小麦转Loxi基因后代外源基因稳定性检测及种子耐储藏性分析

发布时间：2018-05-03 06:30

本文选题：耐储藏性 + 转基因小麦　；参考：《西北农林科技大学》2016年硕士论文

【摘要】：小麦(Triticum aestivum)是重要的粮食作物之一,其产量和品质直接关系到世界粮食安全和人们的生活水平。利用转基因技术改善小麦品质已逐渐成为小麦遗传育种的有效辅助途径之一。小麦种子中脂氧合酶(lipoxygenase,LOX)可催化不饱和脂肪酸的氧化作用,在种子储藏期间产生氢过氧化物,导致小麦种子在储藏过程中种子活力和营养品质降低。因此,选育种子LOX活性低的小麦品种是小麦遗传改良的任务之一。为了获得种子脂氧合酶活性显著降低、外源Loxi(Lox基因的RNAi构件,Lox gene RNAi construct)能稳定遗传且种子耐储藏性增强的转基因小麦新种质,本研究在刘振华等(2014)筛选得到只含Loxi、不含Bar基因且脂氧合酶活性明显下降的转基因TF6代株系的基础上,进一步对TF7和TF8代株系进行了筛选鉴定,研究内容和结果如下:(1)对小麦西农889、闫麦8911和陕优225的TF7和TF8代转基因株系进行PCR检测和Basta涂抹检测,并对TF7代转基因株系进行脂氧合酶活性测定,结果如下:所检测的所有转基因后代植株均无basta抗性;西农889的13个TF7和5个TF8代株系、闫麦8911的7个TF7和5个TF8代转基因株系后代中均检测到外源基因Loxi,而陕优225的转基因株系后代中TF7代有2个株系,TF8代有1个株系所有后代植株均未检测到Loxi,说明经过多代筛选转基因后代已无Bar基因,西农889和闫麦8911转基因后代目的基因稳定遗传,而陕优225转基因后代中还未稳定遗传;脂氧合酶活性测定结果显示西农889、闫麦8911和陕优225转基因后代株系脂氧合酶活性相比亲本均有不同程度的降低,分别为其亲本酶活性的9.97%~91.33%、24.35%~90.67%和26.31%~85.61%,说明外源Loxi能够有效抑制脂氧合酶活性。(2)对部分转基因株系花后28天种子Lox基因家族进行了半定量RT-PCR分析,结果显示转基因后代籽粒中TaLox1基因的表达显著下降,而TaLox2和TaLox3基因的表达量不变,说明转入的外源Loxi可特异性地抑制转基因材料种子中TaLox1基因的表达,最终导致转基因种子酶活性降低。(3)对西农889、闫麦8911和陕优225只含Loxi不含Bar且脂氧合酶活性较低的部分TF7代转基因株系种子进行人工加速老化处理7 d、14 d,测定脂氧合酶活性、萌发7d的发芽率和芽长,并对脂肪酸组分的变化和含量进行分析。结果如下:人工加速老化后,所有材料LOX活性均上升,转基因材料与各自的亲本相比,LOX酶活上升的较少;老化处理后种子发芽率均降低,转基因材料发芽率降低相对缓慢,老化后转基因材料芽长大部分高于各自亲本;种子脂肪酸组分和含量测定分析表明老化处理后转基因株系脂肪酸比其亲本材料氧化损失少。以上结果说明,转基因株系中Lox基因的RNAi可有效抑制其种子LOX活性,使转基因小麦后代种子耐储藏性增强,筛选鉴定得到的种子LOX活性显著降低的转基因小麦株系可为小麦育种提供新的材料。
[Abstract]:Triticum aestivum (Triticum aestivum) is one of the important food crops, and its yield and quality are directly related to the world food security and people's living standards. Improving wheat quality by using transgenic technology has gradually become one of the effective auxiliary ways of wheat genetics and breeding. Lipoxygenase LOX) in wheat seeds can catalyze the oxidation of unsaturated fatty acids and produce hydroperoxide during seed storage, which results in the decrease of seed vigor and nutritional quality during storage. Therefore, the breeding of wheat varieties with low LOX activity is one of wheat genetic improvement. In order to obtain a significant decrease of lipoxygenase activity in seeds, the RNAi component of exogenous Loxi(Lox gene, Lox gene RNAi construction, could stabilize the inheritance and enhance the storage tolerance of transgenic wheat germplasm. On the basis of Liu Zhenhua et al. 2014), transgenic TF6 lines containing only Loxi, no Bar gene and significantly decreased lipoxygenase activity were obtained, and further screening and identification of TF7 and TF8 lines were carried out. The contents and results of the study were as follows: (1) the transgenic lines of wheat Xinong 889, Yanmai 8911 and Shanyou 225 were detected by PCR and Basta smear, and the lipoxygenase activity of TF7 transgenic lines was determined. The results were as follows: all transgenic progenies tested had no basta resistance, 13 TF7 and 5 TF8 lines of Xinong 889, The exogenous gene Loxi was detected in the progenies of 7 TF7 and 5 TF8 generation transgenic lines of Yan Mai 8911, while that of the TF7 generation of Shanyou 225 transgenic lines was not detected in all the progenies of 2 lines of Tf8 line and 1 line in all the progenies, indicating that no Loxi gene was detected in all the progenies of the transgenic lines of Yanmai 8911. After several generations of screening transgenic progenies, there is no Bar gene, The target gene of Xinong 889 and Yanmai 8911 transgenic progenies was stably inherited, while Shanyou 225 transgenic progenies were not. The results of lipoxygenase activity test showed that the lipoxygenase activity of Xinong 889, Yan Mai 8911 and Shanyou 225 transgenic progenies was lower than that of their parents. 9.97% and 26.31%, respectively, of their parents' enzyme activity, indicating that exogenous Loxi can effectively inhibit lipoxygenase activity. The semi-quantitative RT-PCR analysis was carried out on the Lox gene family of seeds 28 days after anthesis of some transgenic lines. The results showed that the expression of TaLox1 gene decreased significantly in the seeds of transgenic progenies, but the expression of TaLox2 and TaLox3 genes remained unchanged, indicating that exogenous Loxi could specifically inhibit the expression of TaLox1 gene in transgenic seeds. Finally, the enzyme activity of transgenic seeds was decreased.) the transgenic seeds of Xinong 889, Yanmai 8911 and Shaanyou 225 with low lipoxygenase activity and no Bar were treated with artificial accelerated aging for 7 days and 14 days to determine the lipoxygenase activity. The germination rate and bud length, the changes and contents of fatty acid components were analyzed at the 7th day of germination. The results were as follows: after accelerated aging, the LOX activity of all materials increased, and the activity of LOX increased less than that of their parents, and the germination rate of seeds decreased after aging, and the germination rate of transgenic materials decreased slowly. After aging, the bud length of transgenic lines was much longer than that of their parents, and the analysis of fatty acid composition and content of seeds showed that the oxidative loss of fatty acids of transgenic lines after aging was less than that of their parents. The results indicated that the RNAi of Lox gene in transgenic lines could effectively inhibit the activity of LOX and enhance the storage tolerance of transgenic wheat seeds. The transgenic wheat lines with significantly decreased LOX activity could provide new materials for wheat breeding.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S512.1

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