【作者】 张君诚；

【导师】 庄伟建；

【作者基本信息】 福建农林大学 ， 作物遗传育种， 2004， 博士

【摘要】 胚胎早期发育是花生产量和品质形成的重要时期，其幼胚发育的正常与否直接关系到以种子为经济指标的花生生产的效益。缺钙花生严重空荚或不饱满而导致减产降质的生产问题长期困扰南方沙质土壤地区花生生产。本研究以高低钙条件下大田和水培花生的种植与观察为基础，成功开展了花生胚胎败育与可育的基因差异显示、差异蛋白质组学方面的研究，初步揭示受钙影响花生胚胎败育的分子机理，主要研究结果如下： 1．开展受钙影响水培和大田（福建平潭）花生植株与幼胚生长发育特性观察与比较，总结空荚发生模式与特征，田间试验表明：大田低钙环境对花生地上部植株的生长发育影响较小，而对荚果发育影响较大，低钙植株烂果或空荚和营养体徒长与返青现象显著；水培实验进一步证实20mg·L^-1Ca²⁺浓度是花生败育的临界浓度。低钙使花生植株矮小，烂果和空荚增多，但不出现营养体徒长。比较认为大田花生更适合作为基因差异显示和差异蛋白质组学等研究的实验材料。提出砂质旱地增施钙肥（石膏、壳灰）以防治花生空荚的方案和注意事项，为指导生产实践提供参考。 2．开展受钙影响花生幼胚基因差异显示研究，摸索出花生幼胚高质量总RNA的提取方法；首次建立和优化了花生幼胚mRNA荧光差异显示实验的技术体系：利用该体系成功筛选得到L^#、1^#、3^#、8^#等四个花生早期胚胎（9d）败育／正常发育的基因差异表达片段，经反向Northern Dot blot和RT-PCR证实为阳性差异片段。测序和检索分析认为L^#差异片段与拟南芥（Arabidopsis thaliana（thale cress））和水稻等的ATP依赖-Clp蛋白酶的ATP捆绑亚单位同源性较高，该基因在低钙组的上调表达提示低钙组幼胚细胞内能量转化和物质的转运受到胁迫。已在Genebank登记该片段（基因号为AY517932）。8^#差异片段与陆地棉（Gossypium hirsutum）MADS box基因mRNA同源性较高，初步分析认为这可能与低钙组幼胚发育调节紊乱相关，该片段已在Genebank登记（基因号为AY517932）。3^#和1#差异片段未能在数据库中检索到较匹配的序列，也可能是个未报道的基因片段。 在此基础上，克隆得到了花生MADS box基因的包含编码区和3’-UTR在内的cDNA序列，长度为785bp，编码261个氨基酸，与已报道多种植物的MADS box的基因和氨基酸序列同源性分别在83%和90%以上，这在花生上国内外尚未有见报道。 3.开展了受钙影响导致花生早期胚胎败育的差异蛋白质组学研究。建立适合花生蛋白质的双向电泳技术体系，结合MALD工一TOF质谱和相关蛋白质数据库等蛋白质组学研究手段首次筛选了花生早期胚胎败青/发育的7个差异表达蛋白。初步检索发现:4.差异蛋白点是与叶绿体被膜休克相关蛋白（来源于SPl’nacia。lerace“L.）高度相似的低钙组表达上调的特异蛋白，这提示低钙胚内膜系统出现生理和或运转障碍。6招差异蛋白与线粒体前体中的ATP合成酶的刀链高度相似，该蛋白在低钙幼胚缺失提示花生幼胚受钙胁迫时ATP合成酶的合成和组装严重受阻。7‘差异蛋白可能与茄属植物（Sola刀姗tuberos二）的肌动蛋白97或100同源，该蛋白在低钙组缺失说明败育胚此时（入土9天）的细胞骨架系统发育或细胞分裂等重要生理活动可能受阻。2，、3.差异蛋白分别与3一磷酸甘油醛脱氢酶和异柠檬酸脱氢酶具一定同源性，这两种蛋白在低钙组幼胚的未表达提示低钙幼胚的氧化还原反应系统可能不正常。而1‘和5件蛋白点得分较低，有可能是未知蛋白。 由以上研究结果初步可以认为:受钙胁迫花生幼胚败育的分子机理可能是与早期幼胚内与能量转化（ATP的合成）、氧化还原系统运转、细胞内膜以及细胞骨架功能维持相关的功能基因的转录和表达异常有关。更多还原

