【作者】 麻文俊；

【导师】 张守攻；

【作者基本信息】 中国林业科学研究院 ， 林木遗传育种， 2013， 博士

【摘要】 楸树（Catalpa bungei）是我国特有珍贵用材树种，分布范围广，材质优良，已有2600多年的栽培历史，目前主要作为高档家具和细木工板材等原材料。然而，目前推广的主栽品种抗旱性较差，在相对干旱的条件下表现不佳。因此，为了提高在相对干旱条件下楸树的生长量，开展种内耐旱和高水分利用效率新品种选育成为重要的研究方向。了解楸树对干旱胁迫的生理和分子响应机理，挖掘关键基因，是开展高效育种的基础。本研究以楸树优良耐旱无性系为试材，利用高通量测序技术建立转录本集合，通过干旱处理和基因芯片技术挖掘胁迫响应关键基因，并结合生理变化，对响应机理进行阐述。主要结果如下：（1）在自然干旱胁迫过程中，当土壤重量含水量下降到23%时，苗高、地径和叶片生长基本停止，成熟叶片的叶面积为正常条件下的53%（93.5cm2），净光合速率（Pn）和光合性能指数（PI）显著下降；当土壤含水量下降到15%时，Pn接近于0，叶片和根系游离脯氨酸、可溶性糖、脱落酸含量在胁迫下均显著升高，茉莉酸含量则轻微升高，可溶性蛋白含量先升高后降低，叶片和根系的SOD、POD活性、以及叶片POD活性均呈现为先下降，后期呈显著升高的趋势。（2）采集正常和干旱胁迫条件下3个楸树基因型的叶片、根、茎等组织进行混合转录组测序（454焦磷酸测序），共获得77,337个Unigene，在Nr数据库中比对，共有33,672个Unigene具有同源序列，其中有19,110个获得GO功能注释。通过KEGG代谢通路富集，共参与了包括碳水化合物、氨基酸、脂肪酸代谢（Carbohydrate，Amino acid，Fatty acidmetabolism）、光合作用（Photosynthesis）等在内的250个代谢通路。发掘了包括双碱基重复（Di-）至六碱基重复（Hexa-），以及混合型在内的2,782个SSR和79,528个SNP。（3）依据转录组测序结果，设计了表达谱芯片，并依据干旱胁迫下生长和生理变化趋势，选择了5个土壤含水量梯度对叶片和根系进行差异基因表达分析。与对照相比，4种胁迫下叶片中差异表达基因共有16,137个，根系中共有14,913个，叶片中有2,233个基因是在4种胁迫下共差异表达的，其中有630个随胁迫加重表达量呈不断升高或降低的趋势（持续差异基因）。根系中有1,006个基因是在4种胁迫下共差异表达的，其中有203个随胁迫加重表达量呈不断升高或降低的趋势（持续差异基因）。对叶片和根系中的持续差异基因进行GO和KOG注释表明，主要涉及到信号转导途径、转录、离子跨膜转运、渗透调节物质合成、抗氧化酶和激素合成等方面。（4）通过功能分析，发掘到与干旱胁迫响应相关的基因，主要包括4个方面：信号转导相关基因：丝氨酸/苏氨酸蛋白激酶（Serine/Threonine-protein kinase）、富含亮氨酸重复序列（leucine-rich repeat，LRR）类受体蛋白激酶、MYB、MYC、bZIP、NAC；抗氧化酶基因：原叶绿素酸酯氧化还原酶（NADPH: protochlorophyllideoxidoreductase）、谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-Px）、锰超氧化物歧化酶（manganese superoxide dismutase，Mn-SOD）、环阿乔醇-C-24位甲基转移酶（S-adenosyl-L-methionine: delta24-sterol-C-methyltransferase）、type II过氧化物酶（type IIperoxiredoxin，POD）、丁酸-辅酶A连接酶(butyrate--CoA ligase AAE11）；渗透调节物质合成相关基因：蔗糖合成酶（Sucrose synthase）、蔗糖磷酸合酶（Sucrosephosphate synthase）、棉籽糖合成酶（Raffinose synthase）、水苏糖合成酶（Stachyosesynthase）、△-吡咯啉-5-羧基合成酶（△1-pyrroline-5-carboxylate synthase）、鸟氨酸-△-氨基转移酶（Ornithine-delta-aminotransferase）、△-吡咯啉-5-羧基还原酶（Pyrroline-5-carboxylate reductase）、谷氨酸脱氢酶（Glutamate dehydrogenase）、乙醛脱氢酶（Aldehyde dehydrogenase）、胆碱单加氧酶（Choline monooxygenase）、水孔蛋白（Aquaporin）；激素合成相关基因：9-顺式-环氧类胡萝卜素双加氧酶（9-cis-epoxycarotenoiddioxygenase，NCED）、ABA-醛氧化酶（ABA aldehyde oxidase）、细胞色素P450、赤霉素-20-氧化酶（Gibberellin20-oxidase）、12-氧-植物二烯酸还原酶（12-oxophytodienoic acid10,10-reductase）。更多还原

