糯谷与非糯谷waxy基因全序列比对与多态性位点分析

Full Sequence Alignment and Polymorphism Loci Analysis of waxy Gene in Non-waxy and Waxy Foxtail Millets

为了明确粳糯谷子waxy基因DNA全序列及其氨基酸序列多态性,以3份糯质和2份非糯谷子品种为材料,通过Primer Premier 5.0引物设计、PCR扩增、目的片段纯化、阳性克隆和菌液PCR测序拼接,以Setaria italica v2.2 (Foxtail millet)基因为参考序列,分析参试材料间waxy基因全序列的多态性位点,利用在线SMART (http://smart.embl-heidelberg.de/)分析预测其waxy氨基酸序列及其二级结构域。结果表明,(1)waxy基因全序列总长度为4 218 bp。5份供试材料与参考基因序列比对共检测到54个突变位点,其中转换、颠换(易位)、插入和缺失突变依次分别为24、19、5和6个。(2)‘公谷68’、‘红粘谷’、‘赤谷4号’、‘赤谷9号’和‘杏黄粘’与参考基因序列比对分别含有36、8、5、5和0个差异位点,其中‘公谷68’与参考基因组序列比对,存在Intron 6第2 052 bp (T)和Intron 9第2 883 bp (AGGTG)等5个插入,在Intron 12 (3 602 bp)缺失T,Intron 6 (2 109~2 125 bp)缺失ACATTCTTTCAGACTGC等6个缺失位点,以及Intron 1 (396, 787 bp)(C-A)等12个颠换,exon 1 (54, 68 bp)(A-G)等12个转换。‘红粘谷’Intron 6在2 078 bp (T-A)等含有3个颠换,以及exon 1第54和68 bp、Intron1第201 bp (A-G)等5个转换。(3)粳糯谷waxy基因序列比对中,‘公谷68’、‘杏黄粘’和‘红粘谷’与非糯‘赤谷4号’分别含有31、5和3个多态性位点。(4)粳糯谷之间检测到70个氨基酸序列差异位点,其中‘公谷68’与其余4份材料存在69个差异位点,‘杏黄粘’第248个氨基酸为S (丝氨酸),其余4份材料均为N (天冬酰胺)。‘公谷68’检测到low complexity (51~67)、Pfam:Glyco_transf_5 (81~341)、Pfam:Glycos_transf_1 (385~524)、Pfam:Glyco_trans_1_4 (396~533)和low complexity (580~604)等5个waxy氨基酸序列二级结构域,‘红粘谷’、‘杏黄粘’、‘赤谷4号’和‘赤谷9号’均含有上述前4个结构域,但与‘公谷68’结构域的氨基酸位置不同,其差异氨基酸数目依次分别为0、1、0和12个。

In order t o clarify the polymorphism of waxy genes in the full-sequence DNA and their amino acid sequences,three waxy and two non-waxy millet cultivars were used to analyze the polymorphic site using the Setaria italica v2.2(foxtail millet)gene as a reference sequence by design primers(Primer Premier 5.0),ordinary PCR amplification,target fragment purification,positive cloning,and sequence splicing of PCR amplification products from bacterial solution,and to predict their waxy amino acid sequences and secondary domains using online SMART(http://smart.embl-heidelberg.de/).The results showed that:(1)The full sequence lengths of the waxy gene were 4218 bp.Compared with reference gene sequences,54 mutation sites were detected among five tested millet cultivars.Of them,conversion,transversion(translocation),insertion and deletion mutations were 24,19,5,and 6,respectively.(2)Compared with reference gene sequences,the numbers of different loci of’Gonggu68’,’Hongniangu’,’Chigu4’,’Chigu9’and’Xinghuangnian’were 36,8,5,5,and 0,respectively.The five insertions of’Gonggu68’compared with the reference genome sequences were 2052 bp(T)and 2053 bp(G)of Intron 6,2883 bp(AGGTG)of Intron 9,6 deletions were Intron 12(3602 bp deleted T),Intron 6(2109~2125 bp,deleted ACATTCTTTCAG ACTGC),12 transversions included intron1(396,787 bp)(C-A)and so on,12 transversions consisted of exon 1(54,68 bp)(A-G)and the like.’Hongniangu’had 3 transversions,such as intron6 of 2078 bp(T-A),5 transversions of A-G in 54 bp and 68 bp from exon 1,201 bp from Intron 1.(3)For sequence alignment of waxy gene between non-waxy and waxy millet strains was concerned,’Gonggu68’,’Xinghuangnian’and’Hongniangu’had 31,5 and 3 polymorphic sites compared by’Chigu4’,respectively.(4)Seventy amino acid sequence different sites were discovered among tested millet cultivars,of them 69 sites existed between’Gonggu68’and the other 4 materials,one site was S(serine)in the 248 th amino acid from’Xinghuangnian’,but that was N(asparagine)from the other 4 millet strains.Five secondary domains of the waxy amino acid sequences were detected in’Gonggu68’,they were low complexity(51~67),Pfam:Glycotransf5(81~341),Pfam:Glycostransf1(385~524),Pfam:Glycotrans14(396~533),and low complexity(580~604).The former four secondary domains also detected in’Hongniangu’,’Xinghuangnian’,’Chigu4’and’Chigu9’,but was different from’Gonggu68’in amino acid positions,the numbers of different amino acids were 0,1,0,and 12 between’Gonggu68’and the other four millet cultivars,respectively.