大米草耐盐基因SaNHX转化红麻及其耐盐性的初步研究
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摘要
土壤盐渍化是一个世界性问题,培育耐盐作物品种已成为一个日益紧迫而意义重大的研究课题。本研究选用福建农林大学作物遗传育种研究所从盐生植物大米草中克隆,并已经证实为大米草Na~+/H~+逆转运蛋白SaNHX基因作为遗传转化目的基因,利用花粉管通道法,将大米草耐盐基因SaNHX导入红麻新品种福红992,并获得了表达,其研究结果如下:
(1)采用2种剂量和3种质粒浓度,通过子房注射法,对福红992进行转导,微注射处理共1933朵花,研究结果表明:两种不同转导剂量T_0结实率和出苗率差异不显著;3种不同质粒浓度T_0代结实率存在显著差异,其中Z-100-8结实率最佳,达65.5%;T_0的出苗率最好的是Z-300-8,为81.1%。
(2)在转导的T_0代群体中,提取282份供试材料DNA,进行耐盐基因的PCR分子检测,检测到具有目的基因特征条带的阳性植株9份,实验转化率为3.19%,结果表明利用花粉管通道法对红麻转基因是行之有效的。
(3)红麻幼苗耐盐性试验的结果表明,转基因红麻T_1代9个株系在0.71%的致死浓度处理下,种子发芽率和生长势明显优于对照红麻。苗高、苗重和根长分别平均比对照提高了2.04cm,0.23g和3.7cm。
(4)三个不同时期的不同盐浓度处理试验的方差分析表明,耐盐转基因T_1代的株系幼苗成活率明显高于普通对照,差异均达极显著水平,其中编号176株系最佳,在0.3%、0.5%和0.71三种浓度下的幼苗成活率分别达100%,85%和60%;在盐胁迫(0.71%)处理下,生长10天的红麻幼苗比较苗高、苗重和根长,转基因耐盐植株与对照存在显著差异。在9个转基因株系中,176号植株最好,其苗高、苗重和根长的增长量分别比对照提高5.47cm、0.72g和4.54cm。
Soil salinization is a cosmopolitism problem. To cultivate and grow salt tolerant crop increasingly become an urgent and substantial research topic. This study selected the SaNHX gene which was cloned from Spartina anglica by Crop Genetics and Breeding Institute of Fujian Agriculture and Forestry University as a genetic transformation of the target gene, used pollen tube pathway, injected salt-tolerant gene SaNHX into new kenaf variety of fuhong992, and it has already expressed. The major results were as follow:
(1) Using Ovary Injection, adopted 2 dosage and 3 concentration of plasmid transducted Fuhong 992, deal with 1933 flowers through microinjection treatment. The results indicated: in transgenic T_0 generation, there was no significant difference between the setting seed rate and the emergence rate of the 2 different dose. The setting seed rate and the emergence rate of T_0 generation of 3 concentration of plasmid have significant difference. The best setting seed was Z-100-8 to reach 65.5%; the best seedling emergence rate of T_0 generation is Z-300-8 to reach 81.1%.
(2) Of transduction To generation, extracted DNA of 282 strains test materials to carry out salt tolerance PCR molecular detection, detected that 9 positive plants had objective gene characteristic bands, The transformation rate was 3.19% through this method. Under 0.71% concentration salt stress treatment, seed germination rate, and growth potential of these 9 positive plants were obviously better than the control, the increment of its seedling height , seedling weight and seedling root length were increased 2.04cm, 0.23g and 3.7cm. The results showed that pollen tube pathway was an effective way in kenaf transgene.
(3) Variance analysis of different concentration of three different periods showed that seedling survival rate of Salt-toerance transgenic T_1 generation were obviously better than common control and differences all reached very significant level; the best was 176 strain, its seedling survival rate were 100%, 85% and 60% respectively under three concentrations: 0.3%、0.5% and 0.71%. Under Salt Stress (0.71%) , the height, the weight and the root length of seedling growth of 10 days were obviously better than the control.Of all the 9 transgenic strains, the best was No. 176 strain, the increment of its height, weight and root length were 5.47cm、0.72g and 4.54cm higher than those of control respectively.
