紫花苜蓿PGIP同源基因克隆与序列核苷酸多态性分析  

Clone of homologous PGIP genes and analysis of the polymorphism of nucleotide sequence in alfalfa

作　　者：桂枝[1] 肖婷[1,2] 皮永硕 袁庆华[3] 杨建翔[1] 刘妍[1] 高建明[1] 

机构地区：[1]天津农学院农学与资源环境学院,天津300384 [2]华南农业大学农学院,广东广州510642 [3]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《草业科学》2015年第10期1603-1611,共9页Pratacultural Science

基　　金：国家自然科学基金(青年)项目(31201840);天津市高等学校创新团队培养计划(TD12-5017);天津市创新训练项目(201410061060)

摘　　要：多聚半乳糖醛酸酶抑制蛋白(PGIP)是植物重要的防卫蛋白之一,了解PGIP基因的核苷酸序列是对其进行分子遗传学与分子生物学研究的前提与基础。本研究采用同源克隆法,从16个紫花苜蓿(Medicago sativa)品种的集群DNA中克隆了7个苜蓿PGIP基因的部分基因组编码序列,分别对应于蒺藜苜蓿(M.truncatula)7个不同的PGIP基因,并命名为Ms PGIP5、Ms PGIP6、Ms PGIP7、Ms PGIP8、Ms PGIP9、Ms PGIP10和Ms PGIP11。其中,Ms PGIP5、Ms PGIP6、Ms PGIP8、Ms PGIP9和Ms PGIP11的单核苷酸多态性(Single Nucleotide Polymorphism,SNP)位点间的平均距离分别为20、74、34、423和21 bp,差异较大。对Ms PGIP5、Ms PGIP6、Ms PGIP8和Ms PGIP11进行荧光标记PCR单链构象多态性(FPCR-SSCP)分析,结果表明,其等位基因数目与PCR片段长度的比值分别为20、56、22和19 bp,变化趋势与这4个基因SNP位点间的平均距离基本一致。比较而言,在发现的7个苜蓿PGIP基因中,Ms PGIP9序列高度保守,Ms PGIP5和Ms PGIP11的变异较大,而Ms PGIP6与Ms PGIP8的变异中等。Polygalacturonase-inhibiting proteins（ PGIP） are one of the most important defensive proteins,and the understanding of the nucleotide sequences of PGIP genes is the premise and basis of the molecular genetics and molecular biology. This study aimed to isolate the genomic coding sequences of PGIP genes in alfalfa（ Medicago sativa） by a homology-based method. Consequently,the part genomic coding sequences of 7 PGIP genes were cloned,and each of these 7 alfalfa PGIP genes had a corresponding homologous PGIP gene from M. truncatula,and they were named as Ms PGIP5,Ms PGIP6,Ms PGIP7,Ms PGIP8,Ms PGIP9,Ms PGIP10 and Ms PGIP11. Of the found 7alfalfa PGIP genes,Ms PGIP5,Ms PGIP6,Ms PGIP8,Ms PGIP9 and Ms PGIP11 possessed a divergent average interSNP distance with a value of 20,74,34,423 and 21 bp,respectively. FPCR-SSCP analysis was performed for Ms PGIP5,Ms PGIP6,Ms PGIP8 and Ms PGIP11,and the results showed that a trend of the ratios between their SSCP allele numbers and their PCR fragment sizes,with a value of 20,56,22 and 19 bp,respectively,and they were basically consistent with those of their average inter-SNP distances. In contrast,of the found 7 alfalfa PGIP genes,the sequence of Ms PGIP9 was highly conserved while Ms PGIP5 and Ms PGIP11 displayed a large sequence variation,and Ms PGIP6 and Ms PGIP8 had a median sequence variation.

关 键 词：紫花苜蓿 PGIP 基因组序列分离 单核苷酸多态性 荧光标记PCR单链构象多态性 

分 类 号：S541.9[农业科学—作物学]