【作者】 武剑；

【导师】 孙日飞； 王晓武；

【作者基本信息】 中国农业科学院 ， 蔬菜学， 2005， 博士

【摘要】 锌是生物体必需的微量金属元素,作为蛋白质的结构和催化成分参与到许多生理生化反应中。缺Zn是严重影响我国人民身体健康的微营养不良症之一,培育Zn强化性作物是克服Zn营养不良症的新策略。缺Zn同时也是严重影响作物生产的植物营养问题之一,培育高Zn效率的作物新品种是解决植物Zn营养不足的一种更具有环境亲和性的途径。另一方面,土壤重金属污染,包括锌污染,日益严重,传统的土壤修复方法耗费很大,植物修复是上世纪九十年代提出的治理土壤污染的新策略,是利用重金属超富集植物吸收和积累土壤中的重金属,达到降低土壤重金属浓度的作用。但是,由于已经发现的重金属超富集植物都存在生物量小,生长周期长的缺点,因此这一策略并没有真正在实践中得以应用。白菜类蔬菜是我国栽培面积和产量最大的蔬菜,包括一些生长周期短,生物量相对较大的栽培种群,因此对白菜类蔬菜进行Zn营养性状的遗传分析,一方面可以为改良白菜类蔬菜的营养品质和培育高Zn效率的白菜类蔬菜新品种提供理论基础;另一方面克隆与植物Zn超富集相关的基因,通过基因工程的手段创建能够超量富集Zn的白菜类植物材料,能够为植物修复技术的实际应用创造条件。 基于以上目的,本论文从以下几个方面对白菜类蔬菜的Zn积累和Zn胁迫下的耐性进行了遗传分析: 1.利用原位噬菌斑杂交法,进行了Zn超富集植物Thlaspi caerulescens的cDNA文库筛选,获得了7个Zn积累相关候选基因的全长cDNA,这些序列与拟南芥的同源基因的序列相似性为86-91%。通过对酵母锌运输缺陷型突变体zrtlzrt2、铁运输缺陷型突变体fet3fet4、锰运输缺陷型突变体smfl的功能互补以及提高了野生型对Cd的敏感性,证明了TcNKAMP3和TcNRAMP4具有Zn、Fe、Mn和Cd的运输活性;TcNRAMP3在Arabidopsis thaliana的nramp3nramp4突变体中过量表达挽救了缺Fe条件下植株的生长。由此证明了TcNRAMP3和TcNRAMP4作为金属转运蛋白参与了植物体的金属积累。 2.利用183个DH株系构建了大白菜的AFLP连锁图谱,该连锁图由10个连锁群组成,包括222个AFLP标记,覆盖基因组长度为1064cM,标记间平均间距为4.8cM。利用这一图谱,对大白菜叶片K、Na、Ca、Mg、Al、Cu、Mn、Fe、Zn和Sr等10种矿质元素含量和总磷含量进行了QTL分析,共找到22个与叶片矿质元素含量相关的QTL和2个与叶片总磷含量相关的QTL,这些QTL对性状的贡献率在6.8-18.8%之间。对这一群体的130个DH株系作了高Zn胁迫下耐性的QTL分析,发现了一个与高Zn胁迫下大白菜生长相关的QTL,和一个仅在相对更高的Zn浓度下出现的QTL。 3.对来自于我国不同地区的分别属于7个栽培种群的184个基因型的白菜类蔬菜和2个来自于荷兰和2个来自于美国的白菜类植物的Zn积累和Zn胁迫条件下的反应进行了调查,结果表明白菜类蔬菜的叶片Zn含量存在显著差异,变化范围在23-160μg g^-1d.wt之间,叶片中Zn、Fe、Mn的积累显著相关。地上部或根部的相对生长量均适合于作为参数比较白菜类蔬菜对高Zn或缺Zn胁迫的耐性差异。白菜类蔬菜中存在高Zn胁迫耐性和Zn效率的显著差异。选出了两个对高Zn胁迫具有相对高耐性的基因型和一个具有高Zn效率的基因更多还原