(1)采用2种剂量和3种质粒浓度,通过子房注射法,对福红992进行转导,微注射处理共1933朵花,研究结果表明:两种不同转导剂量T_0结实率和出苗率差异不显著;3种不同质粒浓度T_0代结实率存在显著差异,其中Z-100-8结实率最佳,达65.5%;T_0的出苗率最好的是Z-300-8,为81.1%。
(2)在转导的T_0代群体中,提取282份供试材料DNA,进行耐盐基因的PCR分子检测,检测到具有目的基因特征条带的阳性植株9份,实验转化率为3.19%,结果表明利用花粉管通道法对红麻转基因是行之有效的。
(3)红麻幼苗耐盐性试验的结果表明,转基因红麻T_1代9个株系在0.71%的致死浓度处理下,种子发芽率和生长势明显优于对照红麻。苗高、苗重和根长分别平均比对照提高了2.04cm,0.23g和3.7cm。
(4)三个不同时期的不同盐浓度处理试验的方差分析表明,耐盐转基因T_1代的株系幼苗成活率明显高于普通对照,差异均达极显著水平,其中编号176株系最佳,在0.3%、0.5%和0.71三种浓度下的幼苗成活率分别达100%,85%和60%;在盐胁迫(0.71%)处理下,生长10天的红麻幼苗比较苗高、苗重和根长,转基因耐盐植株与对照存在显著差异。在9个转基因株系中,176号植株最好,其苗高、苗重和根长的增长量分别比对照提高5.47cm、0.72g和4.54cm。
Soil salinization is a cosmopolitism problem. To cultivate and grow salt tolerant crop increasingly become an urgent and substantial research topic. This study selected the SaNHX gene which was cloned from Spartina anglica by Crop Genetics and Breeding Institute of Fujian Agriculture and Forestry University as a genetic transformation of the target gene, used pollen tube pathway, injected salt-tolerant gene SaNHX into new kenaf variety of fuhong992, and it has already expressed. The major results were as follow:
(1) Using Ovary Injection, adopted 2 dosage and 3 concentration of plasmid transducted Fuhong 992, deal with 1933 flowers through microinjection treatment. The results indicated: in transgenic T_0 generation, there was no significant difference between the setting seed rate and the emergence rate of the 2 different dose. The setting seed rate and the emergence rate of T_0 generation of 3 concentration of plasmid have significant difference. The best setting seed was Z-100-8 to reach 65.5%; the best seedling emergence rate of T_0 generation is Z-300-8 to reach 81.1%.
(2) Of transduction To generation, extracted DNA of 282 strains test materials to carry out salt tolerance PCR molecular detection, detected that 9 positive plants had objective gene characteristic bands, The transformation rate was 3.19% through this method. Under 0.71% concentration salt stress treatment, seed germination rate, and growth potential of these 9 positive plants were obviously better than the control, the increment of its seedling height , seedling weight and seedling root length were increased 2.04cm, 0.23g and 3.7cm. The results showed that pollen tube pathway was an effective way in kenaf transgene.
(3) Variance analysis of different concentration of three different periods showed that seedling survival rate of Salt-toerance transgenic T_1 generation were obviously better than common control and differences all reached very significant level; the best was 176 strain, its seedling survival rate were 100%, 85% and 60% respectively under three concentrations: 0.3%、0.5% and 0.71%. Under Salt Stress (0.71%) , the height, the weight and the root length of seedling growth of 10 days were obviously better than the control.Of all the 9 transgenic strains, the best was No. 176 strain, the increment of its height, weight and root length were 5.47cm、0.72g and 4.54cm higher than those of control respectively.
引文
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