甘蔗乙烯合成酶基因家族三个成员的克隆与序列分析

Cloning and Sequence Analysis of Three Members of ACC Synthase Gene Family in Sugarcane

摘要:

ACC（1-aminocyclopropane-1-carboxylic acid）合成酶是高等植物乙烯生物合成途径中的限速酶.根据已克隆的植物ACS（1-aminocyclopropane-1-carboxylic acid synthase）基因同源序列,设计简并引物,以甘蔗叶片总DNA为模板,通过PCR扩增,得到3条特异性强的扩增片段：Sc-ACS1为1 041 bp、Sc-ACS2为1 345 bp和Sc-ACS3为1 707 bp.将序列在GenBank核酸数据库进行同源性搜索,结果表明,3个片段均为ACS基因,推导编码的蛋白质序列分别包含326、242和310个氨基酸。其中,Sc-A CS1和Sc-ACS3同源性最高,核苷酸序列和蛋白质氨基酸序列分别有98%和96%同源,与禾本科植物玉米Zm ACS6、水稻OS-ACS2、毛竹等ACS基因家族也有很高的同源性,核苷酸序列同源性为88%-98%,蛋白质氨基酸序列同源性为73%-81%。甘蔗Sc-ACS2与水稻OS-ACS5在核苷酸和氨基酸序列上分别有91%和79%同源性,但与甘蔗Sc-ACS1和Sc-ACS3基因成员之间,氨基酸同源性分别只有45%和49%。系统进化分析表明,Sc-ACS1和Sc-ACS3基因与玉米Zm ACS6基因亲缘关系最近,而Sc-ACS2基因与水稻OS-ACS5基因亲缘关系最近。Southern杂交表明三基因在基因组中确实存在而且是多拷贝基因。三个片段已在GenBank数据库中注册,注册号分别为AY620985、AY620986和AY788919。

Abstract:

ACC （1-aminocyclopropane-l-carboxylic acid） synthase （ACS）is one of the key rate-limiting enzymes for ethylene biosynthesis in higher plants. In this paper, two degenerate oligonucleotide primers were designed, coding for conservative amino acid regions in ACC synthase protein family. PCR amplification was performed on sugarcane DNA template, and produced three fragments, 1 041 bp （Sc-ACS1）, 1 345 bp （Sc-A CS2） and 1 707 bp （Sc-A CS3）. By using the program of BLAST on NCBI GenBank database, three sequences were all ACC synthase genes, coded 326, 242 and 310 amino acids, respectively. The identity of Sc-A CS1 and Sc-A CS3 was 98% in DNA sequence and 96% at amino acid level, and that ofA CS genes from other grass plant species Zea mays （Zm A CS6）, Oryza stativa （OS-A CS2） and Phyllostaehys edulis （BA-A CS） reached to 88%-98% in DNA sequence and 73%-81% in amino acid sequence. The identity of Sc-A CS2, compared to OS-A CS5 from Oryza stativa, was 91% in DNA sequence and 79% at amino acid level, which was higher than that for Sc-A CS1 （45%） and Sc-A CS3 （49%） from sugarcane. Phylogenetic analysis showed that the genes of Sc-ACS1 and Sc-ACS3 in sugarcane were closely related to Zm A CS6 in maize, while Sc-A CS2 was closely related to OS-A CS5 in rice. Southern blotting analysis showed that these genes were presented in the genomic DNA with multiple copies. The three sequences have been registered in GenBank with the accession numbers AY620985, AY620986 and AY788919.