【作者】 胡玉玲；

【导师】 姚小华；

【作者基本信息】 中国林业科学研究院 ， 林木遗传育种， 2014， 博士

【摘要】 普通油茶（Camellia oleifera Abel）是我国油茶栽培的主要物种，是典型的花果同时发育多年生常绿树种，开花是油茶产量形成的重要基础。本文分析了普通油茶花发育过程生理生化变化及其影响因子，初步探明了油茶的成花过程生化指标的变化动态，并通过花芽发育过程的转录组和表达谱测序及分析，分离了影响油茶花发育关键基因（FT和PI），并构建了FT基因的超表达和PI基因RNAi干扰植物遗传转化载体以及初步的遗传转化系统，具体结果如下：1.随着普通油茶花芽分化，老叶中可溶性蛋白含量逐渐下降，茎可溶性蛋白含量逐渐上升，新叶中可溶性蛋白含量变化相对平稳；老叶中可溶性糖含量逐渐下降，新梢组织（上叶、下叶和茎）可溶性糖含量变化相对平稳；茎的蔗糖含量上升幅度明显，叶片的蔗糖含量呈“下降-上升-下降”变化趋势；新梢各组织（上叶、下叶和茎）中果糖含量变化趋势相似，呈“下降-上升-下降-上升”趋势，新叶含量比茎高，茎比老叶高；植物激素含量新叶比老叶和茎高，其中含量最高的是脱落酸；新叶的生长素与细胞分裂素比值变化趋势较平缓，而茎和老叶的生长素/细胞分裂素比值变化幅度较大。2.普通油茶花器官形成过程，春梢上各部位组织（上叶、下叶和花芽）中可溶性蛋白和可溶性糖含量呈缓慢上升趋势；蔗糖和果糖变化幅度较大，花芽中的含量普遍比叶片中低；春梢叶片各种激素含量呈“下降-上升-下降”，春梢各部位组织中脱落酸与生长素比值呈“上升-下降-上升-下降”变化，生长素与细胞分裂素比值在花芽中变化平缓，在叶片中呈“下降-上升-下降”变化，上叶和下叶中的比值变化相反。3.在普通油茶花芽转变期，在短日照处理下，可溶性蛋白和可溶性糖新叶部位含量最高，蔗糖在实生苗的老叶部位含量最高；在水分处理条件下，老叶中蔗糖含量明显低于新叶；叶片中的果糖含量在氮素处理时含量最高，在短日照时含量最低；茎中生长素含量普遍比叶片中低，新叶比老叶高，新叶在热处理下生长素含量最高，在光照处理及实生苗中较低；赤霉素含量最高是在生长素处理时的新叶部位，最低为对照处理的茎部位；细胞分裂素含量最高为细胞分裂素处理的新叶部位，脱落酸在春梢不同部位含量变化差异不明显；短日照下的普通油茶茎尖FCA基因相对表达量最高，FLC基因在实生苗茎中表达量最高，FT基因在长日照时茎尖表达量最高。4.转录组测序总共获得28,448,847个reads和5,742,023,480pb碱基，GC含量为46.52%；N50长度为806，普通油茶与葡萄的Unigenes相似度最高，总共SSR数量为8,399个，1kb以上的Unigenes有12,643条，占Unigene库总数的13.38%；Unigenes各数据库功能注释数目依次为：在COG中有9,095条，在GO中有27,201条，在KEGG中有6,431条，在Swissprot中有24,534条，在TrEMBL中有36,393条，在nr中有36,400条，在nt中有30,858条；茎尖中FLC、FCA和FT基因表达量普遍较AP1、AP2和PI基因低，FT基因是油茶成花的关键基因，PI基因与雄蕊发育关系紧密。5.通过对普通油茶花发育的6个时期样品测序，共获得了原始reads数为103,249,386条，平均CycleQ20都为100%，各样品测序reads与转录组部分测序构建的Unigene库的序列对比，对比效率都超过60%，样品间的差异基因分析，共获得了差异表达基因为26,861个。5月9号和8月25日的差异表达基因最少，7月3日和6月15日与其他时期差异表达的基因数量最多。6.普通油茶FT基因编码区序列长度为540pb，编码179个氨基酸，FT基因编码的蛋白有一个磷脂酰乙醇胺结合蛋白家族（BP）的结构域，与中华猕猴桃的FT基因编码蛋白同源性最高；PI基因编码区序列长度为627，共编码208个氨基酸数，PI基因编码的蛋白有一个MADS-box和K-box蛋白结构域，该蛋白和大豆及四季豆PI基因编码的蛋白同源性最高。7. MS培养基是普通油茶体胚再生最适宜的培养基，长林53号品种较易诱导出胚性愈伤组织，7月27日左右的未成熟胚最容易诱导出体胚，TDZ、2.4-D是体胚诱导最重要的激素，2.4-D、KT、TDZ和CH添加物配比最有利于体胚诱导。选择MS培养基，2mg/L浓度的IAA，0.5mg/L浓度的KT，0.5mg/L浓度的TDZ和500mg/L浓度的CH等组合，有适合于体胚萌发和胚状体生长。8.愈伤组织的潮霉素选择压为120mg/L；外植体预培养时间长短、农杆菌侵染时间长短、共培养时间长短及AS浓度大小对抗性愈伤组织选择都有显著影响，侵染时间对抗性愈伤组织选择影响最为明显，其次是共培养时间、AS浓度和预培养时间；侵染时间、共培养时间及AS浓度大小对农杆菌污染都有极显著影响，侵染时间和AS浓度对农杆菌污染影响最为关键，其次是侵染时间；预培养时间为40d，侵染时间为20min，共培养时间为5天，AS浓度为200mg/L时抗性愈伤组织最多，农杆菌污染率最低。更多还原

