利用单片段代换系鉴定水稻抗性淀粉相关基因

目的 发掘稻米淀粉合成相关基因SBEIIb、SSIIa和ISA1的高抗性淀粉等位基因。方法 利用分子标记筛选携带SBEIIb、SSIIa或ISA1的单片段代换系(Single segment substitution lines，SSSLs)，利用改良的AOAC法测定SSSLs材料的抗性淀粉含量(w)，通过Sanger测序分析不同SSSLs的SBEIIb、SSIIa和ISA1基因序列，结合基因型和表型连锁分析，鉴定影响抗性淀粉含量的等位基因。结果 SBEIIb编码区的1个SNP(Ex4-96G/A)引起1个氨基酸的替换(196-Arg/His)，从而产生2种等位基因SBEIIb-1和SBEIIb-2。其中，SBEIIb-1的Ex4-96G导致第196位氨基酸为Arg，表现为高抗性淀粉含量，为1.72%。SSIIa第8外显子的2个SNPs(Ex8–334G/A和Ex8–865C/T)引起2个氨基酸替换(604-Gly/Ser和781-Leu/Phe)，从而产生3种等位基因SSIIa-1、SSIIa-2和SSIIa-3。其中，SSIIa-1的Ex8–334G和Ex8–865C导致第604和781位氨基酸为Gly和Leu，表现为高抗性淀粉含量，为3.37%。ISA1编码区序列的1个Indel(AGG/---)和1个SNP(Ex17–117C/T)导致第70位氨基酸Glu缺失和第717位氨基酸由Thr变为Met，从而产生3种等位基因ISA1-1、ISA1-2和ISA1-3。其中，ISA1-1编码区的AGG插入和Ex17–117C导致第70和717位氨基酸为Glu和Thr，表现为高抗性淀粉含量，为2.09%。结论 SBEIIb、SSIIa和ISA1是影响水稻抗性淀粉形成的重要基因，这3个基因编码区的SNPs和Indels引起了氨基酸的改变，继而影响了抗性淀粉的含量，鉴定到了3个高抗性淀粉含量的等位基因SBEIIb-1、SSIIa-1和ISA1-1。

Identification of resistant starch related genes in rice by single segment substitution lines

Objective To discover the alleles for high resistance starch in starch-synthesis-related genes SBEIIb, SSIIa and ISA1.Method The single segment substitution lines (SSSLs) carrying the starch-synthesis-related genes SBEIIb, SSIIa or ISA1 were detected using molecular markers. Then, the resistant starch contents of the SSSLs were measured using an improved AOAC method. Sanger sequencing and sequence alignment were performed to analyze the sequence variations of SBEIIb, SSIIa and ISA1 in different SSSLs. Through linkage analysis of genotypes and phenotypes, the alleles affecting resistance starch content were identified.Result For SBEIIb gene, a single nucleotide polymorphism (SNP) (Ex4-96G/A) in the coding region results in an amino acid substitution (196-Arg/His), generating two alleles SBEIIb-1 and SBEIIb-2. SBEIIb-1 carrying the Ex4-96G causes Arg at 196th residue, which shows high-resistant starch content of 1.72%. For SSIIa gene, two SNPs (Ex8-334G/A and Ex8-865C/T) in the 8th exon cause two amino acid substitutions (604-Gly/Ser and 781-Leu/Phe), generating three alleles SSIIa-1,SSIIa-2 and SSIIa-3. SSIIa-1 carrying the Ex8-334G and Ex8-865C causes Gly and Leu at 604th and 781th residue respectively, which shows high-resistant starch content of 3.37%. One Indel (AGG/---) and one SNP (C/T) in ISA1 coding region sequence generate three alleles ISA1-1, ISA1-2 and ISA1-3. ISA1-1 carrying AGG-insertion and Ex17-117C causes Glu and Thr at 70th and 717th residue respectively, which shows high-resistant starch content of 2.09%.Conclusion SBEIIb, SSIIa and ISA1 are key genes regulating resistant starch formation in rice. The SNPs and Indels in coding regions of the three genes lead to amino acid variations, which subsequently affects the resistance starch content. The three alleles SBEIIb-1, SSIIa-1 and ISA1-1 for high-resistant starch content are identified.