【作者】 王志伟；

【导师】 哈斯阿古拉；

【作者基本信息】 内蒙古大学 ， 生物与医药， 2023， 硕士

【摘要】 甜瓜（Cucumis melo L.）是一种在世界各地被广泛栽培的园艺经济作物,在我国有着非常悠久的栽培历史,其果实是主要食用部位。甜瓜营养丰富,含有维生素A、B、C以及镁、钠等营养成分,不仅具有多种药用功能,还有非常重要的食用价值。多项研究表明多药与毒性化合物排出转运蛋白（Mutidrug and Toxic Compund Extrusion,MATE）可直接或间接参与调控植物的抗病性、铝解毒和铁稳态。目前,尽管已有大量的MATE蛋白基因被鉴定,但是对于MATE基因调控植物生长发育的研究鲜有报道。本研究以甜瓜品种河套蜜瓜为试验材料,在本实验室前期对甜瓜果实转录组分析的基础上,选择了在甜瓜生长期果实中表达量较高的CmMATE28基因进行了初步的功能分析,主要结果如下:（1）对CmMATE28进行了生物信息学分析,明确了CmMATE28蛋白为酸性亲水蛋白、存在跨膜结构域、无N端信号肽,其二级结构主要由α-螺旋、无规则卷曲、延伸链和β-折叠4种结构组成。（2）对CmMATE28基因进行表达谱分析,结果表明CmMATE28基因在甜瓜各个组织器官中均有表达,其中在两性花中的表达量最高,除此之外在根和子房中也有着较高的表达量。原位杂交结果显示CmMATE28基因在两性花花粉母细胞、雄蕊和雄花花药部位表达。亚细胞定位结果表明CmMATE28定位于细胞膜。（3）以甜瓜果肉总RNA为模板克隆得到长为1602 bp的CmMATE28基因cDNA序列。分别构建了CmMATE28基因的过表达载体pCAM-MATE28和编辑载体pHSE-MATE28。采用子房注射法将重组质粒进行了甜瓜的遗传转化,获得了T₁代过表达载体和编辑载体PCR检测阳性转基因植株。进一步通过自交筛选获得了3个T₃代CmMATE28过表达纯合株系。对CmMATE28过表达植株进行了表型观察,发现过表达CmMATE28基因导致甜瓜植株矮化、茎节变短、叶柄变短、叶面积变小,子房和雄花花托表皮毛卷曲绒毛化,果实变小,种子宽度减小。对CmMATE28-Oe植株和WT植株进行了干旱处理,发现干旱胁迫下CmMATE28-Oe植株高于WT植株,表现出了较强的耐旱性。（4）测量了WT和CmMATE28-Oe植株叶片的光合指标,结果显示CmMATE28-Oe植株叶片的气孔导度、细胞间隙CO₂浓度、蒸腾速率、净光合速率4项光合指标均高于WT。观测了WT和CmMATE28-Oe甜瓜果实的生长发育动态,绘制了生长曲线图,发现两者生长曲线趋势一致,呈现“S”型生长,发育前期果实膨大较快,中期变化平缓,成熟期果实大小趋于稳定。综上所述,本研究选取了在果实生长期特异高表达的CmMATE28为候选基因,明确了甜瓜CmMATE28在甜瓜植株发育、果实发育、雄花花托表皮毛发育、种子发育以及干旱逆境胁迫中的功能。更多还原

【Abstract】 Melon（Cucumis melo L.）is a horticultural cash crop that is widely grown all over the world,and has very long cultivation history in our country,and its fruit is the main edible part.Melon is rich in nutrition,containing vitamins A,B,C,magnesium,sodium and other nutrients,not only has a variety of medicinal functions,but also very important edible value.A number of studies have shown that Mutidrug and Toxic Compund Extrusion（MATE）can directly or indirectly regulate disease resistance,aluminum detoxification and iron homeostasis in plants.At present,although a large number of MATE protein genes have been identified,little is known about the regulation of MATE gene on plant growth and development.In this study,Hetao melon,a melon variety,was used as experimental material.On the basis of previous transcriptome analysis of melon fruits in the laboratory,CmMATE28 gene with high expression level in the flesh of melon during its growing period was selected for preliminary functional analysis.The main results were as follows:（1）The bioinformatics analysis of CmMATE28 showed that CmMATE28 was an acidic hydrophilic protein with transmembrane domain and no N-terminal signal peptide,and its secondary structure was mainly composed ofα-helix,random curling,extended chain and β-folding.（2）The expression profile of CmMATE28 gene was analyzed,and the results showed that CmMATE28 gene was expressed in all tissues and organs of melon.The expression level of CmMATE28 gene was the highest in bisexual flowers.Besides,the expression level of CmMATE28 gene was also high in root and ovary.In situ hybridization results showed that CmMATE28 gene was expressed in the stamens and anthers of bisexual flowers.Subcellular localization results showed that CmMATE28was localized to the cell membrane.（3）The cDNA sequence of CmMATE28 gene with length of 1602 bp was cloned using total RNA of melon pulp as template.The overexpression vector p CAM-MATE28 and editing vector p HSE-MATE28 of CmMATE28 were constructed respectively.Transgenic plants with T₁overexpression vector and modified vector were obtained by PCR after the genetic transformation of melon by ovary injection.Three T₃homozygous CmMATE28 overexpression lines were obtained by self-cross screening.Phenotypes of plants overexpressed by CmMATE28 were observed.It was found that overexpression of CmMATE28 gene resulted in dwarfization of melon plants,shorter stem nodes,shorter petiole,smaller leaf area,curled villous fur on ovary and male receptol,smaller fruit and reduced seed width.The CmMATE28-Oe plants and WT plants were treated by drought,and it was found that CmMATE28-Oe plants were higher than WT plants under drought stress,showing strong drought tolerance.（4）The photosynthetic indexes of WT and CmMATE28-Oe plant leaves were measured.The results showed that stomatal conductance,intercellular CO₂concentration,transpiration rate and net photosynthetic rate of CmMATE28-Oe plant leaves were higher than those of WT.The growth and development dynamics of WT and CmMATE28-Oe melon fruits were observed,and the growth curves were drawn.It was found that the growth curves of both melons showed the same trend,showing"S"type growth.The fruit expanded rapidly in the early stage of development,changed slowly in the middle stage,and the fruit size tended to be stable in the mature stage.In summary,this study selected CmMATE28,which is specifically highly expressed during fruit growth period,as a candidate gene,and clarified the functions of CmMATE28 in melon plant development,fruit development,male receptoid surface fur development,seed development and drought stress.更多还原