【作者】 王琪；

【导师】 战祥强；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2021， 硕士

【摘要】 番茄(Solanum lycopersicum L.)原产于热带和亚热带地区,现在在全球范围内均有广泛的种植,根据联合国粮食及农业组织公布数据全球在2019年有1.82亿吨的番茄产量。在番茄生长过程中经常会遭受到低温胁迫,因此提高番茄对低温的抵抗能力对提高番茄的产量具有至关重要的作用。TGL(Triglyceride Lipase)家族基因在植物、动物、真菌中都有广泛的分布,其对生物体内三酰甘油脂的水解起到至关重要的作用并进而会影响到一系列的生命活动,例如种子的萌发、植物对非生物胁迫的抵抗能力。在本研究中,通过HMMER-BLASTP-Inter Pro Scan策略对番茄中SlTGL的家族成员进行了初步鉴定与分析。首先,通过对保守结构域的检索鉴定出番茄中SlTGL家族一共有129个成员,并对其各成员的基因结构和染色体定位等信息进行比较分析。其次,通过对SlTGL家族成员在番茄低温转录组的数据分析,得到各个家族成员对低温胁迫的响应情况。通过对SlTGL家族成员的鉴定与分析,初步推测SlTGLa10参与到番茄对低温胁迫的响应。因此将番茄中SlTGLa10基因进行克隆并在番茄中进行稳定遗传转化,获得SlTGLa10的过表达系和干涉系,并对SlTGLa10在番茄响应低温胁迫的功能进行初步探究。最后为解释SlTGLa10转基因系的表型,对其进行了转录组测序,并通过酵母单杂交试验、双荧光素酶试验和转录因子活性分析研究了SlTGLa10的作用机制。主要研究结果如下:1.通过生物学分析对番茄中SlTGL家族成员进行了鉴定。在番茄中通过HMMER-BLASTP-Inter Pro Scan策略共鉴定出129个SlTGL家族成员,在12条染色体均有不同数量的分布。通过对氨基酸保守结构域分析,此家族基因均包含有一个保守的α/β水解酶保守结构域,进化树分析表明番茄中SlTGL家族基因由5个亚家族组成。通过对家族成员进行共线性分析,番茄SlTGL家族内共有18个基因对,且都表现为片段重复。结合各个家族成员在低温胁迫下的转录组数据,SlTGL中的a亚家族基因表现为不同程度的受低温胁迫诱导。2.过表达SlTGLa10能增强番茄对低温的敏感性。在受到低温处理时,SlTGLa10表现出受低温胁迫所诱导。SlTGLa10在番茄不同组织特异性表达分析,在番茄的生殖生长器官(果实、种子)中表达较高,而在根、茎中表达量较低。分别对SlTGLa10构建干扰和过表达载体,并将此载体稳定遗传转化至番茄,得到SlTGLa10基因的干涉株系和过表达株系。对SlTGLa10的过表达系、干涉系和AC进行低温胁迫处理,低温处理后SlTGLa10过表达系表现为MDA含量和离子渗透率显著升高,而SOD活性、POD活性等显著低于AC和干涉系,表明SlTGLa10过表达系对冷胁迫更为敏感。基于上述试验结果,SlTGLa10与番茄低温胁迫的抗性密切相关,并且负调控番茄的抗冷性。3.SlTGLa10调控机制的研究。通过对SlTGLa10的干涉系、表达系和AC低温处理后进行转录组测序,并结合qRT-PCR对其中部分基因验证,发现在脂代谢中的一些关键基因的表达受到了影响,说明SlTGLa10是通过影响脂代谢来影响番茄抗冷性。SlTGLa10和Sl MYB16在低温下的诱导表达呈现较高的负相关性,通过酵母单杂交试验、双荧光素酶试验和转录因子活性分析试验系统性的分析了Sl MYB16对SlTGLa10的启动子的调控情况,结果表明Sl MYB16不直接结合SlTGL10的启动子但负调控SlTGLa10的表达。同时为进一步验证的结论,构建并得到Sl MYB16的过表达系,在Sl MYB16过表达系中SlTGLa10的表达量呈现一定程度的下调,进一步说明了Sl MYB16负调控SlTGLa10的表达。更多还原

