【作者】 李春燕；

【导师】 巩慧玲；

【作者基本信息】 兰州理工大学 ， 生物工程（专业学位）， 2021， 硕士

【摘要】 马铃薯(Solanum tuberosum L.)是茄科植物,它是谷物、蔬菜和饲料的高产组合,是世界上四种主要农作物之一。植物蔗糖转运蛋白(sucrose transporter,SUT)主要负责蔗糖从“源”器官到“库”器官的长距离运输,还控制蔗糖在植株体内的分配,影响植物的生长和发育,决定作物的产量和品质。马铃薯SUT家族分别为StSUT1、StSUT2和StSUT4,近期研究表明StSUT1和StSUT4影响马铃薯植株的生长、块茎的产量和光周期调控基因的表达,而StSUT2的生理功能仍不清楚。本研究以马铃薯栽培种“夏波蒂”品种及其StSUT2表达下调的转基因株系RNAi-1和RNAi-2为材料,分析其在应对高盐、干旱、低温、高温等非生物胁迫条件下植株所表现出的耐受性差异,分析了非转基因植株与转基因株系对糖饥饿胁迫的耐受性差异,以及油菜素内酯(Brassinosteroid)对糖饥饿胁迫的影响;用q TR-PCR方法,分析逆境胁迫前后非转基因植株和野生型植株中油菜素内酯信号通路相关基因以及细胞壁形成相关的基因的表达量,从而探讨StSUT2基因对马铃薯逆境耐受性的调控机制。实验结果如下:转基因株系StSUT2-RNAi和非转基因植株在非生物胁迫下的形态学有一定差异。(1)StSUT2-RNAi株系在一定25、50 mmol/L NaCl浓度下,转基因株系StSUT2-RNAi的鲜重、新叶数、茎节数、株高高于野生型。由此初步表明StSUT2-RNAi株系耐盐性高于野生型植株。(2)转基因株系StSUT2-RNAi和非转基因植株盆栽苗经干旱处理,非转基因植株在处理7天时叶片开始卷曲、茎杆倒伏,而转基因株系RNAi-1、RNAi-2分别在9天和12天时叶片开始萎蔫、茎秆倒伏。这一结果初步表明StSUT2-RNAi株系耐旱性高于非转基因植株。(3)转基因株系StSUT2-RNAi和非转基因植株盆栽苗经-2℃低温胁迫处理,非转基因植株在处理1 h时叶片开始萎蔫、出现水渍状、茎杆倒伏,而RNAi-1、RNAi-2株系分别在处理2.5 h、4 h时叶片才开始萎蔫、茎杆倒伏。这一结果初步表明StSUT2-RNAi株系的低温耐受性高于非转基因植株。(4)转基因株系StSUT2-RNAi和非转基因植株盆栽苗经42℃高温胁迫处理,非转基因植株和RNAi-1株系在胁迫2.5 h时叶片开始萎蔫、茎杆倒伏;而RNAi-2株系在5 h后成熟叶片萎蔫、茎杆倒伏。由此初步表明StSUT2-RNAi株系的高温耐受性高于非转基因植株。(5)转基因植株StSUT2-RNAi和非转基因植株在糖饥饿、BR处理下形态学响应表明:耐受糖饥饿胁迫高于野生型;BR处理对野生型植株根的抑制作用较强。(6)在正常生长条件下和盐胁迫处理条件下,两个StSUT-RNAi转基因株系中St BRI基因的表达量显著高于非转基因植株。由此表明StSUT2可能通过影响St BRI基因的表达而提高对盐胁迫的耐受性。在正常生长条件和蔗糖饥饿胁迫下,两个StSUT-RNAi转基因株系中St DHE基因的表达量显著高于非转基因植株。由此表明StSUT2可能通过影响St DHE基因的表达而调节马铃薯细胞壁的形成,这可能是转基因株系的茎在糖饥饿胁迫下易倒伏的主要原因。更多还原

