Identification of two novel nodule-specific genes from Astragalus sinicus L. by suppressive subtractive hybridization  

作　　者：CHOU MinXia WEI XinYuan CHEN DaSong ZHOU JunChu 

机构地区：[1]College of Life Sciences, Northwest Sci-Tech University of Agriculture and Forestry, Yangling 712100, China [2]College of Food Science and Engineering, Northwest Sci-Tech University of Agricuiture and Forestry,Yangling 712100, China [3]State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China

出　　处：《Chinese Science Bulletin》2007年第20期2797-2804,共8页

基　　金：Supported by the National Basic Research Program of China (Grant No. 2001CB108901)

摘　　要：To identify the genes involved in nodule formation and to increase usable molecular probes, a cDNA library of Astragalus sinicus genes specifically expressed in infected roots by Mesorhizobium huakuii 7653R is generated using a PCR-based suppressive subtractive hybridization (SSH) technique with two mRNA populations of infected and uninfected control roots. Two nodule-specific genes, AsIIC259 and AsG2511, are identified from infected roots of A. sinicus. The amino acid sequences deduced from the open reading frames (ORFs) reveal that AsIIC259 and AsG2511 encodes a polypeptide with 134 and 58 amino acids, respectively. A signal peptide sequence is predicted with high probability at the N-termini of the AsIIC259 and AsG2511. The motif searches show that the deduced polypeptide of AsIIC259 con- tains two N-glycosylation sites, a cAMP- and cGMP-dependent protein kinase phosphorylation site and a casein kinase II phosphorylation site. BLASTP searches reveal that AsIIC259 putative protein displays a low degree of similarity to a unique nodulin from Lupinus luteus nodules. No significant identity is displayed over the predicted polypeptides of AsG2511 with any published sequences. Virtual Northern blot and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses indicate that the two genes are expressed exclusively in inoculated roots and that their expression is 2―4 d later than that of the leghaemoglobin (Lb) gene during nodule development.To identify the genes involved in nodule formation and to increase usable molecular probes, a cDNA library of Astragalus sinicus genes specifically expressed in infected roots by Mesorhizobium huakuii 7653R is generated using a PCR-based suppressive subtractive hybridization （SSH） technique with two mRNA populations of infected and uninfected control roots. Two nodule-specific genes, As11C259 and AsG2511, are identified from infected roots of A. sinicus. The amino acid sequences deduced from the open reading frames （ORFs） reveal that AsllC259 and AsG2511 encodes a polypeptide with 134 and 58 amino acids, respectively. A signal peptide sequence is predicted with high probability at the N-termini of the AsllC259 and AsG2511. The motif searches show that the deduced polypeptide of AsllC259 contains two N-glycosylation sites, a cAMP- and cGMP-dependent protein kinase phosphorylation site and a casein kinase Ⅱ phosphorylation site. BLASTP searches reveal that AsllC259 putative protein displays a low degree of similarity to a unique nodulin from Lupinus luteus nodules. No significant identity is displayed over the predicted polypeptides of AsG2511 with any published sequences. Virtual Northern blot and semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） analyses indicate that the two genes are expressed exclusively in inoculated roots and that their expression is 2-4 d later than that of the leghaemoglobin （Lb） gene during nodule development.

关 键 词：根瘤素 共生关系 杂交 紫云英 遗传细胞 

分 类 号：Q943[生物学—植物学]