【Abstract】 Zn is an essential micronutrient required by all organisms for its functions in many physiological processes as a structural or catalytic component of proteins. Unfortunately Zn deficiency is a widespread problem, affecting humans in case of Zn shortage in food, but also affecting crops in case of poor Zn availability in soil. Zn deficiency in food can be improved by Zn supplementation to the daily diet. Soil can be fertilized with extra zinc. Both approaches are not always cost-efficient and in the case of soil fertilization sometimes ineffective because of zinc binding in the topsoil Breeding and growing crops for biofortified Zn content and Zn efficiency are promising and sustainable approaches to solve the Zn deficiency problems in human and soil. On the other hand heavy metal pollution of biosphere has accelerated rapidly since the onset of the industrial revolution and heavy metal toxicity poses major environmental problems. Phyremediation is a relatively new approach to removing contaminants from the environment. It maybe defined as the use of heavy metal hyperaccumulator to remove, destroy or sequester hazardous substances from environment. It has become a topical research field in the last fifteen years since it was proposed in 1991 as it is safe and potentially cheap compared to traditional techniques. However this technology has not been used in a practical way in large scale, mainly due to the shortcomings of hyperaccumulators which are long cycle and low biomass. Brassica rapa vegetable are the most important vegetable in China with the largest consumption. It also included several cultivar groups with relatively short cycling and large biomass. Genetic analysis of Zn accumulation and homeostasis in B. rapa vegetables will provid a genetic bases for the improvement Zn content and Zn efficiency in B. rapa vegetables. The genes related to Zn accumulation cloned from hyperaccumulator T. caerulescens could be used in a GMO approach to construct of materials for phytoremediation.This thesis deals with the genetic characterization of Zn accumulation and homeostasis of B. rapa vegetables and to find out if genes related to Zn accumulation in T. caerulescens can be used in GMO approach to increase Zn content in B. rapa leaves.Chapter 1 gives an overview of progress of genetic mechanism in plant Zn accumulation and QTL analysis in B. rapa.Chapter 2 describes the cloning of candidate genes related with Zn accumulation from T. caerulescens and functional analysis of some of these genes. Totally 7 full-length cDNAs were isolated by T. caerulescen cDNA library screening. Two of seven genes, TcNRAMP3 and TcNRAMP4, were confirmed for their functions in mineral transportion.Chapter 3 describes the construction of genetic linkage map and QTL analysis of leaf mineral and phosphate content in B. rapa. QTL analysis also carried out for excessive Zn toxicity in B. rapa. An AFLP map with 222 AFLPs and 10 linkage group covering 1066 cM was constructed using aheading Chinese cabbage DH population. The average distance of intervals is 4.81 cM. By using the map, 22 QTLs for mineral content and 2 QTLs for phosphate content were detected. The percentages of variation explained were varied between 6.8-18.8%. One QTL controlling tolerance to toxic Zn stress was detected in both 100 uM and 300uM Zn treatment, while one QTL was only detected in 300uM Zn treatment.Chapter 4 describes the characterization of the genotypic variation for Zn accumulation and Zn homeostasis in B. rapa. In total 188 genotypes belonging to 9 cultivar groups, covering the geographical distribution of B. rapa vegetables in China, have been screened for shoot Zn, Fe and Mn content and for Zn tolerance. Leaf Zn content varied between 23.2-155.9(j.g g’’d. wt. Both relative shoot and root dry biomass are suitable indices for evaluation of tolerance to high or deficient Zn stress. Two relative tolerant genotypes to excessive Zn stress and one Zn efficient genotype were selected from the germplasm collection.Chapter 5 describes the establishment of Ecotilling for discovery of DNA polymorphisms in Brassica rapa natural population. We used mung bean nuclease(MBN) instead of routinely used CEL I to cleave single base pair mismatches in heteroduplex DNA templates. Nested set of primers were designed to amplify targeted region to avoid the influence of the variation in quality and quantity of the genomic DNA. To reduce the costs in fluorescently labeled primers, we adopted a strategy universal to all genes by adding Ml3 adapter to 5’end of gene specific primers to make IRD dye labeled Ml3 forward and reverse primers. A Brassica rapa ZIP gene homologue was subjected to the analysis to practice the feasibility of the method in polymorphisms detection. Our experiment showed this method is efficient in discovering DNA polymorphisms in Brassica rapa natural population.更多还原