摘要
植物是人类和牲畜所消耗的蛋白质的主要来源，但其营养品质往往不够完全。一般说，禾谷类作物种子蛋白质中的赖氨酸和色氨酸含量低，而豆类和蔬菜类蛋白质缺乏蛋氨酸和半胱氨基酸等含硫氨基酸。水稻是世界上最主要的粮食作物之一，稻米品质的优劣对人类健康具有重要的影响。稻米所含能量高，其中的贮藏蛋白易被消化吸收，但蛋白质含量较低，赖氨酸含量也缺乏，是稻米蛋白质中的第一限制必需氨基酸。所以，提高稻米中的必需氨基酸含量和蛋白质含量，平衡其营养品质，一直是遗传育种学家追求的目标之一。常规育种技术已在鉴别突变体以改良主要粮食作物蛋白质营养品质等方面获得了一些进展，但在水稻中却收效甚微。因此，寻求更为直接有效的途径去改良稻米的营养品质是非常必要的，分子生物技术的发展为改良稻米种子蛋白质营养品质提供了一条有效的技术路线。本研究即是要通过基因工程技术，在水稻种子胚乳中高效表达来自异源植物四棱豆的一个富含赖氨酸的蛋白质(Lysine-rich protein，LRP)，以提高稻米中的赖氨酸含量，最终达到改良稻米营养品质的目的。为此，重点开展了三个方面的研究工作，包括：(1)水稻主要贮藏蛋白基因启动子的分离及其功能鉴定，(2)LRP在转基因水稻种子中的表达研究，(3)谷蛋白与LRP融合蛋白在转基因水稻种子中的表达。主要研究结果如下：
首先，为了能用种子胚乳特异性表达的启动子来指导异源目标蛋白在转基因水稻种子中高效特异地表达，本研究根据已发表的水稻种子贮藏蛋白基因的核苷酸序列，从我国的水稻栽培品种了克隆了两个谷蛋白基因Gt1(GluA-2)和GluB-1以及一个醇溶蛋白基因RP5的启动子序列。在此基础上，将这3个启动子，连同水稻白蛋白RAG1启动子和玉米Ubi基因启动子，分别与GUS报告基因编码区构
vj扬州大学博士学位论文
建成嵌合基因，并导入同一个水稻品种中。对转基因水稻中的GUS活性分析表
明:(l)所有启动子都能指导GUS基因在转基因水稻种子的胚乳中表达，但不
同启动子的表达强弱有差别，其中醉溶蛋白和谷蛋白基因启动子明显优于RAGI
和〔恤例下万S启动子，玉米Ubi启动子也可高水平地指导GUS基因在转基因水稻
胚乳中表达;(2)谷蛋白启动子的胚乳特异性表达特征最明显，其次为醇溶蛋
白五尹巧，划 Gl几乎没有种子特异性表达特征;(3)在GUS编码区起始密码子
ATG前融合上醇溶蛋白N-端信号肤编码序列后，由尺尸j启动子引导的GUS融合
基因的转录活性明显增强，但却不能检测或只检测到极微量的GUS活性，其机
理尚不清楚。
其次，根据上述对不同启动子表达的比较，选用了谷蛋白和醇溶蛋白基因的
启动子及其5’调控序列与L即cDNA构建了多个嵌合基因，并都导入了同一个水
稻品种中，获得了大量转基因水稻植株。对转基因水稻植株种子中LRP嵌合基因
的表达进行了详细的分析，主要结论如下:(l)在水稻种子贮藏蛋白基因启动
子的指导下，LRP能在转基因水稻种子中高效表达并稳定积累，最高表达量可占
水稻盐溶性种子总蛋白的12%，不同转基因水稻植株种子中的表达量差别较大;
(2)谷蛋白Gtl启动子与醇溶蛋白尺尸J启动子都可指导LRP在转基因水稻种子
中高效表达，两者之间并没有明显的差别;(3)谷蛋白Gtl的信号肤编码序列
融合在LRP eDNA的5’端后，可促进LRP的转录，在翻译后能正确地加工去除
该信号肤序列，但最后的LRI，表达量并没有增多。(4)在转基因水稻种子中高
表达LRP后，对提高种子中的赖氨酸含量确有一定的作用，尤其是在提高蛋白质
结合总氨基酸中的赖氨酸含量有显著的效果;(5) LRP嵌合基因能在转基因水
稻中稳定遗传，Tl代转基因水稻植株间LRP嵌合基因的遗传分离大多符合3:1的
简单遗传模型;(6)为借助于农杆菌介导的共转化法培育可剔除抗性选择标记
转基因水稻，构建了含双T~DNA区的超双元载体，证明用超双元载体法或双载
体法都可将外源基因与抗性选择标记基因同时导入并整合到同一个水稻细胞的基
因组中，但双载体法中的共整合率明显低于超双元载体法;在由双载体法转化获
得的转基因水稻植株的自交后代中，已筛选获得了无抗性选择标记基因但含LRP
嵌合基因的转基因水稻植株。
刘巧泉:基因工程技术提高稻米赖氨酸含量vii
最后，在上述研究的基础上，又采用融合蛋白策略，将富含赖氨酸的L即融
合进水稻种子贮藏蛋白—谷蛋白的大小亚基中，设计并构建了3个Gt::LRP融
合蛋白;这3个融合蛋白基因与水稻本身谷蛋白基因启动子组成完整的融合基
因，经农杆菌介导转入了同一个水稻品种中，并对这些转基因水稻种子中融合蛋
白的表达作了详细的分析，主要结果如下:(1)3个融合蛋白基因在谷蛋白启动
子的指引下，都能在转基因水稻发育的种子中高效转录，形成与预期分子量大小
一样的稳定转录本，LRP cDNA在谷蛋白编码区中的插入位置与其转录效率没有
明显关系。(2)不同融合蛋白都能在转基因水稻种子中正常翻译，产生预期分
子量大小的融合蛋白;但不同融合蛋白的表达及其随后的加工方式有所不同。
LRP融合在谷蛋白的酸性亚基后，表达的融合蛋白能正常加工形?
