【作者】 刘天宇；

【导师】 渠慎春； 庄维兵；

【作者基本信息】 南京农业大学 ， 果树学， 2020， 硕士

【摘要】 苹果(Malus domestica Borkh.)是蔷薇科(Rosaceae)苹果属(Malus)多年生木本植物,是世界上主栽果树之一,同时也是我国最主要的果树树种之一。苹果在生长发育过程中会遇到各种生物和非生物胁迫的影响,其中干早胁迫和盐胁迫会导致植株出现氧化胁迫反应,从而抑制植株的生长发育、降低果实品质。非生物胁迫相关基因在响应植物对干旱、盐害等逆境胁迫过程中,发挥着至关重要的作用。已有研究表明,植物中褪黑素不仅是生长发育调节剂,还是强有力的抗氧化剂,在植物抵御逆境胁迫中发挥着重要作用。本研究构建了珠眉海棠中褪黑素合成酶基因MzASMT1的植物表达载体,通过转入K326烟草和‘嘎啦’苹果中研究该基因在植物抗逆境中的功能,主要研究结果如下:1、成功构建褪黑素合成酶基因MzASMT1的植物表达载体pCAMBIA2300-35s-GUS-CaMVterm-MzASMT1,采用农杆菌介导的遗传转化法,将含MzASMT1基因的表达载体转入模式植物烟草中,通过GUS染色、基因组PCR鉴定筛选获得3株阳性株系。qRT-PCR检测结果表明:和野生型相比,转基因烟草中MzASMT1基因表达量显著增加,且不同转基因株系中MzASMT1基因的表达量有显著差异。通过酶联免疫吸附法测定了转基因株系和野生型的褪黑素含量,结果表明转基因株系的褪黑素含量显著高于野生型,与基因表达结果一致。2、对转MzASMT1基因烟草和野生型烟草进行连续30 d的干旱处理后,通过表型、生理生化指标和干旱相关基因表达等方面的比较分析,结果表明:干旱处理30 d后,转基因植株与野生型相比,植株生长更为健壮,叶片萎蔫和根系受损程度更轻;转基因植株的叶片相对含水量和叶绿素含量均显著高于野生型;同时转基因植株还显著提高了脯氨酸含量,降低了 MDA的积累量,表明转基因植株在干旱胁迫下细胞膜系统受到氧化损伤的程度更轻;进一步研究发现转基因植株提高了抗氧化酶(POD、SOD、CAT)活性,相关基因NtPOD、NtSOD、NtCAT的表达量也显著提高;耐旱相关基因表达量检测结果表明,转基因植株中NtERD10C、NtERD10D、NtLEA5、NtP5CS和NtDREB3的基因表达量提高。以上结果表明:转MzASMT1基因烟草通过提高叶片相对含水量、叶绿素含量、脯氨酸含量、抗氧化酶活性、耐旱相关基因的表达量以及降低MDA的积累量,提高其耐旱能力。3、转MzASMT1基因烟草和野生型烟草进行连续14 d的200 mM NaCl处理后,通过表型、生理生化指标和盐胁迫相关基因表达等方面的比较分析,结果表明:转基因植株与野生型相比,植株生长更为健壮,底部叶片萎蔫和根系受损程度更轻;转基因植株的根、茎和叶片相对含水量分别为野生型的1.6倍、1.5倍、1.2倍,叶绿素含量和叶绿素荧光参数(Fv/Fm)均显著高于野生型,脯氨酸含量平均提高约2.3倍,MDA含量则降低至野生型的75%左右;同时转基因植株显著提高了抗氧化酶(POD、SOD、CAT)活性,相关基因NtPOD、NtSOD、NtAT的表达量也显著提高;用DAB、NBT对盐胁迫处理后的叶片进行化学组织染色,结果显示转基因植株着色更浅;H2O2和O2-含量定量测定结果表明:转基因植株H2O2和O2-含量显著低于野生型;相关基固检测结果表明:转基因株系中NtERD10C、NtERD10D、NtLEA5、NtP5CS和NtDREB3的基因表达量提高。以上结果表明:转MzASMT1基因烟草通过提高根、茎和叶片相对含水量、叶绿素含量、叶绿素荧光参数、脯氨酸含量、抗氧化酶活性、耐盐相关基因的表达量以及降低MDA、H2O2和O2_积累量,提高其耐盐能力。4、利用农杆菌介导的遗传转化法,将含MzASMT1基因的表达载体及空载体分别转入‘嘎啦’苹果,通过GUS染色、基因组PCR鉴定,筛选获得3个转MzASMT1基因嘎啦株系和转空载体嘎啦株系。qRT-PCR检测结果表明,和野生型、转空载体植株相比,转基因植株显著提高了MzASMT1基因表达量,且不同转基因株系间MzASMT1基因的表达量有显著差异。通过酶联免疫吸附法测定转基因植株和野生型、转空载体植株的褪黑素含量,结果表明,转基因植株的褪黑素含量显著高于野生型和转空载体植株。此外,对转基因植株和野生型、转空载体植株进行干旱胁迫和盐胁迫处理,同时测定胁迫相关基因的表达量,结果表明,转MzASMT1基因嘎啦通过上调抗氧化酶相关基因(MdPOD、MdSOD)、ABA合成及信号转导基因(MdNCED3、MdAREB2、MdSnRK2.4)、脯氨酸合成酶基因(MdP5CDH)和胁迫响应基因(MdRD22、MdDREB2A、MdRD29A)的表达丰度,提高其耐旱和耐盐能力。更多还原