【Abstract】 Catalpa bungei, one of the endemism and valuable timber tree species in China, whichhas been cultivation more than2600years. This species was widely distribution in China andprimarily be used as the raw materials of high-end furniture and musical instruments cause ofthe excellent material quality. However, the recently promotion main cultivar has been shown adiscrepancy drought resistance, the performance was misadventure under the relativelydroughty condition. Therefore, how to selection breeding the excellent new varieties withrelatively drought-tolerant and high water use efficiency has become an important orientationfor improvement the mass growth of Catalpa bungei under the relatively droughty condition.Understanding the physiological and molecular response mechanism to drought stress andmining the key genes in Catalpa bungei is the foundation of effective breeding. This studychoose the excellent drought resisting clone of Catalpa bungei as test materials, usinghigh-throughput sequencing technology to build the assemblage of transcription, adopting thegene chip technology to excavate the key response gene after drought treatment, combinedwith the physiological changes to elaborate the response mechanism. The main results were asfollows:（1）In the process of natural drought stress, the height, diameter and leaf growth basicallystopped when the gravimetric water content drop to23%, the leaf area of mature leaves was53%of that under normal conditions（93.5cm2）. Net photosynthetic rate （Pn）and photosyntheticperformance index（PI）decreased significantly, Pn was close to0when the soil moisturecontent down to15%, no significant change in the efficiency of light energy conversion（Fv/Fm）. The content of free proline, soluble sugar, abscisic acid in leaves and roots weresignificantly elevated under drought stress treatment, the content of jasmonic acid was slightlyincreased, the content of soluble protein was firstly increased and then decreased; the activity of SOD, POD in leaves and roots and leaf POD activity were shown a trend of decline at firstand significantly increased in late.（2）Leaves, roots, stems, and other tissue of three Catalpa bungei genotypes which undernormal and drought stress treatment were acquisition and mixed for transcriptome sequencing（454pyrosequencing）, a total of77,337unigene were obtained, Clustering with NR database,a total of33,672unigene have been found homologous sequences, among which19,110acquired GO functional annotation.250metabolic pathways including Carbohydrate, Aminoacid, fatty acid metabolism, Photosynthesis, citrate cycle, glycolysis were got by KEGGmetabolism pathways enrich.2,782SSR including double-nucleotide repeat（Di-）to six baserepeat （Hexa-）, and mixed type were explored, a total of79,528SNP were excavated.（3）Based on the transcriptome sequencing results, expression profile chip were designed.According to the growth and physiological change trends under drought stress treatment,5soilmoisture gradient（including controls）were chosen to analysis the gene differences expressionin leaves and roots. Comparing with the control, totally16,137differentially expressed genes inleaves and14,913in the roots were discovered under four kinds of drought stress treatments.2,233genes in leaves were commonly differentially expression under the four drought stresstreatments, including630genes（continuous differential gene）have the trend of increasing ordecreasing expression quantity with the drought stress degree increased;1,006genes in rootswere commonly differentially expression under the four drought stress treatments, including230genes（continuous differential gene）have the trend of increasing or decreasing expressionquantity with the drought stress degree increased; results of GO and KOG annotation of thecontinuous differential gene in leaves and roots showed that the differential gene in leaves androots were mainly involved in signal transduction pathway, transcription, transmembrane iontransport, osmotic adjustment substances sythesis, antioxidant enzyme and hormone sythesisother functions and processes.（4）According the functional analysis, the droughts stress response-related genes wereexplored, including four aspects. Signal transduction related genes: Serine/threonine-protein kinase, leucine-rich repeat（LRR）receptor-like protein kinase, MYB, MYC, bZIP, NAC;Antioxidant protective enzyme gene: NADPH（protochlorophyllide oxidoreductase）,glutathione peroxidase（GSH-Px）, manganese superoxide dismutase（Mn-SOD）, S-adenosyl-L-methionine（△24-sterol-C-methyltransferase）, type II peroxiredoxin（POD）, butyrate-theCoA ligase AAE11;Adjustment substances synthesis related gene: sucrose synthase, sucrose phosphatesynthase, Raffinose synthase, and stachyose synthase,△1-pyrroline-5-carboxylate synthase,ornithine-△-aminotransferase, pyrroline-5-carboxylate reductase, glutamate dehydrogenase,aldehyde dehydrogenase, choline monooxygenase;Hormone synthesis related genes:9-cis-epoxycarotenoid dioxygenase（NCED）, ABAaldehyde oxidase, cytochrome P450, gibberellin20-oxidase,12-oxophytodienoic acid10,10-reductase. The differential expression gene above-mentioned were the molecular basis ofCatalpa bungei clones2-8respond to drought stress, the mining and function analysis of suchgene have supported for the selection and breeding of superior drought resistant Catalpabungei clones更多还原