二沟黄芪富硒基因smt1转化紫花苜蓿初步的研究

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1、摘要硒代半胱氨酸甲基转移酶（Selenocysteinemethyltransferase,SMT）可将硒代半胱氨酸转化为非蛋白氨基酸，即硒甲基半胱氨酸储存起来，从而提高植物体内硒的含量。本论文对紫花苜蓿中苜一号的组培再生技术进行了优化，同时从富硒植物二沟黄芪（Astragalusbisulcatus）中克隆出smt1基因，用农杆菌介导法将其转化紫花苜蓿，旨在获得硒含量提高的转基因材料，主要研究结果如下：（1）紫花苜蓿愈伤诱导最适培养基为SH培养基+2.0mg/L2,4-D+0.25mg/LKT+0.25mg/LTDZ+7.

2、5g/L琼脂+30g/L蔗糖；（2）紫花苜蓿不定芽诱导最适培养基为MSO培养基+2.0mg/L2,4-D+0.5mg/L6-BA+2g/L酪蛋白水解物+7.5g/L琼脂+30g/L蔗糖。（3）紫花苜蓿最适外植体为5天苗龄的子叶和胚轴。（4）采用化学合成方法获得二沟黄芪smt1基因的编码区cDNA，它由1017bp组成，推测其编码蛋白含338个氨基酸残基，分子质量为36.7kDa，等电点为4.69。（5）将smt1编码区cDNA片段构建到植物表达载体pCAMBIA1301上，并将其转入农杆菌LBA4404感受态细胞，获得工程菌

3、株。（6）通过农杆菌介导法对中苜一号的胚轴和子叶进行了遗传转化，经筛选和再生培养获得15株抗性再生植株。（7）通过PCR、RT-PCR和GUS活性检测，证明获得了6株转基因紫花苜蓿植株，且smt1基因有较高水平的表达。上述工作为获得硒含量提高的转基因苜蓿材料奠定了基础。关键词：硒；smt；紫花苜蓿；遗传转化PrimarystudyonMedicgoSativatransformationoftheSelenium-hyperaccumulationGenesmt1fromAstragalusbisulatusAbstract

4、Selenocysteinemethyltransferase(SMT)specificallymethylatesselenocysteinetoproducemethylselenocysteinwhichisanon-proteinaminoacidandaccumulatesinplantcell,thereforethecontentofseleniuminplantincreased.Inthispaper,theregenerationtechnologyofalfalfainvitrowasoptimized

5、,andsmt1,animportantgeneforseleniumhyperaccumulationinAstragalusbisulcatus,wasclonedfromthisplant.Thenthesmt1genewasintroducedintoalfalfabyAgrobacterium-mediatedtransformationsoastoimprovetheselenium-accumulatingabilityofthetransgenicplants.Themainresultsareasfollo

6、ws:(1)ThesuitablemediumtoinducecallusfromalfalfawasSHmedium+2.0mg/L2,4-D+0.25mg/LKT+0.25mg/LTDZ+7.5g/Lagar+30g/Lsucrose.(2)ThesuitablemediumtoinduceadventitiousbudsfromalfalfawasMSOmedium+2.0mg/L2,4-D+0.5mg/L6-BA+2g/Lcaseinhydrolyzate+7.5g/Lagar+30g/Lsucrose.(3)The

7、suitableexplantswerecotyledonandhypocotylfrom5-dayoldseedlingsofalfalfa.(4)ThecodingregioncDNAofAstragalusbisulatussmt1wassynthesizedartificialy.Itcontains1017basepairsandencodesapolypeptidecomposedof338aminoacidresidues,withamolecularweightof36.7kDandanisoelecricp

8、ointof4.69.(5)ThecDNAfragmentofsmt1wasinsertedintoplantexpressionvectorpCAMBIA1301,andthentherecombinantplasmidwasintroducedintothestrainLBA4404o