【Abstract】 Apple(Malus domestica Borkh.)is a perennial woody plant of the genus Malus of the Rosaceae family.It is one of the main fruit trees in the world.It is also one of the most important fruit tree species in China.Apples will encounter various biological and abiotic stresses during the growth and development process.Among them,drought stress and salt stress will cause severe oxidative stress to the plant,thereby inhibiting plant growth and reducing fruit quality.Abiotic stress-related genes play a vital role in responding to plant stresses such as drought and salt damage.Studies have shown that melatonin in plants is not only a growth regulator,but also a powerful antioxidant,so it plays an important role in plants against stress.Therefore,this study constructed the plant expression vector of the melatonin synthase gene MzASMT1 in Malus zumi Mats to study the function of this gene in plant stress resistance by transferring into K326 tobacco and ’Gala’ apple.The main results were as follows:1.Successfully constructed the plant expression vector pCAMBIA2300-35s-GUS-CaMVterm-MzASMT1 of the melatonin synthase gene MzASMT1.Using the Agrobacterium-mediated genetic transformation method,the expression vector containing the MzASMT1 gene was transferred into model plant tobacco and stained by GUS.Three positive lines were obtained by genomic PCR identification and screening.The results of qRT-PCR showed that compared with wild type,the expression of MzASMT1 gene in transgenic tobacco increased significantly,and the expression of MzASMT1 gene in different transgenic lines was significantly different.The melatonin content of transgenic lines and wild type was determined by enzyme-linked immunosorbent assay.The results showed that the melatonin content of transgenic lines was significantly higher than that of wild type,which was consistent with the gene expression results.2.After a 30-day drought treatment of MzASMT1 transgenic tobacco and wild-type tobacco,a comparative analysis of phenotype,physiological and biochemical indicators,and drought related gene expression showed that after 30 days of drought treatment,the transgenic plants and wild-type plant growth is more robust,leaf wilting and root damage are less severe.The relative water content and chlorophyll content of leaves of transgenic plants are significantly higher than that of wild type.At the same time,transgenic plants also significantly increased proline content and reduced MDA.The amount of accumulation indicates that the transgenic plants have a lesser degree of oxidative damage to the cell membrane system under drought stress.Further research found that the transgenic plants increased the activity of antioxidant enzymes(POD,SOD,CAT)and the expression levels of related genes NtPOD,NtSOD,NtCAT.The expression of drought-tolerance related genes showed that the gene expression of NtERD10C,NtERD10D,NtLEA5,NtP5CS and NtDREB3 in transgenic plants increased.The above results indicate that the MzASMT1 transgenic tobacco increased its drought tolerance by increasing the relative water content,chlorophyll content,proline content,antioxidant enzyme activity,drought-tolerance related gene expression and reducing the accumulation of MDA.3.After transgenic MzASMT1 tobacco and wild-type tobacco were treated with 200 mM NaCl for 14 days,the phenotype,physiological and biochemical indicators and salt-stress related gene expression were compared and analyzed.The results showed that:transgenic plants compared with wild-type plants,the growth is more robust,the wilting of the bottom leaves and the damage to the root system are lighter.The relative water content of the roots,stems and leaves of the transgenic plants is 1.6 times,1.5 times,1.2 times of the wild type,chlorophyll content and chlorophyll fluorescence parameters(Fv/Fm)were significantly higher than the wild type,the proline content increased by about 2.3 times on average,and the MDA content was reduced to about 75%of the wild type.At the same time,the transgenic plants significantly increased the antioxidant enzyme(POD,SOD,CAT)activity,related The expression levels of genes NtPOD,NtSOD,and NtCAT also increased significantly.Chemical tissue staining of leaves after salt stress treatment with DAB and NBT showed that the transgenic plants were lighter in color.The quantitative determination of H2O2 and O2-content showed that:the transgenic plants H2O2 And O2content is significantly lower than that of wild type.Related gene test results show that:NtERD10C,NtERD10D,NtLEA5,NtP5CS and NtDREB3 in transgenic lines increased gene expression.The above results show that:MzASMT1 transgenic tobacco can increase the relative water content of roots,stems and leaves,chlorophyll content,chlorophyll fluorescence parameters,proline content,antioxidant enzyme activity,salt-tolerance related gene expression and reduce MDA,H2O2 and O2-accumulate the amount to improve its salt tolerance.4.Using the Agrobacterium-mediated genetic transformation method,the expression vector containing the MzASMT1 gene and vacant vector were transferred into ’Gala’ apples respectively.Through GUS staining and genomic PCR identification,three MzASMT1 gene-transmitted Gala lines and vacant vector Gala lines.The results of qRT-PCR showed that the transgenic plants significantly increased the expression of MzASMT1 gene compared with wild-type and transgenic vector plants,and the expression of MzASMT1 gene was significantly different among different transgenic lines.The melatonin content of transgenic plants and wild type and vacant vector plants was determined by enzyme-linked immunosorbent assay.The results showed that the melatonin content of transgenic plants was significantly higher than that of wild type and vacant vector plants.In addition,drought stress and salt stress treatment were carried out on transgenic plants and wild-type and vacant vector plants,and the expression levels of stress-related genes were also measured.The results showed that Gala transgenic MzASMT1 gene up-regulated antioxidant enzyme related genes(MdSOD,MdCAT),ABA synthesis and signal transduction genes(MdNCED3,MdAREB2,MdSnRK2.4),proline synthase gene(MdP5CDH)and stress response Genes(MdRD22,MdDREB2A,MdRD29A)expression abundance,improve their drought and salt tolerance.更多还原