及小黑麦遗传多样性及分子标记辅助育种奠定了基础。同时，为稳定和培养小黑麦研究人员，提升课题组小黑麦研究与育种技术水平，扩大国际影响具有重要意义。