摘要
通过根癌农杆菌C58C1ATHVRifR 介导法 ,利用烟草花叶病毒 35S双启动子和苜蓿花叶病毒引导序列控制下的含抗新霉素磷酸转移酶基因 (nptⅡ )和哈兹木霉几丁质酶基因(ThEn 4 2 )的质粒pBin19ESR为载体 ,对 3个核桃体细胞胚系进行遗传转化 ,结果获得 4 1个抗卡那霉素的体细胞胚系。经PCR和复式PCR检测 ,4 1个转化系均含有nptⅡ基因 ,其中 38个转化系含有ThEn 4 2基因。Southern杂交分析表明 ,ThEn 4 2基因已被整合到核桃体的基因组中。几丁质酶活性检测结果表明 ,转化的体细胞胚系的几丁质酶活性比对照高几十至几千倍。
Somatic embryos of English walnut( Juglans regia L.)were Agrobacterium mediately transformed with the Trichoderma endochitinase gene ThEn 42 under the control of a double 35S CaMV promoter and Alfalfa Mosaic Virus leader sequence.The selectable marker gene neomycin phospotransferaseⅡ( npt Ⅱ)driven by the nopaline synthase promoter was also used.A total of 41 putatively transformed somatic embryo lines were evaluated for expression of the npt Ⅱ and ThEn 42 genes by PCR and multiplex PCR.All of the 41 somatic embryo selections expressed the npt Ⅱ gene,among which 38 somatic embryo selections expressed the ThEn 42 gene.Analysis of chitinase activity by using a fluorometric assay showed dozens to 1000 fold higher chitinase activity in transformed somatic embryos than that in non transformed ones.All of the tested chitinase postive somatic embryo lines analyzed by Southern blotting had an intact copy of the ThEn 42 gene.Chitinase expressing somatic embryos were further germinated and are being propagated for disease resistance assays.
出处
《园艺学报》
CAS
CSCD
北大核心
2001年第1期12-18,共7页
Acta Horticulturae Sinica
关键词
核桃
几丁质酶
转基因
体细胞胚
农杆菌介导
Walnut( Juglans regia )
Chitinase
Genetic transformation
Somatic embryos