Plants are the primary source of all proteins consumed by humans and livestock. However, most plant proteins are nutritionally unbalanced, because they are deficient in certain essential amino acids. In general, cereal proteins are low in lysine and tryptophan while legume and most vegetable proteins are deficient in methionine and cysteine. Rice (Oryza sativa L.), one of the leading food crops and the staple food of over half the world's population, is a very good and relatively cheap source of energy and protein. However, like other cereals, rice proteins are nutritionally incomplete due to their deficiency in threonine, tryptophan, especially lysine. Traditional breeding approach has been attempted to increase the lysine content in a few food crops, but so has not been successful in rice. Therefore, the development of a more efficient approach to enhance the lysine content of rice protein is of extreme importance. Recent advancements in molecular biotechnology offer new opportunities to improve the nutrit
ional quality of rice grains.
In this study, we attempt to genetically engineering rice to over-express a gene encoding the lysine-rich protein (LRP) from winged bean. LRP contains 10.7 mol% lysine; its expression in the endosperm of transgenic rice should raise the content of lysine in rice grains. The research work includes three complementary parts: 1) isolation and analysis of the expression of promoters for rice seed storage proteins; 2) expression of LRP in transgenic rice seeds; and 3) expression of glutelin and LRP fusion protein in transgenic rice grains.
For Part 1, three endosperm-specific promoters, namely, the glutelin Gt1 (also known as GlnA-2) and GluB-1 and the prolamin RP5 promoters, were isolated from the
Chinese rice varieties by PCR technique. These promoters, together with the rice albumin RAG1 and maize Ubi promoter, were fused transcriptionally to the GUS coding sequences. All these GUS chimeric genes were introduced into the same rice variety by /Igroforcterwm-mediated transformation. Results from GUS activity in transgenic rice plants showed that all the tested promoters could drive GUS expression in the endosperm of transgenic rice plants, but the expression level differed. The GUS activity driven by the promoter of rice glutelin or prolamin was significantly stronger than that by the promoter of rice RAG1 or CaMV 35S. The maize Ubi promoter also directed high GUS activity in the endosperm of transgenic rice plants. Analysis of the GUS activity in various tissues of transgenic rice plants showed that the control of endosperm-specific expression by glutelin promoters were more stringent than that by the prolamin RP5 promoter. In the case of RAG1 promoter, high GUS activity was detected in the stem and leaf of transgenic rice plants, indicating that the expression of RAG1 promoter is not endosperm-specific. It was interesting to find that the transcription of GUS chimeric gene could be enhanced after inserting the RP5 signal peptide coding sequence between the RP5 promoter and the GUS coding sequence; however, there was no or very low GUS activity in the endosperm of transgenic rice plants. Based on these results, the promoters of rice glutelin and prolamin are suitable for driving foreign proteins express in the rice endosperm.
For Part II, the 477-bp cDNA coding sequence of LRP was fused to the 5' regulatory sequence of the rice glutelin Gtl gene or prolamin RP5 gene with or without the signal peptide coding sequence. These chimeric genes were all transferred into a Chinese elite rice variety Wuxiangjin 9 via Agrobacterium. Many transgenic rice plants were regenerated. Northern and Immunoblot analysis showed that the LRP was highly expressed in the grains of transgenic rice plants. Stable accumulation of the 18-kDa LRP in transgenic rice seeds was demonstrated by Tricine SDS-PAGE as well as Western blot, with the highest expression amounting to 12% of the total salt-soluble seed protein. No significant difference in LRP expression, at both mRNA and protein levels, was obse
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