【作者】 王刚；

【导师】 王刚； 王勇；

【作者基本信息】 四川农业大学 ， 森林培育， 2023， 博士

【摘要】 红豆杉是当今工业化生产抗癌药物紫杉醇及其人工半合成所需原料10-去乙酰基巴卡亭Ⅲ（10-DAB）的唯一材料。云曼红豆杉（Taxus madia×Taxus yunnanensis‘Yunman’）是课题组人工杂交选育出的高含量10-DAB红豆杉药用植物新品种,其F₁代实生苗木（树龄<5）杂种优势显著,但仅有4～5年的可利用期,致使采收时单株生物量及主要次生代谢物累积量低,单位面积产量不足,杂种优势利用及品种推广受到阻碍。人工调控促进苗木生长是新品种大面积推广种植最简单方便的措施,揭示人工调控对苗木生长及次生代谢物的响应机理,寻求利用周期内提高单株主要次生代谢物累积量的有效调控措施,是云曼红豆杉杂种优势利用及品种推广亟待解决的科学与技术问题。在前期对云曼红豆杉生物学特性研究的基础上,以出圃前1年生容器苗为试验材料,运用稳态营养施肥方式,开展生长调查、生理生化指标检测及次生代谢物动态跟踪测定,研究养分对其生长、营养生理和主要次生代谢物的影响,揭示其生长需肥规律和养分累积分布特征;以出圃后2年生苗木为试验材料,在光肥互作条件下,开展苗木生长、光合与抗逆生理及主要次生代谢物累积响应的研究,基于模糊Borda法的光肥互作模式优选模型构建,以及转录组测序与分析,探索云曼红豆杉生长与次生代谢物对光肥复合调控的生理和分子响应机制,寻求人工调控促进苗木生长的有效措施,为其杂种优势利用及品种推广提供理论与技术支撑。得出以下主要研究结果。（1）稳态营养施肥显著促进云曼红豆杉1年生苗木生物量和营养元素N、P和K的积累（P<0.05）,其生物量和养分积累在稳态营养施肥下以施氮量1.6 g/株最大,施氮量高于3.2 g/株或低于2.4 g/株均会抑制其生长和紫杉烷类化合物的合成,枝叶中紫杉醇和10-DAB均在5～9月呈上升趋势,9～11月表现为下降趋势。云曼红豆杉1年生苗木各器官中N、P和K营养元素的分配依次为叶>根>茎,叶片吸收营养元素含量总体呈现出N>K>P,各器官中P和K、P和N以及叶、根中的N与K均呈显著的正相关（P<0.05）。（2）光肥复合调控显著改善云曼红豆杉2年生苗木光合作用,促进可溶性蛋白、可溶性糖和脯氨酸的合成以及紫杉烷类化合物的累积（P<0.05）。在40～70%光照与3.6 g/株施氮量条件下,枝叶中紫杉醇和10-DAB的单株累积量显著增加（P<0.05）。云曼红豆杉2年生苗木生长快速期为3～6月,最佳采收利用期为9～12月,紫杉醇和10-DAB单株累积量分别在9月（9.010 mg）和12月（85.228 mg）达最大值。（3）从生理、生长和次生代谢3个方面,选取净光合速率（P_n）、茎容量指数（SVI）和紫杉醇累积量（APTX）等12个关键指标,建立综合评价指标体系,采用改进CRITIC赋权法、灰色关联度法、TOPSIS法和隶属函数分析法,对16种光肥互作组合进行综合评价和排序,利用λ²检验法,验证四种评价方法的一致性,构建基于模糊Borda法的云曼红豆杉光肥组合优选模型。结果表明,模糊Borda组合评价值在90分以上的依次为S3F3（40%光照+3.6 g/株施氮量）>S2F3（70%光照+3.6 g/株施氮量）>S3F2（40%光照+1.8 g/株施氮量）。（4）通过对光肥最优组S3F3和对照组（未处理）的云曼红豆杉2年生苗木叶片转录组分析,共鉴定到1,334个DEGs,包括上调基因752个,下调基因582个。DEGs中鉴定出b HLH、NAC和MYB等569个转录因子。21,944个转录本在Nr数据库中的注释率最高,为75.40%。差异基因的GO富集主要在信号转导、跨膜转运蛋白活性、防御应答等方面;KEGG富集主要在苯丙烷、二萜、萜类骨架生物合成等方面。光肥复合调控诱导了b HLH、NAC等转录因子基因差异表达,主要通过苯丙烷代谢、淀粉和蔗糖合成、萜类生物合成途径等影响云曼红豆杉的生长生理及次生代谢物合成,实现对光肥复合调控的响应,其中鉴定到影响紫杉醇合成的5个CPX基因。稳态营养施肥对云曼红豆杉1年生容器苗生物量积累的提高影响显著;光肥组合S3F3（40%光照+3.6 g/株施氮量）可显著提高云曼红豆杉2年生苗木紫杉醇及10-DAB单株累积量。研究结果揭示了云曼红豆杉苗木在光肥调控下的生长与代谢响应机理,为药用植物云曼红豆杉杂种优势利用及品种推广提供了理论与技术支撑。更多还原

