摘    要

SPL(SQUAMOSA promoter-binding protein-like)基因作为植物所特有的一类转录因子，在植物发育进程中起着多方面地调控作用。前期实验证实黄瓜CsSPL9和CsSPL13基因在雌花芽中积累量较高，很可能参与了性别分化调控，但其具体功能尚未明确。本研究分析和克隆这两个黄瓜SPL基因，构建相应的表达载体并导入农杆菌，利用花序浸润法遗传转化模式植物拟南芥，获取和验证纯合转基因拟南芥植株。最终，成功克隆了这两个基因并构建了相应的过表达载体，共筛选和鉴定出CsSPL9稳定高效表达的纯合转基因拟南芥株系7个，CsSPL13稳定高效表达的纯合转基因拟南芥株系10个。这为后续探索黄瓜CsSPL9和CsSPL13基因对拟南芥生长发育的影响提供了研究基础。85132

As one of the special plant transcription factors, SPL (SQUAMOSA promoter-binding protein-like) genes which are only found in plant play important roles in the process of plant development。 Previous experiments had verified that cucumber CsSPL9 and CsSPL13 genes accumulated more in female flowers bud than in male ones, which illustrated that these two genes were likely to be involved in Sex differentiation。 However, their specific function remains unclear。 In this project, we analyzed and cloned these two SPL genes, constructed over-expression vectors and introduced them into the agrobacterium。 Floral-dip method was used to transform model plant Arabidopsis and the transgenic homozygous plants were obtained and verified。 As a result, these two genes and over-expression vectors were acquired, 7 CsSPL9 homozygous transgenic plants and 10 CsSPL13 homozygous transgenic plants which expressed these genes stably and efficiently were selected。 This study provides research foundation to further exploration of the influence of cucumber CsSPL9 and CsSPL13 genes。

毕业论文关键词： 黄瓜； CsSPL； 转基因； 生长发育

Keyword: cucumber; CsSPL; transgene; plant development

目    录

1 引言 4

1。1 SPL基因的研究现状 4

1。2 研究的引出 4

2 材料与源Q于W优H尔J论K文M网WwW.youeRw.com 原文+QQ75201.,8766 方法 5

2。1 材料 5

2。2 主要试剂和仪器 5

2。3 基因克隆 5

2。3。1黄瓜培养及RNA提取 6

2。3。2 逆转录获取cDNA 6

2。3。3 以cDNA为模板进行基因克隆 7

2。3。4 PCR产物的T-A克隆 9

2。3。5 克隆载体鉴定 9

2。4 植物表达载体构建及验证 10

2。5 表达载体转化农杆菌 10

2。6 拟南芥的遗传转化 11

2。6。1 拟南芥的育苗及培养 11

2。6。2 花序浸润法转化拟南芥 11

2。6。3 筛选纯合拟南芥植株 12

2。7 转基因植株分子鉴定 12

3 实验结果 12

3。1 黄瓜CsSPL9和CsSPL13基因克隆 12

3。1。1 黄瓜CsSPL9和CsSPL13基因特异性PCR扩增 12

3。1。2 克隆载体酶切验证