【作者】 王飞；

【导师】 车代弟；

【作者基本信息】 东北农业大学 ， 园林植物与应用， 2020， 硕士

【摘要】 观赏甜菜（Beta vulgaris var.cicla）,外观艳丽,色泽诱人,是潜在的观叶类植物。主要分布于西南地区及长江黄河部分流域,在东北地区露地栽培不能越冬。如果培育适应东北地区气候的园林绿化新品种,则需评估多种观赏甜菜抗寒性并研究耐寒相关分子调节机制。本研究测定了低温胁迫下两种观赏甜菜叶片电导率、SOD、CAT、POD的活性、可溶性糖、脯氨酸含量,评估了两种观赏甜菜的抗寒性。通过低温胁迫下高通量测序数据分析,筛选获得冷胁迫响应的糖基转移酶基因Bv GT708和Bv GT74。糖基转移酶（GT）是甜菜素糖基化过程的一类重要修饰酶,参与甜菜素的合成途径,是甜菜呈现红色并具有观赏性的重要因素。同时,糖基转移酶基因调控植物响应多种非生物胁迫,特别是低温胁迫。本研究利用生物信息学和分子生物学技术,预测Bv GT708、Bv GT74的生化特性并克隆两个基因,构建植物表达载体,并将Bv GT708基因转入模式植物烟草和拟南芥中,以期在模式植物中研究基因功能,最终为开发耐寒观赏甜菜新品种及观赏植物色素合成途径研究奠定基础。主要研究结果如下:（1）在-10℃下,随着低温胁迫时间的延长,红甜菜和黄甜菜相对电导率、脯氨酸含量呈逐渐上升的趋势;SOD、CAT、POD的活性呈先上升后下降的趋势;红甜菜可溶性糖含量逐渐上升,而黄甜菜可溶性糖含量则逐渐下降。通过隶属函数法综合评价得出红甜菜的抗寒性强于黄甜菜,为观赏甜菜抗寒性提供参考数据。（2）通过连续6周测定红甜菜叶片中甜菜素含量,发现随着红甜菜不断生长发育,甜菜素含量逐渐增加。q PCR结果显示Bv GT708相对表达量随着红甜菜发育期的逐渐增加而逐渐上升,Bv GT74则呈先下降后上升的趋势,这表明Bv GT708与甜菜素积累呈正相关,而Bv GT74与甜菜素含量无明显相关性。推测Bv GT708可能参与甜菜素的合成。（3）通过测定观赏甜菜在-10℃甜菜素含量,发现甜菜素含量随低温胁迫时间的延长呈先升高后降低的趋势,表明甜菜素可能响应低温胁迫。通过荧光定量PCR发现低温处理下,红甜菜Bv GT708相对表达量变化趋势和甜菜色素相似,也呈先升高后降低的趋势,推测Bv GT708可能通过参与甜菜色素合成来响应低温胁迫。（4）为了进一步分析Bv GT708、Bv GT74基因在甜菜色素合成中的作用,从红甜菜叶片中克隆Bv GT708、Bv GT74基因,并进行生物信息学相关分析。Bv GT708开放阅读框共494个氨基酸,等电点为6.20,分子量为38450.37 KD。总平均疏水指数（GRAVY）为-0.255,属于亲水蛋白。Bv GT74开放阅读框共471个氨基酸,等电点为6.21;分子量为36.1116 KD脂肪数为72.66,总平均疏水指数（GRAVY）为-0.130,亲水性蛋白。（5）成功构建了Bv GT708、Bv GT74植物表达载体:p BI121-Bv GT708、p BI121-Bv GT74,以及Bv GT708的荧光蛋白双元表达载体p BI121-GFP-GT708。通过花序侵染法将Bv GT708转入拟南芥,通过抗性筛选初步筛选出转基因拟南芥幼苗T₀代4株,阳性率为57.1%,为该基因的抗寒性研究奠定基础。利用农杆菌介导将Bv GT708转入烟草,获得T₀代转基因烟草6株,阳性率为75.0%,为该基因在植物中甜菜素合成研究提供基础。更多还原