【Abstract】 Potato(Solanum tuberosum L.)is a genus of Solanaceae.It is a high-yield combination of grains,vegetables and feed,and is one of the four main crops in the world.Plant sucrose transporter(SUT)is mainly responsible for the long-distance transportation of sucrose from the "source" organ to the "sink" organ.It also controls the distribution of sucrose in the plant body,affects the growth and development of the plant,and determines the yield and quality of the crop.Potato SUT families are StSUT1,StSUT2,and StSUT4.Recent studies have shown that StSUT1 and StSUT4 affect potato plant growth,tuber yield and photoperiod-regulating gene expression,while the physiological functions of StSUT2 are still unclear.In this study,the potato cultivar "Sharpotie" and its transgenic lines RNAi-1 and RNAi-2 with down-regulated expression of StSUT2 were used as materials to analyze its response to abiotic stress conditions such as high salt,drought,low temperature,and high temperature.The tolerance differences shown by non-transgenic plants and transgenic lines were analyzed for the tolerance differences of sugar starvation stress,as well as the effect of Brassinosteroid on sugar starvation stress;the q TR-PCR method was used to analyze The expression levels of brassinolide signaling pathway-related genes and genes related to cell wall formation in non-transgenic plants and wild-type plants before and after stress,so as to explore the regulation mechanism of StSUT2 gene on potato stress tolerance.The experimental results are as follows:There is a certain difference in morphology between the transgenic line StSUT2-RNAi and the non-transgenic plant under abiotic stress.(1)StSUT2-RNAi strain under a certain concentration of 25,50 mmol/L NaCl,the fresh weight,number of new leaves,number of stem nodes,and plant height of the transgenic strain StSUT2-RNAi are higher than those of the wild type.This preliminarily shows that the salt tolerance of StSUT2-RNAi strain is higher than that of wild-type plants.(2)The potted seedlings of the transgenic line StSUT2-RNAi and non-transgenic plants were treated with drought.The non-transgenic plants began to curl and the stems fell after 7 days of treatment,while the transgenic lines RNAi-1and RNAi-2 were treated at 9 days and At 12 days,the leaves began to wilt and the stems fell.This result preliminarily indicates that the drought tolerance of StSUT2-RNAi lines is higher than that of non-transgenic plants.(3)The transgenic line StSUT2-RNAi and the potted seedlings of non-transgenic plants were treated with-2℃ low temperature stress.When the non-transgenic plants were treated for 1 h,the leaves began to wilt,appear water-stained,and the stems fell,while RNAi-1,RNAi The leaves of the-2 line began to wilt and the stems fell when treated for 2.5 h and 4 h,respectively.This result preliminarily indicates that the low temperature tolerance of StSUT2-RNAi strain is higher than that of non-transgenic plants.(4)The transgenic line StSUT2-RNAi and the potted seedlings of non-transgenic plants were treated with 42℃ high temperature stress.The leaves of the non-transgenic plants and RNAi-1 lines began to wilt and the stems fell when the stress was 2.5 h;while the RNAi-2 lines After 5 h,the mature leaves wilted and the stems fell.This preliminarily shows that the high temperature tolerance of StSUT2-RNAi strain is higher than that of non-transgenic plants.(5)The morphological response of transgenic plants StSUT2-RNAi and non-transgenic plants under sugar starvation and BR treatments showed that the tolerance to sugar starvation stress was higher than that of wild type;BR treatment had a stronger inhibitory effect on the roots of wild type plants.(6)Under normal growth conditions and salt stress treatment conditions,the expression of St BRI gene in the two StSUT-RNAi transgenic lines was significantly higher than that of non-transgenic plants.This indicates that StSUT2 may increase the tolerance to salt stress by affecting the expression of St BRI gene.Under normal growth conditions and sucrose starvation stress,the expression of St DHE gene in the two StSUT-RNAi transgenic lines was significantly higher than that of non-transgenic plants.This indicates that StSUT2 may regulate the formation of potato cell wall by affecting the expression of St DHE gene,which may be the main reason why the stems of transgenic lines are prone to lodging under sugar starvation stress.更多还原