【Abstract】 Tomato(Solanum lycopersicum L.)is native to tropical and subtropical regions and are now widely planted worldwide.According to data released by the Food and Agriculture Organization of the United Nations,there were 182 million tons of tomato production in the world in 2019.Tomato is often subjected to chilling stress during the growth process,so improving the resistance of tomato to chilling stress plays a vital role in increasing the yield of tomato.The TGL family genes are widely distributed in plants,animals,and fungi.They play a vital role in the hydrolysis of triacylglycerides in organisms and affect a series of life activities,such as seed germination,plant resistance to abiotic stress.In this study,by analyzing the gene family of triglyceride lipase in tomato,firstly,a total of 129 members in tomato were determined,and their chromosome location and gene structure were analyzed.Second,analyze its response to chilling stress through transcriptome data.Based on the identification and expression analysis of the SlTGL family genes,it is speculated that SlTGLa10 may be involved in the response of tomato to chilling.Therefore,this gene in tomato was cloned,and stable genetic transformation was performed in tomato to obtain the overexpression line and knockdown line of SlTGLa10,and the function of SlTGLa10 in response to chilling stress in tomato was preliminarily explored.Finally,in order to explain the chilling resistance phenotype of the SlTGLa10 transgenic line,we sequenced the transcriptome and studied the mechanism of SlTGLa10 through yeast one-hybrid experiment,dual-luciferase experiment,and transcription factor activity analysis.The main findings in our study are as follows:1.The members of the TGL family in tomato were identified by bioinformatics analysis.In tomato,a total of 129 SlTGL family members were identified through the HMMERBLASTP-Inter Pro Scan strategy,and they have different numbers of distributions on 12 chromosomes.Through the analysis of amino acid conserved domains,all genes in this family contain a conserved α/β hydrolase conserved domain.Phylogenetic tree analysis shows that the SlTGL family genes in tomato can be divided into 5 subfamilies.Through collinearity analysis of family members,there are a total of 18 pairs of genes in the tomato SlTGL family,and all of them appear to be fragment duplications.Combining the transcriptome data of each family member under low temperature stress,the genes of subfamily a in SlTGL appear to be induced by low temperature stress to varying degrees.2.SlTGLa10 can enhance the sensitivity of tomato to chilling stress.Under chilling stress,SlTGLa10 responds to chilling stress.The tissue-specific expression analysis of SlTGLa10 showed that the expression was higher in the reproductive growth organs,but lower in the roots and stems.The SlTGLa10 interference and overexpression vectors were constructed and genetically transformed into tomato,and the knockdown and overexpression lines of SlTGLa10 were obtained respectively.SlTGLa10 overexpression lines,knockdown lines,and AC were treated with chilling stress.After chilling stress treatment,SlTGLa10 overexpression lines showed a significant increase in MDA content and ion permeability,while SOD activity and POD activity were significantly lower than AC and knockdown lines.It shows that SlTGLa10 overexpression lines are more sensitive to cold stress.It is concluded that SlTGLa10 participates in the response of tomato to cold stress and negatively regulates the cold resistance of tomato.3.Research on the regulation mechanism of SlTGLa10.Through transcriptome sequencing of SlTGLa10’s knockdown line,expression line,and AC chilling stress treatment,combined with qRT-PCR to verify some genes,it was found that the expression of some key genes in lipid metabolism was affected,indicating that SlTGLa10 affects lipid metabolism affects tomato cold resistance.Under the chilling stress,the induced expression of SlTGLa10 and Sl MYB16 showed a high negative correlation,through the yeast single hybrid experiment,dual-luciferase experiment,and transcription factor activity analysis experiment systematically analyzed the regulation of Sl MYB16 on the promoter of SlTGLa10,The results indicate that Sl MYB16 does not directly bind to the promoter of SlTGL10 but negatively regulates the expression of SlTGLa10.At the same time,in order to further verify our conclusions,we constructed and obtained an overexpression line of Sl MYB16.In the overexpression line of Sl MYB16,the expression level of SlTGLa10 showed a certain degree of down-regulation,further indicating that Sl MYB16 does not directly bind to the promoter of SlTGa L10,but negative Regulate the expression of SlTGLa10.更多还原