甜瓜果实蔗糖合成酶基因(SS)的克隆、表达分析及遗传转化

Cloning and Characterization of the Sucrose Synthase-encoding Gene from Muskmelon Fruit and Transformation

作者: 专业:蔬菜学 导师:于喜艳 年度:2010 学位:硕士  院校: 山东农业大学

Keywords

Muskmelon, Sucrose Synthases, Cloning, Expression, Transformation

        甜瓜(Cucumis melon L.)是世界性的重要园艺作物,糖分组成及含量是衡量甜瓜品质的主要依据之一。蔗糖、葡萄糖和果糖是甜瓜果实中主要的可溶性糖,其含量和组成对果实品质起重要作用,它们的含量和组成严格受蔗糖代谢酶调控,而蔗糖代谢酶包括蔗糖合成酶(SS)、蔗糖磷酸合成酶(SPS)和转化酶(Invertase),其中SS在蔗糖代谢中调控可逆反应,主要是起分解作用。SS活性的降低是蔗糖积累的前提条件,改变SS的活性能改变蔗糖的代谢模式。因此,利用现代分子生物学的方法改变蔗糖合成酶的活性对甜瓜品质育种具有重要的意义。本研究首先通过RT-PCR及3′、5′RACE技术克隆到了甜瓜果实蔗糖合成酶基因全长cDNA,研究了该基因在组织器官和果实发育过程中的表达特异性,然后构建了甜瓜果实SS基因的反义表达载体。以甜瓜自交系M01-3为材料,通过子房注射法将甜瓜果实反义SS基因进行了甜瓜遗传转化,目前已经收获转基因种子;同时用农杆菌介导法转化甜瓜,经PCR检测已经检测出5株转基因植株。研究的主要结果如下:1.通过RT-PCR及3′、5′RACE技术克隆到了甜瓜SS基因全长cDNA。测序结果表明该基因全长为2585bp,编码804个氨基酸,命名为CmSS1。2. Real-Time PCR结果表明:CmSS1在甜瓜根、茎、叶、雄花、雌花和成熟果实中均有表达。其中在根中的表达量最高,在果实中的表达量最低;在甜瓜果实发育不同时期中,随着果实发育时间的延长,CmSS1的表达量逐渐降低,在花后5 d时表达量最高,花后30 d表达量最低。3.将克隆在pMD18-T载体上的甜瓜CmSS1基因用BamHI和SalI酶切,酶切产物经0.1%琼脂糖凝胶电泳后回收1.4 kb的片段,将该片段与经相同的酶切后的植物表达载体pBI121进行连接,这样获得的重组质粒片段为反向插入35S启动子之后,TNOS终止子之前。经酶切和PCR检测表明,成功构建了甜瓜反义CmSS1基因的植物表达载体。4.利用子房注射法将反义CmSS1基因进行了甜瓜的遗传转化,现在已经收获种子大约1500粒,正在鉴定中。5.利用农杆菌介导法将反义CmSS1基因进行了甜瓜的遗传转化,经PCR检测,初步鉴定出5株转基因植株。
    Muskmelon (Cucumis melon L.) is one of the major horticultural crops. The content and composition of soluble sugars in fruit play key roles in muskmelon quality. Sucrose, glucose and fructose are the main soluble sugars。In melon fruit, the accumulation and composition of the soluble sugars in melon fruit are controlled by sucrose metabolism enzymes including sucrose synthase (SS), sucrose phosphate synthase (SPS) and acid invertase (AI). The previous studies showed that the SS played a key role in sucrose metabolism in muskmelon fruit. Therefore, cloning and characterization of SS encoding gene from muskmelon is necessary to toward the genetic improvement of the quality of muskmelon fruit.In this study, we cloned the SS full-length cDNA from muskmelon fruit by RT-PCR and 3’, 5’ RACE and this clone was named CmSS1. The expression levels of CmSS1 in the tissue-organ and during fruit development in muskmelon were analyzed by Real-time RT-PCR. We constructed the antisense expression vector of CmSS1 and transformed it to muskmelon by ovary injection method and Agrobacterium-mediated method . Five transgenic plants were obtained by PCR screening. The main results are as follows:1. The full-length cDNA of SS gene from muskmelon fruit were cloned by RT-PCR and 3′, 5′RACE. The sequence analysis showed that SS gene contained 2585 bp and coded 804 amino acid residues.2. Real Time PCR analysis indicated that CmSS1 transcripts are easily detected in the leaves, stems, mature fruit, roots and flowers. The highest expression level existed in root, However, in mature fruit showed the lowest level. With the development of fruit, the expression level of CmSS1 showed a decreased trendy. CmSS1 mRNA strongly expressed in 5 DAP fruit, the lowest level existed in 30 DAP fruit.3. A 1.4 Kb fragment of the CmSS1 was removed from pMD18-T vector by BamHⅠand SalⅠdigested and the fragment was ligated into the plant expression vector pBI121 in the antisense orientation. We constructed the antisense expression vector of CmSS1 successfully by PCR and digested with restricted enzymes.4. Anti-CmSS1 was transformed to muskmelon by ovary injection, at present we got the injected seeds.5. Anti-CmSS1 was transformed to muskmelon by Agrabactria-mediated and five transgenic plants were obtained by PCR screening.
        

