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Functionally analysis of legume symbiotic specific genes in transgenic Lotus japonicus

转基因百脉根豆科植物共生特异性基因的功能分析

基本信息

批准号：
16580047
负责人：
TAJIMA Shigeyuki
金额：
$ 2.37万
依托单位：
Kagawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16580047/
关键词：
Lotus japonicus transgenic plant nodule SNARE promoter analysis root cortex PEPC C N metabolism 二次元電気泳動共生窒素固定

项目摘要

(1)For surveying symbiotic differentiation of key organelle, mitochondria, soybean nodule and root mitochondria were compared after two dimensional electrophoresis and the proteins were characterized in combination with MALDI-TOF MS. We also surveyed the soybean nodule bacteroid by the methods of proteome analysis.(2)We identified a Sed5-like clone LjSYP32-1 which contributes to nodule tissue formation and plant growth in Lotus japonicus. Promoter analysis and in situ hybridization revealed that LjSYP32-1 expressed significantly in inner cortex cell layer surrounding the infected zone of young nodules and at meristem area of developing lateral root. The anti-sense plants showed the nodules were much smaller and some nodules fused each other by sharing the vascular bundles. Such irregular nodules were observed in the rate of two times more comparing to those of wild type plant. The data suggest that LjSYP32-1 is contributing to support plant growth and normal nodule tissue differentiation.(3)Using antisense technique, we diminished the expression levels of the nodule enhanced PEPC gene (Ljpepc1) in a determinate legume plant (Lotus japonicus) for surveying the influence to symbiotic phenotype and biochemical parameters. In nodules of Asppc plants, PEPC activity reduced to ca. 10% of non-transformants and the plants showed a typical nitrogen deficient symptom without nitrogen nutrient supply, and returned to normal growth with supply of 2.5 mM nitrate. Significant reduction of sucrose, succinate, asparagine, asparatate and glutamate contents also decreased in Asppc nodules. The data was discussed on a role of PEPC for the carbon/nitrogen metabolic flux in nodules.(4)We isolated the some novel protein kinase possessing the subdomain VIB sequences characteristic of Ser/Thr kinase. To determine the function of the gene, we have made transgenic Lotus japonicus by RNAi methods.

(1)为了研究关键细胞器的共生分化，通过二维电泳比较了线粒体、大豆根瘤和根系线粒体，并结合MALDI-TOF ms对蛋白质进行了鉴定，并采用蛋白质组学分析方法对大豆根瘤类细菌进行了调查。(2)鉴定了一个与sed5类似的克隆LjSYP32-1，该克隆参与了莲藕组织形成和植株生长。启动子分析和原位杂交表明，LjSYP32-1在幼根瘤感染区周围的内皮层细胞层和侧根发育的分生组织区有显著表达。反义植物的根瘤较小，部分根瘤通过共享维管束相互融合。这种不规则根瘤的发生率是野生型植物的2倍以上。数据提示LjSYP32-1参与支持植物生长和正常根瘤组织分化。(3)利用反义技术，在确定的豆科植物荷花（lotusjaponicus）中降低结核增强PEPC基因（Ljpepc1）的表达水平，考察其对共生表型和生化参数的影响。在Asppc根瘤中，PEPC活性降低到非转化体的10%左右，在没有氮素营养供应的情况下，植株表现出典型的缺氮症状，在提供2.5 mM硝酸盐后恢复正常生长。蔗糖、琥珀酸、天冬酰胺、天冬氨酸和谷氨酸含量显著降低。讨论了PEPC在根瘤碳氮代谢通量中的作用。(4)分离到具有丝氨酸/苏氨酸激酶特征的VIB亚结构域的新型蛋白激酶。为了确定该基因的功能，我们采用RNAi方法对日本莲进行了转基因。