【Abstract】 Seeds is the most economic index of peanut ( Arachis hypogaea L.) production and the early development of embryo is the important stage for the yield and quality of peanut.For many years now , there has been a general problem existing in peanut production areas in the red and yellow soil region of south China,which barepods and falles in yield of peanut resulting from deficient calcium in soil.Baseing on the measures and comparisions between water culturing and field experiment in Pingtan(Fujian province) in different levels of calcium, the project deeply and systematically dealt with the experiment about DDRT and proteome in order to reveal the molecular biology mechanism of peanut embryo abortion mediated by calcium. Some valuable results were as follows:1. Revealed the mode of the peanut plant growth and development and empty pod occurrence. In field experiment, leaves and stems of peanut were influenced gently, but pods were influenced badly by calcium. Lacking of calcium caused changes of photosynthetic product and ill-filled pods and empty pods increasing, which significantly lowered the yield. But under calcium deficiency stress in water culture ,both vegetatives and pods of growth in water culture were influenced seriously.In our opinions, the peanut materials for molecular experiment from the field experiment were more suitable than that from water culture .The field production managements against empty pods were also presented.2. Succeeded firstly in conducting the research of DDRT of peanut early embryoes between in deficient and sufficient levels of calciumion .8 differentially displayed cDNA fragment were obtained and four of them (L#, 1#, 3#, 8#)were confirmed by reverse northern dot blot and RT-PCR . B1AST was used to search the NCBInr database.The result were as follows: Fragment L# shows high homology with the ATP-dependent Clp protease ATP-binding subunit CIpX from Arabidopsis thaliana (thale cress) .We registered it in Genebank(AY517932); Fragment 8s shows 87% homology with the MADS box protein GHMADS-1 (MADS-1) mRNA complete cds ( AF538965 ) from Gossypium hirsutum L.. Fragment 8# was also registered in Genebank( AY572857) by us. Fragment 1# and 3# could not be found homologous with any valuable sequences,thus,maybe a new gene.Basing on above,we cloned the cDNA of peanut MADS genes and the structural characteristics was preliminarily analysized firstly.The cDNA of peanutMADS genes showed good homology with the MADS box gene or amino acid seguences from many plants.3. Established firstly a preliminary 2-D electrophoresis system for proteins from peanut early embryoes with two calcium levels. The peptide mass fingerprinting(PMF) of Seven differentially expressed proteins were got by MALDI-TOF-MS. Mascot was used to search the protein database.The results were as follows: 4# protein.which up-regulated in embryoes from sufficient calcium group, showed high homology with chloroplast envelope membrane 70 kDa heat shock-related protein from Spinacia oleracea L.6# protein(down-regulated in embryoes with sufficient calcium) showed high homology with (ATPB)ATP synthase beta chain from Hevea brasiliensis L.And 7# protein (disappeared in embryoes in sufficient calcium group)was considered similar to Actin 97 from Solarium tuberosum. 2#\3# protein showed some homology with Glyceraldehyde 3-phosphate dehydrogenase\ Isocitrate dehydrogenase.All the results indicated:the molecular mechanism for embryoes abortion mediated by calcium may be relevant to some genes, which controlling assenergy transfering (ATP synthesis) oxidoreduction system endomembrane system and cytoskeleton structure system ,transcripted or expressed unnomally.更多还原