基于引物扩增受阻突变技术开发水稻耐冷基因CTB4a特异性分子标记  

Development of Specific Molecular Marker of Cold Tolerance Gene CTB4a in Rice Base on PARMS

作　　者：梁海福 潘英华 刘开强[2] 邓国富[2] 黄娟[2] 卿东进 高菊 伍豪[2] 高利军[2] Liang Haifu;Pan Yinghua;Liu Kaiqiang;Deng Guofu;Huang Juan;Qing Dongjin;Gao Ju;Wu Hao;Gao Lijun(Key Laboratory of Guangxi for Rice Genetics and Breeding,Rice Research Institute,Guangxi Academy of Agricultural Sciences,Nanning,530007;Guangxi Crop Genetic Improvement and Biotechnology Laboratory,Guangxi Academy of Agricultural Sciences,Nanning,530007)

机构地区：[1]广西壮族自治区农业科学院水稻研究所,广西水稻遗传育种重点实验室,南宁530007 [2]广西壮族自治区农业科学院,作物遗传改良生物技术重点开放实验室,南宁530007

出　　处：《基因组学与应用生物学》2021年第4期1719-1724,共6页Genomics and Applied Biology

基　　金：广西自然科学基金重点项目(2018GXNSFDA281053);广西水稻优质化育种研究人才小高地青年人才培养基金(桂农科水稻QN-05);广西重大科技创新基地,广西水稻遗传育种重点实验室开放课题(2018-05-06-KF08)共同资助。

摘　　要：水稻(Oryza sativa)作为热带与亚热带起源的作物对低温敏感。对水稻种质进行耐冷性鉴定,能筛选出耐冷性强的种质,发展耐冷基因分子标记,能够有效鉴别种质中耐冷基因的基因型。本研究使用芽期4℃低温处理10 d对41份水稻材料进行芽期耐冷鉴定,对品种的芽期耐冷能力进行评价,获得了参试材料中除了‘昆明小白谷’之外的芽期耐冷性最强的品种‘南特号’。对已克隆的耐冷基因CTB4a开发分子标记,能够辅助选择水稻的耐冷育种。水稻孕穗期耐冷基因CTB4a来源于‘昆明小白谷’,能够影响水稻抵抗低温的能力。参照公布的CTB4a序列信息,从中挑选出序列中的作用位点SNP (单核苷酸多态性, single nucleotide polymorphism),结合引物扩增受阻突变技术(Penta-primer amplification refractory mutation system,PARMS),用Primer 6.0设计引物,建立CTB4a基因荧光分子标记GM-CTB4a,使用荧光分子标记GM-CTB4a对41份水稻品种进行鉴定,使用酶标仪在‘昆明小白谷’中检测到利用标记扩增产物中包含‘昆明小白谷’特异SNP、T碱基引物携带的FAM荧光信号,在另外40份品种的扩增产物中检测到包含作用位点的C碱基引物携带的HEX荧光信号。本研究利用设计的分子标记,鉴定了41份水稻品种的耐冷性和基因型。比对分析耐冷性和基因型鉴定结果,说明我们开发的分子标记GM-CTB4a特异性较强,具有实际应用价值。研究结果为利用水稻孕穗期耐冷基因CTB4a培育强耐冷水稻品种奠定坚实基础。Rice(Oryza sativa L.) is a cold-sensitive species crop that originated from tropical or sub-tropical areas. It is important to select cold tolerance germplasm and develop the molecular markers of cold tolerance genes for improving resistance breeding of rice. The study identified the cold tolerance of 41 varieties under low temperature at 4 ℃ f or 10 d at germination stage. After then the nantehao with the coldest tolerance stood out from those varieties except Kunming xiaobaigu. The cold tolerance gene CTB4 A from Kunming xiaobaigu at booting stage can affect the low temperature resistance ability of rice. Primer 6.0 was used in CTB4 a for developing a GM-CTB4 a gene marker of a SNP(single nucleotide polymorphism) with PARMS(penta-primer amplification refractory mutation system). Then 41 cultivars were detected to possess the intact CTB4 a gene by using the GM-CTB4 a marker. And that it can be used in checking 41 cultivars for allele of CTB4 a. The fluorescence signal of T-FAM in GM-CTB4 a was so specific that it was detected in Kunming xiaobaigu only but fluorescence signal of C-HEX was detected in other 40 varieties by microplate reader TECAN SparkTM10. The study identified cold tolerance and CTB4 a genotype of 41 varieties by cold treatment at germination stage and PARMS. The results of cold tolerance and genotype identification showed that the molecular marker GM-CTB4 A developed by us has strong specificity and practical application value. These results will greatly facilitate the utilization of CTB4 a in rice breeding programs by marker-assisted selection.

关 键 词：耐冷基因 CTB4a 分子标记 

分 类 号：S511[农业科学—作物学]