【Abstract】 Taxus is the only material required for the industrial production of the anticancer drug paclitaxel and 10-Deacetylbaccatin III（10-DAB）,the ingredient required for artificial semi synthesis.Taxus madia×Taxus yunnanensis‘Yunman’（YM）is a new medicinal plant variety of Taxus with high concentration of 10-DAB selected and bred by the research group through artificial hybridization.The F₁ hybrids seedling（<5 years old）has obvious heterosis,but only has 4～5 years of available period,resulting in low biomass of individual plant,low accumulation of major secondary metabolites at harvest time and insufficient yield per unit area,which hinders the utilization of heterosis and promotion of the variety.Boosting the growth of seedlings by artificial regulation is the most simple and convenient measure to promote the large-scale planting of new varieties,which is revealing the response mechanism of artificial regulation to the growth of seedlings and secondary metabolites.Therefore,exploring an effective regulation measure to increase the accumulation of major secondary metabolites per plant during the utilization cycle is a scientific and technical problem to be settled urgently in terms of the utilization of heterosis and promotion of the variety for YM.Based on the research of the biological characteristics of YM in the early stage,and taking 1-year-old container seedlings before leaving nursery as experimental materials,this paper carries out growth surveys,physiological and biochemical indicators detection,and dynamic tracking measurement of secondary metabolites with the method of steady state nutrient fertilization,so as to study the influence of nutrients on the growth,nutritional physiology,and main secondary metabolites and reveal the laws of fertilizer requirements for growth and the characteristics of nutrient accumulation and distribution.2-year-old seedlings after leaving nursery are used as experimental materials for the study on the growth,photosynthesis,stress resistance physiology,and accumulation response of major secondary metabolites of seedlings under the conditions of light and fertilization interaction.Based on the construction of the optimized model of the light and fertilization interaction mode with the fuzzy Borda method,as well as transcriptome sequencing and analysis,it explores the physiological and molecular response mechanism of the growth and light and fertilization compound control of the secondary metabolites of YM,and seeks effective measures for boosting growth by artificial regulation.Our studies provide theoretical and technical support for the utilization of heterosis and variety promotion.The main research results are as follows.（1）Steady state nutrient fertilization can significantly promote the biomass and accumulation of nutrient elements N,P,and K in 1-year-old seedlings of YM（P<0.05）.The biomass and nutrient accumulation reach highest with a nitrogen application rate of 1.6g/plant under steady state nutrient fertilization.When the nitrogen application rate is higher than 3.2 g/plant or lower than 2.4 g/plant,the growth and the synthesis of taxanes will be inhibited.Taxol and 10-DAB in branches and leaves present an upward trend from May to September,and a downward trend from September to November.The distribution amount of nutrients N,P,and K in various organs of 1-year-old seedlings of YM is in the order of leaves>roots>stems.The overall trend of nutrient element absorption in leaves is displayed as N>K>P,and there is a significant positive correlation between P and K,P and N in various organs,as well as N and K in leaves and roots（P<0.05）.（2）The combined regulation of light and fertilizer significantly improves the photosynthesis of 2-year-old seedlings of YM,promotes the synthesis of soluble proteins,soluble sugars,and proline,as well as the accumulation of taxanes（P<0.05）.Under conditions of 40～70%light and nitrogen application of 3.6 g/plant,the cumulative amount of paclitaxel and 10-DAB in branches and leaves of single plant significantly increases（P<0.05）.The rapid growth period of 2-year-old seedlings of YM is from March to June,and the optimal harvesting and utilization period is from September to December.The cumulative amount of paclitaxel and 10-DAB of single plant reach their maximum values in September（9.010 mg）and December（85.228 mg）,respectively.（3）From three aspects of physiology,growth,and secondary metabolism,12 key indicators such as net photosynthetic rate（P_n）,stem volume index（SVI）,and paclitaxel accumulation（APTX）are selected to establish a comprehensive evaluation index system.The improved CRITIC method,Grey Relational Analysis,TOPSIS method,and Membership Function Analysis method are used for the comprehensive evaluation and ranking of 16 light fertilizer regulation combinations.Theλ² testing method is used to verify the consistency of the four evaluation methods,and construct a optimized model of the light and fertilization combination of YM with the fuzzy Borda method.According to the result,the fuzzy Borda combination evaluation values above 90 points are in an order of S3F3（40%light+3.6 g/plant nitrogen application）>S2F3（70%light+3.6 g/plant nitrogen application）>S3F2（40%light+1.8 g/plant nitrogen application）.（4）Through the analysis on the leave transcriptome of 2-year-old seedlings of YM in the light and fertilization optimal group S3F3 and the control group（untreated）,a total of1,334 DEGs（differentially expressed genes）are identified,including 752 up-regulated genes and 582 down-regulated genes.All 569 transcription factors such as b HLH,NAC,MYB are identified in DEGs.21,944 transcripts have the highest annotation rate in the Nr database,saying 75.40%.The gene ontology（GO）enrichment of differential genes mainly lies in signal transduction,transmembrane transport protein activity,defense response,etc.Kyoto encyclopedia of genes and genomes（KEGG）enrichment mainly occurs in the aspects of biosynthesis of phenyl-propane,diterpene,and terpenoid frameworks.The light and fertilization compound control induces differential expression of transcription factor genes like b HLH and NAC,which affects the growth physiology and secondary metabolite synthesis of YM mainly through phenylpropanoid metabolism,starch and sucrose synthesis,and terpenoid biosynthesis pathways,in response to the effect of light and fertilization interaction.Among them,five CPX genes affecting paclitaxel synthesis are identified.In summary,there is a significant effect of steady-state nutrient fertilization on the improvement of biomass accumulation of 1-year-old container seedlings of YM.The light and fertilization combination S3F3（40%light+3.6 g/plant nitrogen application）can significantly promote the cumulative amount of paclitaxel and 10-DAB of single plant in 2-year-old seedlings of YM.The research result reveals the growth and metabolic response mechanism of YM seedlings under light and fertilization regulation.This finding provides theoretical and technical support for the utilization of the heterosis of the medicinal plant YM,and the promotion of varieties.更多还原