| 番茄红素合成调控基因 SISGR1的分子特征及sgRNA分析 |
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| 引用本文：刘江娜,张西英,李荣霞,张小伟,白云凤,张爱萍.番茄红素合成调控基因 SISGR1的分子特征及sgRNA分析[J].西北农业学报,2022,(8):1017~1024 |
| DOI:10.7606/j.issn.1004-1389.2022.08.009 |
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| 基金项目:国家自然科学基金 (32160710，31760571);兵团科技人才创新计划(2021CB037)；第六师科技计划(1903)。 |
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| 中文摘要:旨在明确番茄 SlSGR1基因的分子特征及其sgRNA的分布，为利用CRISPR/Cas9 技术对 SlSGR1及其上游启动子序列进行定点突变或片段删除、调控 SlSGR1的表达为提高番茄果实的番茄红素含量提供技术支持。利用Blast分析番茄红素合成调控基因 SlSGR1的分子特征，结果表明 SlSGR1基因位于番茄第8号染色体，含4个外显子和3个内含子，编码区长度为819 nt，编码272 aa，其中48～200 aa为典型的staygreen superfamily保守结构域。基于对RNA-seq建立的 SlSGR1数字表达谱分析表明， SlSGR1呈果实特异性表达，在植株的根、茎、叶等部位不表达。利用在线工具CRISPRdirect 分析表明， SlSGR1外显子区域含有PAM为NGG的sgRNA有64条，其中1条为番茄全基因组上唯一邻近PAM 12 nt 特异性最高的种子序列，预示该sgRNA可用于番茄不同品种 SlSGR1基因的靶向编辑并可有效避免脱靶效应。对 SlSGR1上游1 500 bp启动子序列分析表明，该序列含有2条番茄全基因组上特异性较好的唯一sgRNA序列，7条分别含有不同顺式元件的sgRNA，意味着选用这些sgRNA进行基因编辑，可使所含的启动子元件序列发生突变致使功能变化，以提高番茄红素含量。 |
| 中文关键词:番茄 SlSGR1 分子特征 启动子 sgRNA |
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| Molecular Characteristics and sgRNA Analysis of SlSGR1 Gene in Tomato |
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| Abstract:The aim of this study is to clarify the molecular characteristics of SlSGR1 gene and its distribution of sgRNA in tomato,which is expected to use CRISPR/Cas9 technology for site-directed mutation or fragment deletion of SlSGR1 and its upstream promoter sequences ,and for improvement of lycopene content in tomato fruits by regulation of SlSGR1 expression.Blast was used to analyze the molecular characteristics of lycopene synthesis regulation gene SlSGR1.The results showed that SlSGR1 gene was located on chromosome 8 of tomato,which contained 4 exons and 3 introns,with encoding region 819 nt,encoding 272 aa，48-200 aa was a typical staygreen superfamily conserved domain.Based on RNA-Seq,the digital expression profile of SlSGR1 showed that SlSGR1 was specifically expressed in fruits,and was not expressed in roots,stems and leaves of plants.The results of CRISPRdirect analysis showed that there were 64 SgRNA,with PAM as NGG in the exon region of SlSGR1,among which one was the only seed sequence adjacent to PAM 12 nt which had the highest specificity in the whole tomato genome.The results indicated that the sgRNA could be used for SlSGR1 gene targeted editing in tomato varieties and could effectively avoid off-target effect.The analysis of 1 500 bp upstream promoter sequence of SlSGR1 showed that the sequence contained 2 unique sgRNA sequences which had good specificity in the whole genome of tomato,and 7 sgRNA sequences with different cis-elements,respectively.It was suggested that the selection of these sgRNA sequences for gene editing could cause mutations in the promoter sequences,and resulted in functional changes. |
| keywords:Tomato SlSGR1 Molecular characteristics Promoter sgRNA |
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