甜瓜果实蔗糖合成酶基因(SS)的克隆、表达分析及遗传转化

中文摘要10-12
Abstract12-13
1 引言14-36
    1.1 甜瓜果实糖的变化及关键代谢酶14-17
        1.1.1 甜瓜果实发育过程中糖含量的变化14-15
        1.1.2 甜瓜果实不同部位糖分的分布15
        1.1.3 蔗糖代谢相关酶15-17
    1.2 蔗糖合成酶基因的研究进展17-22
        1.2.1 蔗糖合成酶(SS)的基本性质17-18
        1.2.2 目前公布的SS 基因家族18-19
        1.2.3 SS 基因的生物学功能19-22
            1.2.3.1 影响库强、调控输入蔗糖多少和代谢蔗糖的能力20
            1.2.3.2 参与细胞分化与纤维细胞壁合成20-21
            1.2.3.3 调节淀粉合成21
            1.2.3.4 提高植物抗逆性21-22
        1.2.4 SS 基因的遗传转化22
    1.3 反义技术的应用22-25
        1.3.1 反义基因的基本概念22-23
        1.3.2 反义 RNA 技术的作用机制23-24
        1.3.3 反义 RNA 技术在植物中的应用24-25
    1.4 甜瓜组织培养与遗传转化研究进展25-35
        1.4.1 转化方法25-28
            1.4.1.1 农杆菌介导的基因转移25-26
            1.4.1.2 外源 DNA 直接导入26-27
            1.4.1.3 种质系统的基因转移27-28
        1.4.2 甜瓜的组织培养28-34
            1.4.2.1 甜瓜再生体系建立的影响因素28-31
            1.4.2.2 甜瓜组织培养国内外研究现状31-32
            1.4.2.3 对甜瓜的遗传转化32-34
        1.4.3 转基因植株的鉴定34
        1.4.4 存在的问题与展望34-35
    1.5 研究目的与意义35-36
2 材料与方法36-53
    2.1 材料36-38
        2.1.1 材料、试剂及仪器36
        2.1.2 再生体系培养基36-38
        2.1.3 PCR 引物38
    2.2 试验方法38-53
        2.2.1 甜瓜果实总RNA 的提取38-39
            2.2.1.1 提取RNA 所用器皿的处理及溶液配制38-39
            2.2.1.2 异硫氰酸胍—酚—氯仿一步法提取RNA的方法和步骤39
        2.2.2 甜瓜蔗糖合成酶(SS)基因的克隆39-41
            2.2.2.1 CmSS1 基因中间片段的克隆39-40
            2.2.2.2 CmSS1 基因3′端的克隆40
            2.2.2.3 CmSS1 基因5′端的克隆40-41
        2.2.3 琼脂糖凝胶电泳41
        2.2.4 目的片段的回收41-42
        2.2.5 连接反应42
        2.2.6 转化42-45
            2.2.6.1 用CaCl_2 法制备感受态细胞42-43
            2.2.6.2 热激法转化E.coli.DH5α43-44
            2.2.6.3 重组子的PCR 及酶切鉴定44-45
        2.2.7 测序及序列分析45
        2.2.8 CmSS1 表达特异性分析45-46
        2.2.9 反义表达载体的构建46-49
            2.2.9.1 目的基因片段的获得47-48
            2.2.9.2 表达载体PBI121 的酶切48
            2.2.9.3 目的基因和植物表达载体酶切后的片段回收48
            2.2.9.4 连接反应48
            2.2.9.5 热激法转化E.coli.DH5α48
            2.2.9.6 PCR 扩增鉴定反义表达载体48-49
            2.2.9.7 酶切鉴定49
        2.2.10 农杆菌感受态细胞的制备与转化49-50
            2.2.10.1 根癌农杆菌LBA4404 细胞的制备49-50
            2.2.10.2 转化农杆菌50
        2.2.11 农杆菌介导法转化甜瓜50-51
        2.2.12 子房注射法转化甜瓜51
            2.2.12.1 提取重组载体质粒51
            2.2.12.2 甜瓜子房的处理51
            2.2.12.3 子房注射51
        2.2.13 鉴定转基因植株51-53
            2.2.13.1 CTAB 法提取基因组51-52
            2.2.13.2 PCR 鉴定52-53
3 结果与分析53-62
    3.1 甜瓜果实RNA 的提取53
    3.2 CmSS1 基因的克隆53-54
        3.2.1 CmSS1 基因中间片段的克隆53-54
        3.2.2 CmSS1 基因3′端的克隆54
        3.2.3 CmSS1 基因5′端的克隆54
    3.3 CmSS1 基因的氨基酸序列同源性分析54-55
    3.4 CmSS1 表达特性分析55-57
        3.4.1 CmSS1 在甜瓜不同组织器官中的表达特性分析55-56
        3.4.2 CmSS1 基因在甜瓜果实不同发育时期的表达特性分析56-57
    3.5 反义表达载体的构建57-58
    3.6 反义表达载体转化农杆菌58-59
    3.7 甜瓜不同外植体部位的再生能力59
    3.8 外植体的再生59-60
    3.9 转基因甜瓜的DNA 鉴定60-62
4 讨论62-66
    4.1 关于甜瓜果实总RNA 的提取及对策62
    4.2 反转录反应62
    4.3 RT-PCR 优化及Real-time PCR62-63
    4.4 载体的构建63-64
    4.5 关于甜瓜的转化因素64-65
        4.5.1 不同外植体和年龄64
        4.5.2 植物激素64
        4.5.3 农杆菌的浓度和侵染的时间64-65
    4.6 子房注射法获得转基因植株65-66
5 结论66-67
参考文献67-78
致谢78-79
攻读硕士期间发表论文79
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