利用CRISPCAS9技术改良水稻YN6的香味品质分析研究 生物技术专业VIP免费

摘要稻米的香味品质是稻米品质的重要内容之一。对稻米品种的香味品质改良，有利于增加其市场经济价值。研究表明，稻米的香味性状主要由水稻第8染色体上的甜菜碱醛脱氢酶（BetaineAldehydeDehydrogenase，OSBadh2）控制。该基因失活使芳香物质2-乙酰基-1-吡咯啉（2-acetyl-1-pyrroline，2AP）在稻米中大量积累，产生香味。本次实验使用了最近几年新兴的定点基因敲除技术CRISP/CAS9，本实验通过基因敲除技术来对北方粳稻品系YN6进行改良，通过敲除YN6水稻基因组中OSBadh2基因改良基因香味品质。具体操作结果如下：1实验过程中实现了对具有定点敲除功能的CRISP/CAS9载体的构建；2转化CRISP/CAS9载体，获得了1000个独立抗性愈伤，从中分化出732个独立的转基因植株。3通过对CRISP/CAS9修饰的OSBadh2目标位点处的序列进行测序分析，从中获得了两株发生了基因编辑。目标基因经修饰后产生了移码突变，经加代纯合后，产生了香味表型。关键词香味基因；CRISP/CAS9；定点突变技术；转基因AbstractFragranceisoneofthemostsignificantricequalities.Improvementofricefragrancequalitywillundoubtedlyenlargericemarketingvalue.Ricefragrancewassuggestedtobecontrolledbyabetainealdehydedehydrogenase(OsBadh2)gene，whichislocatedinricechromosome8.Thedisfunctionofthisgenewillleadtomassiveproductionofanaromaticcompound2-acetyl-1-pyrroline(2AP)inrice，whichconsequentlygeneratericefragrancephenotype.Thisstudyappliedarecentlydevelopedgene-editingtechnology，whichismediatedbyatranscriptionactivator-likeeffectornuclease(CRISP/CAS9）andis，inresult，capableofmodifyingthetargetgene.ThroughCRISP/CAS9technology，wesuccessfullydisenabletheOsBadh2geneinanelitejaponicavarietyYN6，stronglyaddingthematerialthericefragrance.Themainresultswereconcludedasfollows:1.AfunctionalCRISP/CAS9vectorswasestablished.2.ThroughtransformationofCRISP/CAS9viaAgrobacteriummediatedgenetictransformation，weobtained1000independenthygromacin-resistantcalli，andamongthem，732independenttransgenicplantsweredifferentiated.3.ThroughanalysisbysequencingtheCRISP/CAS9aimedsiteinthetargetgeneOsBadh2，weidentified2plants，inwhichseveralbaseswereoccurreddeletion，leadingtogeneframeshiftmutation.Thehomologouslineswereabletoproducereadilyperceivablefragrance.KeywordsFragrancegene;CRISP/CAS9;geneeditingtechnology;geneticallymodified目录摘要............................................................................................................................................IAbstract....................................................................................................................................II1.前言......................................................................................................................................11.1香稻米的定义及我过香稻米的现状..........................................................................11.2水稻香味的研究进展.................................................................................................11.2.1水稻香味的形成机制......................................................................................11.2.2香味的鉴定方法...............................................................................................21.3CRISP/CAS9技术原理................................................................................................21.4实验目的及意义..........................................................................................................32.材料与方法...............................................................................