超声波介导Mz_2NHX_1基因遗传转化提高珠美海棠耐盐性  

The research on ultrasonic mediated genetic transformation of the Mz_2NHX_1 gene enhancing salt tolerance of Malus zumi

作　　者：孙婷梅[1] 王璐平 张永利[1,2] 秦家慧[1] 于梦楠[1] 李爱[1] 彭立新[1] 

机构地区：[1]天津农学院园艺园林学院果树学重点实验室,天津300384 [2]天津市汉邦植物保护剂有限责任公司,天津300381

出　　处：《果树学报》2016年第9期1043-1051,共9页Journal of Fruit Science

基　　金：国家自然科学基金(30671440;31300564)

摘　　要：【目的】提高珠美海棠的耐盐性,建立更高效的珠美海棠遗传转化体系。【方法】在转化液中利用超声波直接转化无菌条件下横切主脉的珠美海棠试管苗幼叶,通过愈伤组织培养诱导植株再生、抗性筛选,GUS染色鉴定,RT-PCR分析转基因植株的耐盐性。【结果】较利于珠美海棠愈伤组织诱导分化及抗性筛选的培养基为MS+6-BA 2.0 mg·L^(-1)+NAA 0.1 mg·L^(-1)+Kana 50 mg·L^(-1)。超声波处理的最优工作条件为:处理6 s,间歇10 s,工作重复20次,功率80 W,转化效率为31.3%。GUS染色与RT-PCR鉴定结果一致率为100%,RT-PCR结果分析显示,Mz2NHX1基因在转化苗中的表达量约为对照的3倍。荧光定量PCR结果显示转化苗Mz2NHX1基因的表达量为对照组的6.21倍,盐处理试验结果显示转化苗耐盐性得到了显著地提高。【结论】建立了超声波介导的珠美海棠高效遗传转化体系,获得珠美海棠耐盐新材料,为该耐盐基因的研究和盐碱地的改良奠定了基础。【Objective】In order to improve the salt-tolerance of Malus zumi and establish a more efficientgenetic transformation system for Malus zumi, the study established an efficient ultrasonic-mediated ge-netic transformation system for the Mz2NHX1 gene from Malus zumi by connecting the gene with thep RI201-AN-GUS DNA to build a plant expression vector. The Mz2NHX1 gene is a full-length Na＋/H＋anti-porter gene which can be found on the vacuolar of Malus zumi. The establishment of the ultrasonic- medi-ated efficient genetic transformation system of Malus zumi can be applied to the improvement of soil salini-zation and further analysis of the function of the Mz2NHX1 gene. The method of ultrasonic-mediated genet-ic transformation of Malus zumi is more efficient as well as less false-positive descendants than the meth-od of Agrobacterium-mediated genetic transformation and it represented the first time for the study of theultrasonic-mediated genetic transformation of Malus zumi.【Methods】Cut the main vein of the leaves of Malus zumi with 3-4 incisions perpendicularly under aseptic conditions. Inoculate the leaves on the MSmedium with different concentration ratios of 6-BA and NAA. Cultivate the leaves first in darkness for 2weeks at 24 ℃, then in 3 000 lx of light for 12 hours every day at 24 ℃ for 4 weeks to study the optimalmedium components of the callus induction and differentiation of Malus zumi. Cultivate leaves of Malus zumi on MS medium containing different concentrations of kanamycin： 0, 20, 30, 40, 50, 60, 70 mg·L-1re-spectively to study the sensitivity of the callus of Malus zumi to kanamycin. Prepare the reaction buffer ofthe ultrasonic-mediated genetic transformation for 50 m L containing DMSO（1.1 mg·L-1） for 5m L, recombi-nant DNA（Mz2NHX1-p RI201-AN-GUS DNA） for 200 ng, sodium chloride for 3 mg and sodium citratefor 2 mg. Leaves of Malus zumi with 3-4 incisions were immersed in the reaction buffer. The probe of theultrasonic was 5mm underneath the fluid level of the reaction buffer pr

关 键 词：珠美海棠 遗传转化体系 超声波 植株再生 耐盐基因 过表达 

分 类 号：S661.4[农业科学—果树学]