不同化感型杉木无性系连栽过程根际酚类物质动态变化及其代谢机制

Chinese fir is one of the most important timber tree species planted in southern China, and autotoxicity is one of the main reasons for productivity decline in continuous-planting Chinese fir plantations. Base on the large research achievements, different allelopathic types of Chinese fir clones (tolerant-autotoxins clone, middle-autotoxins clone and sensitive-autotoxins clone ) were acted as experimental materials, the soils with different generation Chinese fir plantation（include first, second and third generation）and evergreen broad-leaf forest(CK) were acted as substrates, field and pot experiments were done in this study.Through determining ecological factor, microbial groups, the key enzyme activity, concentration and types of phenolic compounds in rhizospheric soils of different types of allelopathic Chinese fir clones, the study would further to determine the key enzyme activity of phenolic compounds on the root tip of different types of allelopathic Chinese fir clones. Combining with molecular biology and bioinformatics technology,the study would extract,isolate and identify differentially expressed proteins in the root tip of different types of allelopathic Chinese fir clones, searching differentially expressed proteins encoding genes, constructing transformation expression vector,transformating of differentially expressed proteins encoding genes into Arabidopsis thaliana to elucidate the function. The study could reveal the mechanisms of changes of rhizosphere soil phenolic compounds of different types of allelopathic Chinese fir clones and response mechanism of changes of rhizosphere soil phenolic compounds to rhizosphere environment, expound metabolic mechanism of rhizosphere soil phenolic compounds of different types of allelopathic Chinese fir clones through differential proteomics and transgene.

杉木是我国南方最重要的用材树种之一，自毒作用是导致杉木连栽地力衰退的主要原因之一。该项目在众多研究成果的基础上，以课题组筛选的不同化感型（耐自毒型、自毒中间型、自毒敏感型）杉木无性系为材料，通过大田及盆栽试验，以不同栽杉代数人工林（1代、2代及3代）土壤及常绿阔叶林土壤（对照）为基质，通过测定不同化感型杉木无性系根际土壤生态因子、微生物类群、土壤关键酶活性、土壤酚类物质种类及含量的基础上，进一步测定不同化感型杉木无性系根尖酚类物质代谢关键酶活性，并结合分子生物学和生物信息学技术，提取及分离不同化感型杉木无性系根尖差异蛋白，鉴定特异蛋白及寻找特异蛋白编码基因，构建转化表达载体，并导入拟南芥中，进一步验证其功能，从而从机理上揭示不同化感型杉木无性系连栽过程根际酚类物质变化及其对根际微环境的响应，并从差异蛋白组学及转基因角度阐述不同化感型杉木无性系根际酚类物质代谢机制。

1．不同连栽代数杉木人工林及阔叶林土壤理化性质差异.随着杉木连栽代数增加，土壤容重、速效钾含量呈现出逐渐增加趋势，含水率、最大持水量、最小持水量、毛管持水量、总孔隙度、pH值、全氮、全碳、全钾则呈逐渐降低的趋势。.2．不同连栽代数不同化感型杉木无性系生长量差异.同一化感型杉木无性系苗高及地径生长量均表现为随着连栽代数增加其生长量呈现下降趋势。同一土壤培养基质条件下，杉木忍耐性无性系苗高及地径增长量大于杉木敏感型。.3．连栽地不同化感型杉木无性系根际及非根际土壤酶活性测定.忍耐型根际土壤脲酶、蔗糖酶活性CK在春秋冬季均高于其他类型土壤；忍耐型根际土壤纤维素酶活性春夏冬季TCF活性值最大；敏感型根际土壤纤维素酶活性，春夏秋冬季分别为TCF、SCF、FCF和CK最高；忍耐型根际和非根际土壤酸性磷酸酶活性SCF最高。.4．连栽地不同化感型杉木无性系根际及非根际土壤微生物功能多样性测定.PBP土壤微生物总体活性（AWCD）、Shannon指数、 Simpson指数及McIntosh指数值均高于其他3种类型；AWCD值、土壤pH、全C、全N、微生物生物量 C、N的大小顺序为PBP > FCF > SCF > TCF。.5. 杉木连栽地杉木土壤微生物组成鉴定及相关分析.土壤细菌优势门类为酸杆菌门、变形菌门、浮霉菌门、绿弯菌门；Ace、Chao1、Simpson、Shannon指数分别与土壤容重、Total P、Total N呈显著相关。真菌门类有子囊菌门、担子菌门、接合菌门等；Chao1指数和总孔隙度正相关关系；Shannon指数与pH、全氮和全碳显著正相关。.6．连栽地忍耐型杉木无性根尖及根际土壤酚酸种类鉴定.根尖共鉴定出13种酚酸物质，其中儿茶素和奎尼酸的相对含量较高。根际土壤中共鉴定出12种酚酸物质，阿魏酸、咖啡酸、香草酸相对含量较高。.7. 不同连栽代数土壤化感忍耐型杉木无性系根尖差异蛋白鉴定.共鉴定到10333个蛋白。连栽3代的与1代相比，102个上调表达蛋白，108个下调表达蛋白；而连栽3代与2代相比，77个上调表达蛋白，91个下调表达蛋白。.构建两个差异蛋白基因A和B植物表达载体，获得转基因拟南芥。基因A和B与内参基因GADPH相比，在根、茎、叶中表达量较低。基因A在根中表达最高，而基因B在叶中有相对高的表达。