谷子6号染色体雄性不育基因的克隆

Cloning Male Sterility Gene on Chromosome 6 of Foxtail Millet

克隆谷子雄性不育材料1066A的不育基因,分析不育基因与可育基因存在的突变位点,为揭示谷子雄性不育分子机制、利用分子标记辅助选择方法选育多用途的不育材料奠定基础。利用谷子全基因组测序数据及前人不育基因定位结果克隆谷子雄性不育材料1066A的雄性不育基因,发掘导致不育的突变位点,旨在为从分子水平揭示谷子不育机制、利用分子标记辅助选择方法选育多用途的不育材料奠定基础。首先利用生物信息学方法从豫谷1号6号染色体找到1个雄性不育基因位点(Si015780m.g),该基因全长5 027个碱基,编码479个氨基酸,且位于前人用分子标记定位的基因组区间内。根据豫谷1号不育基因序列设计2对特异引物在雄性不育材料1066A的基因组DNA进行PCR扩增。将扩增产生的2个基因组片段进行拼接后在谷子不育材料1066A中获得2 561 bp的基因序列,包含了下游部分编码区。通过对豫谷1号、张谷、1066A的不育基因部分编码序列及推定的蛋白质序列进行比对分析,结果发现谷子不育材料1066A的不育基因编码序列存在3处突变:2处单碱基替换和1处单碱基插入,这3处突变导致谷子不育材料1066A的不育基因蛋白的第402,403个氨基酸由异亮氨酸和亮氨酸替换成缬氨酸和异亮氨酸,同时导致其不育基因编码的蛋白在第466个氨基酸处发生提前终止。3处突变中2处氨基酸替换对编码蛋白的功能影响不大,因此,认为谷子不育材料1066A的不育基因蛋白翻译提前终止可能是导致其产生不育的原因。

Cloning a male sterility gene from foxtail millet male sterility material 1066 A,analyzing the mutation sites between sterility gene and fertile gene,which could provide foundation for exploring molecular mechanism of male sterility,breeding multi-purpose male sterility materials by marker assisted selection method. A putative male sterility gene was cloned from 1066 A based on the complete foxtail millet genome sequence data and location results of predecessors to explore mutation sites leading to sterility,which could provide foundation for uncovering male sterility mechanism at molecular level and breeding multi-purpose male sterility materials by marker assisted selection method. Firstly,a male sterility gene with total length of 5 027 bp（ coded 479 aa,Si015780 m. g） was found on chromosome 6 of foxtail millet cultivar Yugu 1 by bioinformatics method,and the gene was in the same genome region as that located by molecular markers. Two pairs of specific primers were designed according to male sterility gene sequence of Yugu 1 to amplify corresponding male sterility gene of 1066 A（ a male sterility material）. Totally 2 561 bp gene sequence was obtained after assembling of two amplification fragments,which contained part code region downstream of the gene. After sequence alignment of part coding regions and putative protein sequences of male sterility gene from Yugu 1,Zhanggu and 1066 A,three mutation sites including two single base substitutions and one base insertion were found in coding region of male sterility gene from 1066 A. Two base substitutions led to Ile（ 402） and Leu（ 403） from protein of Yugu 1 and Zhanggu were replaced by Val and Ile from protein of 1066 A,and one base insertion led to premature translation termination of protein from 1066 A when reached the 466 th aa. Of the three mutation sites,two aa substitutions had limited effect on function of coding protein,so we suggested that premature translation termination of protein from 1066 A male sterility gene would be the reason for its sterility phenotype. This study provided foundation for uncovering molecular mechanism of male sterility in foxtail millet.