【Abstract】 Camellia oleifera Abel. is a typical flower and fruit co-development perennial evergreenplants. Blossom is an important basis for plant production and breeding. This paper analyzedthe physiological and biochemical variation during flower development of C. oleifera and mainfocused on the effect of environmental factors on the blossom. Based on the data results fromtranscriptome sequencing of flower, differentially expressed genes especially those related toflowering process were detected and the preliminary mechanism of C. oleifera flowering waselucidated. By RT-qPCR, the expression profiles of the key flower genes (FT and PI) wereanalysis and then the above genes were successfully cloned into the over-expressing and RNAinterfering vector. Moreover, gene transformation system was analysted and optimized. Theresults were listed as follows:1. During the flower differentiation, the content of soluble proteins gradually decreased in oldleaves, increased in stems. The content of soluble sugar were reduced in old leaves. Thesucrose content was sharply elevated in the stems and kept steady in leaves. The variation offructose content displayed similar change method in different organs of spring shoots.Comparing with the stem and old leaves, the content of fructose in young leaves was the mostabundant while was the least abundant in old leaves. In young leaves, the ratios of abscisic acidand auxin kept constant while in the stems and old leaves they varied largely.2. During floral formation, the contents of soluble protein and soluble sugar rised slowly indifferent parts of spring shoots. The content of sucrose and fructose varied dramatically, andthose were lower in flower buds than in leaves. The level of hormone in leaves showed atendency of “down-up-down”. The ratio of abscisic acid and gibberellic acid showed atendency of “up-down-up-down” in different parts of the spring shoot, The ratio of abscisicacid and auxin showed different tendencies in different parts of the spring shoot.3. During the period of flower differentiation, the contents of soluble protein and soluble sugar in the leaves were the highest in the short-day treatment. The content of sucrose was thehighest in the old leaves. The content of fructose in leaves was the highest in the nitrogentreatment and it was the lowest in the short day treatment. The content of auxin was lower instems than that in leaves. The content of auxin was highest in the heat treatment than that inothers treatments, and the lowest in light treatment. The content of gibberellin was the highestin new leaves under auxin treatment, and the lowest in stems of the control. The content ofcytokinin was the highest in young leaves under cytokinin treatment than that in othertreatmenyts. The relative gene expression of FCA in shoot tips was the highest under differentlight intensity treatments. The relative gene expression of FLC was the highest in seedling andthat of FT was the highest under the long-day treatment.4. The transcriptome sequencing obtained total of28,448,847reads and5,742,023,480bp. Thelength of N50was806bp. The number of unigenes longer than1kb was12,643. With24,534Unigenes being annotated in Swissprot,36,393in TrEMBL,36,400in Nr. The annotatedunigenes were then further classified into different processes and pathways by COG, GO andKEGG. RT-qPCR results showed that the expression levels of FLC, FCA and FT in the stemapex were lower than those of AP1, AP2, PI showed close relationship with the stamendevelopment. The digital sequencing of samples from six periods obtained a total of103,249,386reads. Alignment of the above data with transcriptome sequences. The analysisdetected26,861differentially expressed genes among the tested samples.5. The ORF of FT gene in C. oleifera was540pb that encoding179amino acids with themolecular weight was20KD and isoelectric point was7.74. The protein was most homologousto the FT gene in Actinidia chinensis Planch. The ORF of PI in C. oleifera was627bp andencoded208amino acids with the molecular weight of24KD and isoelectric point of9.11.That was most homologous to PI in soybean and sauteed green beans.6. MS medium was the most suitable medium for the regeneration of somatic embryosinduction and Changlin53variety was most easy for callus induction. TDZ,2.4-D areimportant factors influencing somatic induction. The combination of MS culture medium with 2mg/L IAA,0.5mg/L KT,0.5mg/L TDZ and500mg/L CH was most suitable to somaticinduction.7. Hygromycin concentration for callus selection pressure was120mg/L. The effect of explantpreculture time, Agrobacterium infection and co-culture time and AS concentration onHygromycin resistant callus was significant. Pre-culture time, infection time, co-culture timeand AS concentration had significant effect on the callus of pollution, and infection time andAS concentration had important effect on the pollution, secondly was the time of infection andpre-culture time. The conversion efficiency was the highest and the rate of pollution was thelowest under the condition of40days preculture,20min infection,5days co-culture and the200mg/L AS concentration.更多还原