【Abstract】 Beta vulgaris var.cicla,with a beautiful appearance and attractive color,is a potential foliage plant.It is mainly distributed in the southwest region and part of the Yangtze and Yellow River basins,and it can not be overwintered in open cultivation in the northeast region.If new varieties of landscaping adapted to the climate of Northeast China are to be cultivated,it is necessary to evaluate the cold resistance of various Beta vulgaris var.cicla and study the molecular regulation mechanism of cold tolerance.In this study,the leaf electrical conductivity,SOD,CAT,POD activity,soluble sugar,and proline content of two ornamental sugar beets under low temperature stress were determined,and the cold resistance of the two Beta vulgaris var.cicla was evaluated.Through high-throughput sequencing data analysis under low-temperature stress,the glycosyltransferase genes Bv GT708 and Bv GT74 that obtained cold stress response were screened.Glycosyltransferase（GT）is an important class of modified enzymes in the glycosylation process of betalains.It is involved in the synthesis of betalains and is an impor tant factor for beet to appear red and ornamental.At the same time,glycosyltransferase genes regulate plants in response to a variety of abiotic stresses,especially low temperature stress.This study uses bioinformatics and molecular biology techniques to predict the biochemical properties of Bv GT708 and Bv GT74 and clone two genes to construct a plant expression vector,and transfer the Bv GT708 gene into model plants tobacco and Arabidopsis in order to be in model plants The study of gene function ultimately lays the foundation for the development of new cold-resistant Beta vulgaris var.cicla varieties and the synthesis of ornamental plant pigments.The main findings are as follows:（1）At-10℃,with the prolongation of low temperature stress,the relative c onductivity and proline content of Red chard and Yellow chard showed a gradual upward trend;the activities of SOD,CAT,POD showed a trend of first increasing and then decreasing;The soluble sugar content of Red chard gradually increased,while the soluble sugar content of Yellow chard gradually decreased.According to the comprehensive evaluation of the membership function method,it is concluded that the cold resistance of Red chard is stronger than that of Yellow chard.Provide reference data for the cold resistance of Beta vulgaris var.cicla.（2）By measuring the content of betalains in Red chard leaves for 6 consecutive weeks,it was found that the content of betalains increased gradually as the Red chard continued to grow and develop.The q PCR results showed that the relative expression level of Bv GT708 increased gradually with the gradual increase of the development period of Red chard,Bv GT74 showed a trend of first decreasing and then increasing,which indicated that Bv GT708 was positively correlated with betalains accumulation,but Bv GT74 had no significant correlation with betalains content..It is speculated that Bv GT708 may be involved in the synthesis of betalains.（3）By measuring the content of betalains in Beta vulgaris var.cicla at-10℃,it was found that the content of betalains increased first and then decreased with the prolongation of low temperature stress,indicating that betalains may respond to low temperature stress.Fluorescence quantitative PCR found that under low temperature treatmen t,the relative expression of Red chard Bv GT708 changed similarly to betalains,and also showed a trend of increasing first and then decreasing.It is speculated that Bv GT708 may respond to low temperature stress by participating in betalains synthesis.（4）In order to further analyze the role of Bv GT708 and Bv GT74 genes in betalains synthesis,Bv GT708 and Bv GT74 genes were cloned from Red chard leaves and bioinformatics related analysis was performed.Bv GT708 has an open reading frame of 494 amino acids,an isoelectric point of 6.20,and a molecular weight of 38450.37 KD.The total average hydrophobic index（GRAVY）is-0.255,which is a hydrophilic protein.The Bv GT74 open reading frame has a total of471 amino acids,an isoelectric point of 6.21;a molecular w eight of 36.1116 KD,a fat number of72.66,a total average hydrophobic index（GRAVY）of-0.130,and a hydrophilic protein.（5）Successfully constructed Bv GT708 and Bv GT74 plant expression vectors:p BI121-Bv GT708,p BI121-Bv GT74,and Bv GT708 fluorescent protein binary expression vector p BI121-GFP-GT708.Transgenic Bv GT708 was transformed into Arabidopsis thaliana by inflorescence infestation,and transgenic Arabidopsis seedlings were screened initially through resistance screening.The four T₀ generations had a positive rate of 57.1%,which laid the foundation for the cold resistance research of this gene.Agrobacterium-mediated transformation of Bv GT708 into tobacco yielded 6 strains of T₀ transgenic tobacco with a positive rate of 75.0%,providing a basis for the gene synthesis of betalains in plants.更多还原