【作者】 王婷；

【导师】 李强；

【作者基本信息】 山东农业大学 ， 蔬菜学， 2021， 硕士

【摘要】 番茄(Solanum lycopersicum L.)是世界上广泛种植的蔬菜作物之一,在全球农业生产中占有重要地位。RAV(Related to ABI3/VP1)家族属于B3超家族,是植物特有的转录因子。RAV家族基因包含一个B3 DNA结合域和/或AP2 DNA结合域,在植物信号转导、逆境胁迫以及生长发育过程中起着非常重要的作用。目前,番茄RAV基因的生物学功能知之甚少。本研究系统分析了番茄RAV基因家族,利用转录组、过表达和CRISPR敲除等技术初步分析了SlRAV1、SlRAV2、SlRAV3和SlRAV4在番茄果实、花和叶片发育中的功能。主要研究结果如下:1、从番茄基因组中鉴定到了9个RAV基因家族成员。番茄RAV基因(SlRAV)被分为4个亚家族。基因结构分析表明SlRAV基因含有1-3个外显子,同一亚家族内的基因结构相对保守。保守基序分析表明,所有SlRAV家族成员均含有motif-5,同一亚家族内的基序种类与数量相对保守。启动子顺式作用元件分析表明,SlRAV基因主要与激素调控、生长发育及逆境胁迫的响应有关。共线性分析发现,SlRAV基因家族中只有2对基因来源于片段复制事件(SlRAV1/SlRAV2和Sl NGA2/Sl NGA3),片段复制在基因家族进化及成员扩张过程中发挥着重要作用。2、番茄果实发育早期的转录组分析发现,SlRAV1、SlRAV2、SlRAV3和SlRAV4在番茄果实发育早期的表达量较高。共表达基因鉴定和GO富集分析表明,SlRAV1、SlRAV2、SlRAV3和SlRAV4所在的共表达模块分别主要与器官发育、激素代谢、光合作用和细胞周期等生物学过程相关。3、亚细胞定位结果表明,SlRAV2、SlRAV3和SlRAV4均定位在细胞核中,SlRAV1定位在细胞核和细胞质中。4、构建了SlRAV1、SlRAV2、SlRAV3和SlRAV4的过表达和CRISPR敲除载体。SlRAV1功能缺失突变体不能产生花粉。SlRAV4过表达株系的叶片和保卫细胞形状指数及叶片表皮细胞面积均显著高于对照。综上,本研究对番茄RAV转录因子家族进行了系统的生物信息学分析并对番茄SlRAV1和SlRAV4的功能进行了初步分析,为进一步研究SlRAV基因在番茄发育中的功能并阐明其机制奠定了基础。更多还原

【Abstract】 Tomato(Solanum Lycopersicum L.)is one of the most widely cultivated vegetable crops in the world and plays an important role in global agricultural production.The RAV(Related to ABI3/VP1)family belongs to the B3 superfamily and is a specific gene family in plants.The RAV family gene contains a B3 DNA binding domain and/or APETALA2(AP2)DNA binding domain,which is involved in a series of plant growth and development processes,stress responses,and signal transduction.At present,there are few studies on the RAV gene family in tomato,and little is known the functions of SlRAVs in tomato.The present study performed comprehensive analyses of tomato RAV gene family.Transcriptome analysis,over-expression and gene knockout mediated by CRISPR were performed for preliminary analysis of the functions of SlRAV1,SlRAV2,SlRAV3 and SlRAV4 in the development of fruit,flower and leaf in tomato.The main results are as follows:1.Nine RAV genes were identified from tomato genome.The tomato RAV genes were divided into four subfamilies(Subfamily I,II,IV and V).Gene structure analysis showed that SlRAVs contained one to three exons,and the gene structure of the genes in the same subfamily was relatively conserved.Motif analysis showed that all SlRAVs contained motif-5,and the motif type and number were relatively conserved within the same subfamily.The cis-regulatory elements analysis showed that SlRAV genes was mainly involved in hormone regulation,growth and development,and stress responses.Synteny analysis showed that two pairs of SlRAV were derived from segmental duplication events(SlRAV1 / SlRAV2 and Sl NGA2 / Sl NGA3),indicating that segmental duplication played an important role in SlRAV family evolution and expansion.2.Transcriptomic analyses indicated that SlRAV1,SlRAV2,SlRAV3 and SlRAV4 showed high expression levels in the early stages of tomato fruit development.Co-expression and GO enrichment analyses showed that the co-expression modules containing SlRAV1,SlRAV2,SlRAV3 and SlRAV4 were related with organ development,hormone metabolism,photosynthesis,cell cycle and other biological processes.3.Subcellular localization analysis showed that SlRAV2,SlRAV3 and SlRAV4 were localized in the nucleus,and SlRAV1 was localized in nucleus and cytoplasm.4.The over-expression and CRISPR constructs of SlRAV1,SlRAV2,SlRAV3 and SlRAV4 were constructed,which were genetic transformed into tomato.The knockout mutant of SlRAV1 can not produce pollen.Overexpression SlRAV4 significantly increased the shape index of leaflets and guard cells as well as the area of epidermal cells of leaves compared with Control.In conclusion,we conducted a comprehensive analysis of the tomato RAV gene family and analyzed the functions of tomato RAV1 and RAV4,which lay a foundation for further study the function and mechanisms of SlRAV genes in tomato.更多还原